36

Chapter 3 - Immunodiagnosis
Frixos Paraskevas
John Foerster
Immunoassays
Basic Principles
Assays Based on Primary Binding of Antibody to Antigen
Assessment of Changes in Serum Proteins
Serum Immunoglobulin
Complement
Autoantibodies and Autoimmune Diseases
Investigation of Connective Tissue Disease
Evaluation of Disease Activity
Circulating Immune Complexes
Monoclonal Antibodies
Lymphocyte Function
Mitogenic Stimulation
Mixed Lymphocyte Reaction
Lymphocyte Cytotoxicity

Immunoassays
Basic Principles
All immunologic assays are based on either the primary binding of
antibody to antigen or the secondary phenomena resulting from such
interaction. Some assays are designed to detect the presence of
antibodies against known antigens, such as DNA or red cell antigens. In
other instances, tests are designed to identify specific antigens by
using antibodies of defined specificity, such as anti-immunoglobulin
(Ig), anticomplement, and antiviral antibodies.
Methods that depend on the primary binding of antibody to antigen
include radioimmunoassays (RIA), enzyme-linked immunoassays
(ELISA), and fluorescent antibody techniques. In all of these methods,
the binding of the antibody to a given antigen is evaluated by an easily
identifiable label attached to the antibody, including radioactive
elements, enzymes, or fluorescent dyes. Among the most important
secondary phenomena used in immunoassays are precipitation,
agglutination, and complement fixation.
Precipitation

This process occurs when antibody binds to antigen and forms large
insoluble aggregates. In a typical quantitative precipitin reaction,
various concentrations of antigen are added to a series of tubes
containing a constant amount of antibody and the precipitate that
forms is collected and measured. When the amount of precipitate is
plotted as a function of the antigen dose, a precipitin curve is obtained.
As the antigen concentration increases, the amount of precipitate

increases, until it reaches a plateau; it then decreases as more antigen

is added (Fig. 3.1) . The shape of the precipitin curve is understood if
we analyze the supernatant in each tube for the two immunologic
reagents. Before peak activity is reached, the supernatants contain
free antibody, whereas the supernatants in the tubes with decreasing
amounts of precipitate after peak activity contain free antigen. Neither
free antigen nor free antibody are detected in the supernatants from
the tubes with the maximal amount of precipitate.
The first zone is known as the antibody-excess zone, the
37

Figure 3-1 The precipitin reaction. Increasing amounts of antigen are added in a
series of tubes containing a constant amount of antibody. At low antigen
concentrations, the amount of precipitate (ppt) progressively increases and the
supernatant contains free antibody (Y) (antibody excess zone). At high antigen
concentrations, precipitation decreases and the supernatant contains free antigen (o)
(antigen excess zone). At intermediate antigen concentrations, the precipitate
reaches a maximum level and no antibody or antigen is present in the supernatant
(equivalence zone). The precipitin curve is explained on the basis of the size of
complexes formed according to the "lattice theory."
second as the equivalence zone, and the third as the antigen-excess zone. The nature of the precipitin curve
is explained by the lattice theory, which states that each antibody molecule can bind to more than one
antigen molecule and vice versa. With increasing amounts of antigen, the aggregates increase in size in the
antibody-excess zone, as antibody and antigen molecules form large lattices that precipitate when they
reach a critical size. In the antigen-excess zone, each antibody molecule is surrounded by many antigen
molecules, and lattices are not formed or they are too small to precipitate; therefore the amount of
precipitate decreases. Thus, the exact quantity of antibody in an antiserum can be measured only at
equivalence, because in the two other zones, soluble complexes containing antibody will not precipitate.
When the amount of aggregate is too small or the antibody is nonprecipitating, precipitation is facilitated by
the addition of ammonium sulfate at 50% saturation (Farr assay).

Many immunoassays are based on precipitin reactions occurring in gels

rather than in liquids. In 1946, Oudin described a precipitation reaction
in which antigen was diffused into tubes filled with agar that had been
impregnated with antibody. In this single immunodiffusion technique, a
precipitin band is formed at a point where antigen and antibody are at
optimal concentrations. The position of the band moves as antigen
diffuses into the gel, and its final position is a function of the antigen
concentration. The final position of the band is influenced also by the
size and shape of the antigen, which determine the diffusion coefficient
of the molecule as well as the concentration and affinity of the
antibody in the gel. The single radial (as opposed to the Oudin linear)
immunodiffusion technique has found wide application in techniques
for the quantitation of human serum proteins (see subsequent
discussion). In contrast to the single diffusion, antigen and antibody
both may be allowed to diffuse into a gel simultaneously. This double
diffusion technique was developed by Ouchterlony and bears his name.
In the Ouchterlony technique, the antigen and antibody are placed in

wells punched into agar gels and form a precipitin line as they diffuse
against each other. This technique is used also to determine the
identity of unknown antigens by comparing the fusion pattern of the
precipitin lines formed by a known and an unknown antigen. When two
precipitin lines fuse, they are considered to be generated by
substances that are largely immunologically identical (pattern or
identity), whereas lines that cross are considered to be derived from
substances that are nonidentical. Partial identity is suggested when
only one line continues beyond the meeting point.
Agglutination

The essential difference between agglutination and precipitation is the

size of the antigen. Precipitation involves soluble macromolecules,
whereas agglutination involves a particle or cell. Agglutination occurs
when an antibody simultaneously crosslinks at least two particles, such
as red cells. When antibody is in great excess, crosslinking of particles
may not take place, because the antibody binds only to one particle, a
reaction known as a prozone phenomenon. A prozone reaction
sometimes results from interference by an anti-Ig antibody. Antibodies
that bind to particles but do not cause agglutination are sometimes
referred to as incomplete antibodies (Chapter 46) , but this term does
not indicate a structural defect of antibodies. Nonagglutination also
may be caused by low affinity antibodies, or antibodies bound to the
same particle by means of both combining sites, as a result of which
no crosslinking between adjacent particles occurs (monogamous
binding). Nonagglutination also may be the result of physicochemical
conditions existing at the surface of the particle, because incomplete
antibodies can agglutinate cells treated by enzymes. Bacteria or red
cells may be agglutinated by antibodies directed against their own
antigens. In other tests, red cells simply serve as indicators or carriers
of chemicals or proteins that have been coupled to them by treating
the red cells with tannic acid or chromium chloride to facilitate their
adsorption on the surface of the red cells. The former agglutination is
called active, and the latter is known as passive. Passive
hemagglutination is more sensitive than precipitation techniques and
can detect antibodies at concentrations of 0.001 g/ml. Both active and
passive agglutination may be brought about by one of two
mechanisms. In direct agglutination, the particles are agglutinated
38

directly by the antibodies in question; indirect agglutination is mediated by a second antibody directed
against the first antibody (i.e., by an anti-Ig antibody).

Agglutination techniques are considered semiquantitative, and the last

dilution of the serum that causes agglutination is defined as the titer of
the antiserum. When using particles coated with specific antigens,
inhibition of agglutination may be used as a sensitive assay for the

presence of the specific antigen in solution, because such antigens

bind to antibodies and prevent them from interacting with the same
antigen coupled to the indicator particle. Agglutination reactions find
their widest application in the detection of antibodies against red cells
(Coombs' test, Chapter 46 ) and immunoglobulins (see the section
addressing rheumatoid factor). Passive hemagglutination is used to
detect antibodies to drugs, chemicals, and other substances.
Hemolytic Assays and Complement Fixation

Antibodies capable of fixing complement inflict damage to cell

membranes and cause lysis of the cell (Chapter 21) . A typical
hemolytic assay involves the use of sheep red blood cells sensitized
with rabbit anti-red cell antibody. For the detection of complement
levels, serial dilutions of the patient serum are added to the red cells.
The degree of lysis is calculated by the spectrophotometric
measurement of the hemoglobin released. The complement level is
reported in CH50 units. One CH50 unit corresponds to the amount of
complement necessary to lyse 50% of the red cells. The CH50 level is
used because, at this point, a nearly linear relationship exists between
the degree of hemolysis and the amount of complement in solution.
The complement fixation tests are sensitive and can be used for
measuring antigen or antibody concentrations. They are based on the
fact that an antigen-antibody system consumes ("fixes") complement,
which therefore is unavailable to lyse sensitized red cells. The
remaining active complement is titrated with sensitized red cells and
the degree of lysis (measured by the amount of hemoglobin released)
is inversely proportional to the amount of complement fixed, which in
turn depends on the antibody or antigen concentration in the mixture.
Cytotoxicity assays are used widely in HLA typing procedures (see
Chapters 26 and 28 ) and in other immunoassays.
Assays Based on Primary Binding of Antibody to Antigen
Some of the most important advances in immunoassays have been
made as a result of the ability to detect the primary antigen-antibody
interaction in the absence of any secondary effects. This result is
achieved by using labels attached to either of the two reactants, but
most often to the antibody. Although a large variety of labels are used,
they fall into three main categories: those that emit light, those that
emit radioactivity, and enzymes that generate an easily identifiable
product from their substrates. These reagents distinguish the following
broad categories of assays: immunofluorescence assays,
radioimmunoassays (RIA), enzyme-linked immunoassays (ELISA), and
Western blotting.
Immunofluorescence

Certain substances have the capacity to absorb light at a certain

wavelength and emit light of a characteristic longer wavelength in the
visible part of the spectrum. These substances, fluorochromes,
therefore have characteristic absorption and emission spectra.
Fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate, phycoerythrin, and others are some of the
fluorochromes commonly used. FITC can be attached to protein
molecules such as antibodies through free amino groups of the lysine
molecules at alkaline pH. FITC has an absorption maximum at 490 to
495 nm and emits green light of 517 nm; rhodamine absorbs at 550
nm and emits red light at 580 nm. In fluorescence microscopy,
excitation filters are used to select an exciting wavelength as close to
the excitation maximum as possible, and barrier filters are interposed
to remove all but the emitted light. For greater sensitivity in
fluorescence microscopy, the excitation light is allowed to reach the
specimen from above through the objective lens (epifluorescence of
Ploem) while the emitted light is allowed to reach the eye of the
observer through a dichroic mirror. Epifluorescence is particularly
useful when the intensity of fluorescence is weak. Two variations of
immunofluorescence techniques are recognized (Fig. 3.2) . In the direct
method, the conjugated specific antiserum is applied directly to the
tissue under investigation. In the indirect method, which is used to
detect antibodies in sera or body fluids, the binding of the antibody is
demonstrated by the use of an anti-Ig antiserum conjugated with a
fluorochrome.
Radioimmunoassays (RIA)

The RIA is based on competitive binding between a ligand (which could

be a protein, drug, etc.) and its binder, which most often is an antibody.
In RIA, the substance analyzed, technically known as a ligand or
analyte, competes with a radioactively labeled substance of similar
structure, known as the label or tracer, for binding to a limited quantity
of specific antibody. The higher the concentration of the ligand in the
sample, the more label remains unbound (Fig. 3.3) . The bound and
free label can be separated by a number of methods, such as salting
out with ammonium sulfate or the
Figure 3-2 The principles of fluorescent antibody techniques. Direct method:
Specific antibody (Ab) (Y) labeled with a fluorochrome (* ) is applied over tissues or
cells to detect an antigen. Indirect method: Serum from patients containing an
antibody is layered over tissues. The bound antibody is then detected by an anti-Ig
conjugated with a fluorochrome.

39

use of a second antibody, usually an anti-Ig, both of which precipitate the bound fraction of the ligand. Free
ligand also can be removed by absorption on charcoal or ion exchange resins. In other forms of RIA, the
ligand is bound to plastic surfaces or beads (solid phase RIA).

An important aspect of all RIA is the construction of a calibration curve

with standards of known concentrations. Specimens with known
concentrations of ligand are always incorporated into an RIA test for
quality control. The greatest sensitivity that can be achieved by RIA is
10-14 mol/L corresponding to approximately 6 106 molecules/ml.
Enzyme Immunoassays

Radioactive labels have been replaced by enzymes in many

competitive ligand binding assays. The enzymes used most commonly
are horseradish peroxidase and alkaline phosphatase. This type of
assay provides an advantage over RIA in that exposure to radioactivity
is avoided and no large and expensive instruments are needed to
measure radioactivity. We distinguish two categories of enzyme
immunoassays: heterogeneous when the separation of free from bound
ligand is required, and homogeneous when it is not required.
A heterogeneous enzyme immunoassay method that is widely used is
the enzyme-linked immunosorbent assay (ELISA). In this method, the
antigen or the antibody is adsorbed to a plastic surface, such as a
microtiter well, or magnetic particle or plastic bead. The attachment
facilitates separation of bound and free labeled reactants. In the
indirect ELISA technique, the antigen is coupled to the solid surface
and is used to bind antibody present in the sample ( Fig. 3.4 A). An
enzyme conjugated anti-Ig antibody then detects the antibody bound
to the antigen. The use of class-specific anti-Ig antibodies allows the
detection of antibodies of various isotypes.
Figure 3-3 The principles of radioimmunoassay (RIA). In fluid phase RIA, specific
antibody is added to a fluid that contains the antigen under detection, together with
a known concentration of the same antigen labeled radioactively ( * ). The antigen in
the fluid competitively displaces the labeled antigen and its concentration is
estimated from a standard curve. In the solid phase RIA, the antibody is fixed on a
solid surface or plastic bead.

Figure 3-4 The principles of the enzyme-linked immunosorbent assay (ELISA). Two
basic variations are the indirect method (A), and the double antibody or sandwich
method (B).

A second approach is commonly used for the detection of antigens. In

this double antibody or sandwich variation, the antibody is coated on
the solid phase ( Fig. 3.4 B), and residual "sticky" areas are covered by
albumin to prevent any subsequent non-specific attachment of the
antigen. The sample is added to the well and is allowed to react with
the antibody, and the binding of the antigen (ligand) is detected by a

second antibody directed against a different epitope of the antigen.

This second antibody is labeled with an enzyme and an appropriate
substrate is added that is converted to a product usually measured by
a spectrophotometer. The amount of the product generated is a
function of the concentration of the enzyme which depends on the
concentration of the ligand bound to the antibody.
A homogeneous enzyme immunoassay known as enzyme multiplied
immunoassay technique (EMIT) is widely used for drug, hormone, or
metabolite assays. In this method, the antibody against the analyte
(ligand) is added together with the enzyme substrate to the patient's
sample. The antibody binds to the analyte. In a second step, an
enzyme-analyte conjugate is added that also binds with the antibody
(present in excess). This binding affects the enzyme activity either as a
result of conformational change of the enzyme or interference of the
access of the substrate to the enzymatic active site. The change in
activity of the enzyme is measured and is directly proportional to the
concentration of the analyte.
40

Assessment of Changes in Serum Proteins

In the detection of serum protein abnormalities by immunoassays, the
serum protein serves as the antigen and is quantitated by the use of
specific antibodies. Serum Ig, complement components, and other
proteins usually are quantitated by using such techniques. In other
instances, the serum provides the antibody directed against
endogenous or exogenous antigens, such as nuclear antigens, blood
cells, bacteria, or viruses.
Serum Immunoglobulin (Ig)

[1]

Abnormalities of serum Ig can be either quantitative or qualitative.

Quantitative abnormalities of serum Ig are either increases (polyclonal
or monoclonal) or decreases involving all Ig classes or only some of
them. Qualitative abnormalities involve abnormal physicochemical
properties, such as aggregation leading to an increase of blood
viscosity, or a decrease in solubility such as is found in cryoglobulins.
Protein Separation
Zone Electrophoresis.

This nonimmunologic technique forms the basis for many immunologic

tests. It depends on the observation that proteins can be separated in
an electric field on the basis of their electric charges. Proteins exhibit
an amphoteric behavior; that is, they act both as acids and bases. The
-COOH and -NH2 groups are ionized, the former acquiring a negative
charge (loss of H+ ) and the latter a positive charge (acquisition of H+ ).

The net charge of the protein (sum of negative and positive charges)
depends on the pH of the solution. At a certain pH known as the
isoelectric point, the protein has an equal number of positive and
negative charges. At a pH higher than the isoelectric point, the protein
carries a net negative charge, and a pH lower than the isoelectric point
results in a net positive charge. The isoelectric point of
immunoglobulins is 6.4 to 7.2; that of albumin is 4.8. Therefore, when
serum electrophoresis is carried out with a buffer of pH 8.0, albumin is
strongly negatively charged and the Ig only weakly. As a result,
albumin moves further toward the positive electrode than the Ig.
Electrophoretic mobility also depends on the size and shape of the
molecule, the ionic strength of the buffer, and the nature of the
supporting medium. Serum may be subjected to electrophoresis in the
free liquid form or on a supporting medium. The latter is known as
zone electrophoresis and is the method used most commonly in clinical
laboratories. The most commonly used supporting media are agar or
agarose gels. After migration of the proteins, the separated fractions
are stained by dyes that bind to the protein (such as amido black or
ponceau S) and the density of the band is measured and recorded.
Normally, a given Ig class is represented by a wide band, because it is
electrophoretically heterogeneous. A single plasma cell synthesizes a
distinct Ig molecule; therefore, the heterogeneity of Ig is based on the
heterogeneity of contributing plasma cells ( Fig. 3.5 A). Increased
serum levels may reflect increased activity by all clones of plasma cells
Figure 3-5 The electrophoretic heterogeneity of serum Ig. A, Each plasma cell
in the body synthesizes an electrophoretically distinct Ig molecule, which
explains the electrophoretic heterogeneity of normal serum Ig. B, In proliferative
diseases of plasma cells, one of the clones is affected, resulting in the accumulation
of large amounts of Ig molecules with the same mobility. This accumulation creates
the well-known "spike" observed during electrophoresis of serum of patients with
multiple myeloma (monoclonal gammopathy). ALB, albumin.
normally present, leading to a polyclonal increase such as is seen in association with rheumatoid arthritis or
infections. Monoclonal increases result from expansion of a single clone, such as occurs in multiple
myeloma ( Fig. 3.5 B). A polyclonal increase is seen as a wide band, similar to a normal band in shape but
of greater width and density. In contrast, a monoclonal protein forms a heavy band with narrow mobility
and is called an M-band (M for myeloma). Zone electrophoresis is a more accurate technique for the
quantitation of IgG M-bands, because immunologic techniques
41

quantitate not only the monoclonal but also the residual normal IgG. For monoclonal IgA and IgM,
quantitation is more accurate with immunologic methods since M-bands from these Ig classes overlap with
beta-globulins and errors due to normal residual IgA or IgM are negligible.
Capillary Electrophoresis (CE).

CE is a recently developed method in which electrophoresis is carried

out in a small-bore (25-75 mum) fused silica capillary tube . Proteins
[2]

are separated within a liquid phase much like that used by Tiselius, the
founder of electrophoresis, except that the tube is much smaller in
diameter. Reduction of the capillary bore improves resolution and
decreases separation times. The capillary tube is open on both ends,
which are immersed in two buffer reservoirs and high voltage is
applied (about 10,000 volts). During separation, the negatively
charged proteins move towards the anode. However, the ionizable
groups of the fused silica capillary interact with the positively charged
ions of the buffer, and when the voltage is applied the cations move
toward the cathode creating a flow of buffer known as
electroendosmotic or electro-osmotic flow. Because the
electroendosmotic flow is stronger than the electrophoretic mobility,
the proteins are carried toward the cathode. Peaks with lower mobility
will be in front (i.e. Ig), while those with higher mobility will be located
at the end.
The sample is introduced either by pressure or by electrokinetic
injection. Boundaries formed from the protein fractions are detected by
light from a light source directed across the capillary tube. The
wavelength for protein separation is near 200 nm, which corresponds
to the absorptivity of the peptide bond of the protein molecules. The
absorbance is proportional to the number of peptide bonds and thus
correlates to the protein concentration. There is no visual record of the
protein separation (as in agar gel electrophoresis), except for the
graphic tracing.
When paraproteins are detected by agar gel electrophoresis, they are
classified by immunoelectrophoresis or by immunofixation. With CE,
identification of paraproteins is achieved by
immunofixation/subtraction. This method involves incubation of the
sample with antibodies of known specificity bound to a solid surface.
After absorption, the sample is electrophoresed again and the
paraprotein is identified by the specific antibody that has removed the
paraprotein peak. Comparison of the tracings before and after
immunofixation provides the concentration of the paraprotein. For
complete identification of the paraprotein, the serum is absorbed with
antibodies specific for IgG, IgA, IgM, kappa, and lambda light chains.
A commercially available system (Beckman) consists of seven
capillaries and electronic signal processing components that send the
data to the operator's computer where they are converted to graphic
or numeric formats. Overall running time is about 6 minutes.
Immunoelectrophoresis (IEP).

A more satisfactory separation of serum proteins is achieved by IEP, a

technique that permits differentiation of proteins on the basis of their
electrophoretic and immunologic properties. IEP of human serum is
carried out in two stages. During the first stage, human serum proteins
are separated by zone electrophoresis in agar gels. Electrophoresis is

run with the reservoirs containing barbital buffer of pH 8.0 and 0.05
ionic strength, and the agar gel is made in a barbital buffer of pH 8.0
and 0.025 ionic strength. Agar or agarose possesses ionized groups
that are fixed in contrast to the ions of the solution. When the current
is applied, the mobile ions, which are highly hydrated, are free to move
and give rise to a movement of the solvent as well. This movement of
the buffer is known as electroendosmosis. At pH 8.0, the
electrophoretic mobility of the serum proteins carries them toward the
positive electrode, while electroendosmosis carries the buffer in the
opposite direction. Since Igs are only weakly charged and their
electrophoretic mobility is weak, electroendosmosis carries them
behind the well of origin. The spread of Ig by electroendosmosis
actually facilitates detection of M-proteins present in low
concentrations. After this separation, a trough is cut into the gel and is
filled with an antiserum to human serum proteins. These antibodies are
allowed to diffuse into the gel and react with the separated human
serum protein fractions. Within a few hours, precipitin lines or arcs
form at points of contact between the electrophoretically separated
proteins and the specific antisera that have diffused from the trough
(Fig. 3.6) . Whether a line or an arc forms depends on the homogeneity
of the protein in terms of its electrophoretic mobility (Fig. 3.7) . A
homogeneous M-protein forms a precipitin arc whereas normal,
heterogeneous Ig forms a straight precipitin line only slightly curved at
the cathodic end.
Immunoelectrophoresis has many useful applications, the most
important of which is the detection of myeloma proteins (M-proteins).
These proteins are electrophoretically homogeneous and are present in
the serum of patients suffering from plasma cell dyscrasias such as
multiple myeloma and macroglobulinemia, lymphoreticular neoplasms,
autoimmune diseases, and epithelial or connective tissue neoplasms.
When a myeloma protein is present, the straight line of the normal
immunoglobulin is replaced by a prominent arc similar to that given by
albumin. Use of a class-specific antiserum allows identification of the
class of the myeloma protein. With specific antisera, the light chain
type (kappa or lambda) also can be identified. Typical examples of the
investigation of an M-protein are shown in Figure 3.8 ; others are
provided in Chapters 98 and 99 .
Another important application of IEP is in the identification of Bence
Jones protein in the serum and urine in certain cases of myeloma in
which no M-band is apparent on serum electrophoresis. This form of
myeloma (sometimes also called light chain disease) accounts for
about 20% of all cases of multiple myeloma (Chapter 99) . Free light
chains (Bence Jones proteins) in the serum often cannot be detected
by zone electrophoresis, especially if they are present in small amounts
or overlap in mobility with other fractions.

Immunoelectrophoresis is useful as a screening test for various

immune deficiency diseases. In extreme cases, the normal precipitin
line of IgG, IgA, or IgM does not form at all; in other cases, the line of
one or more of the Igs is greatly diminished.
Immunofixation.

Immunofixation combines the techniques of electrophoresis and

immunoprecipitation. After separation of the serum proteins by
electrophoresis, monospecific
42

Figure 3-6 Immunoelectrophoresis of normal human serum. The IgG (G) and
IgA (A) lines of normal human serum (NHS) (top pattern) are straight and only
slightly curved at their cathodic (-) end. Middle pattern is that of serum from a
patient with IgG myeloma (IgG-MM). Arrow, the myeloma arc. Bottom pattern is that
of serum from a patient with IgA myeloma (IgA-MM) showing a prominent myeloma
arc (arrow) while the IgG line is normal.

Figure 3-7 The two basic patterns of precipitin lines in immunoelectrophoresis.

A precipitin line forms an arc when the protein is electrophoretically
homogeneous, i.e., albumin, myeloma proteins. With proteins that are
heterogeneous (i.e., spread over a wider area during electrophoresis), such as normal
Ig, the precipitin line is straight. The change from a straight line to an arc of the Ig
precipitin lines forms the basis for the diagnosis of the presence of a myeloma
protein.
antiserum is placed over the separated fraction under investigation to form a precipitate. Nonprecipitated
proteins are removed by washing, and the immune precipitate that remains is stained (Fig. 3.9) . This
method has broad applications in the identification of M-proteins present in small amounts that are difficult
to detect by other methods, even by immunoelectrophoresis. Sensitivity of the method has been reported to
range between 5-10 mug/ml. The antiserum must be used at an optimal dilution to ensure that antigen and
antibody are at equivalence.
Quantitative Measurements
Single Radial Immunodiffusion

[3] [4]

This technique is used most commonly for the quantitation of a

number of serum proteins. Highly specific antisera are incorporated
into an agar gel and human serum is placed in a small well. As the
serum diffuses into the gel, it reacts with the antibody to form a circle
of precipitation around the well. Two methods can be used to calculate
the concentration of protein in the test material. The end-point method
requires that precipitation rings reach their maximal size. In this case,
the area of precipitation is proportional to the protein concentration .
If the method depends on ring measurements taken at a fixed time
before the end point, the diameter of the ring is proportional to the
logarithm of the protein concentration. The values of the unknown sera
are calculated from a standard curve. The advantage of this method
lies in its simplicity and its ability to quantitate a single protein derived
[3]

[4]

from a complex mixture such as human serum. The method, however,

is not always reliable. Because high molecular weight substances
diffuse more slowly, falsely low values may be obtained in the
presence of polymeric Ig, such as those found in association with
certain plasma cell dyscrasias, or in the presence of high molecular
weight immune complexes, such as those found in patients with
cryoglobulinemia (Chapter 103) or rheumatoid arthritis. Falsely high
concentrations may be obtained in the presence of monomeric IgM
molecules, which diffuse more rapidly than their normal pentamers, or
in the presence of anti-Ig molecules, as occurs, for instance, in patients
suffering from IgA deficiency. Such antibodies cause the formation of
immune complexes that are unrelated to the intended measurements.
The sensitivity of this technique is in the range of 1 to 3 mug/ml of
antigen.
Electroimmunodiffusion (EID).

Several variations of this technique are in use. In one type, the antigen
and antibody are electrophoresed simultaneously in opposite directions
(one-dimensional double EID). In another, the antibody is
43

Figure 3-8 Examples of the detection of "myeloma proteins" in human

sera. Serum is fractionated by electrophoresis and the values of the
main
fractions are measured (boxes, upper part) while the levels of the three
main Ig classes are determined by nephelometry (boxes, lower part). The
identification of the "myeloma protein" (M) is accomplished by
immunoelectrophoresis. T.P., total protein; NHS, normal human serum; Alb, albumin.
A, IgG-kappa; B, IgA-kappa; C, IgM.
incorporated into the gel and the antigen moves into the gel during electrophoresis, forming a "rocket"
(one-dimensional single EID or "rocket electrophoresis") (Chapter 101) . The height of the rocket is
proportional to the protein concentration.
Nephelometry

[5] [6] [7]

When light interacts with particles in suspension, it is absorbed,

reflected, or scattered. The decrease in the intensity of incident light
passing through the solution is measured and forms the basis of
turbidimetry, which is carried out at a 180 angle with respect to the
incident light. Turbidimetry should be distinguished from nephelometry
(Gr nephele = cloud), in which light scattered by particles is measured
at some angle between 0 and 90. Nephelometry is more sensitive
than turbidimetry and works best at low concentrations of antigen
when the formation of antigen-antibody complexes is facilitated by the
44

Figure 3-9 Immunofixation. Two "M proteins" are detected in the serum: both
are IgG ( strip 2), one kappa ( strip 3) and one lambda ( strip 4). The
electrophoretic pattern of the serum is shown in strip 1. Arrowheads, position of
the M-bands. Arrow, position of the application of the serum.
addition of polyethylene glycol. The degree of light scatter correlates well with the concentration of antigen
in solution. Nonspecific light-scattering particles may be present in the serum, such as lipids, or in the
reagents, and must be removed first by filtration. Results from nephelometry, however, are not affected by
variations in molecular weight. Two types of nephelometric assays are in common use. In static or endpoint nephelometry, measurements are taken at the end of the antigen-antibody reaction, whereas in rate
nephelometry, measurements are taken at the peak rate of reaction between antigen and antibody. The
kinetics of immune complex formation is different in the three phases (antibody excess, equivalence, and
antigen excess) and computer algorithms have been developed that flag antigen excess automatically. The
nephelometric methods in general are simpler, faster, and more sensitive than the immunodiffusion
techniques. The development of automated nephelometers and high-quality reagents have led to the
widespread use of nephelometry in the quantitation of serum proteins and have largely replaced the radial
immunodiffusion techniques.
Applications.

Quantitative determinations of serum Ig levels are indicated in the

diagnosis of immune deficiency diseases and in their follow-up to
assess the results of therapy; in plasma cell dyscrasias, such as
multiple myeloma, they provide quantitation of myeloma protein at the
time of diagnosis and follow-up to monitor the effect of therapy.
Increased levels of myeloma proteins usually are associated with the
presence of a large tumor cell mass, and a decreasing level of the
myeloma protein is indicative of successful therapy (see Chapter 99 ).
Serum Ig levels also are determined in a variety of other conditions,
such as rheumatoid arthritis (RA); lupus erythematosus (SLE); chronic
as well as acute hepatitis or cirrhosis of the liver; and infections, such
as tuberculosis. In people susceptible to recurrent bacterial infections
with normal IgG levels, assessment of the serum levels of the IgG
subclasses is indicated. In all of these conditions, levels of one or all
major Ig classes are elevated. In none of them, however, is a pattern
sufficiently distinct to lead to a specific diagnosis. With successful
treatment, the Ig level tends to return to normal values.
Cryoglobulins

[8] [9] [10]

The term defines the ability of Ig (or other serum proteins) to form
insoluble precipitate at 4C that can be redissolved when the sample is
re-warmed to 37C. The three categories of cryoglobulins (see Chapter
103 ) are as follows: type I consists of monoclonal Ig only; type II or
mixed cryoglobulins are composed of two different classes of Igs, one
monoclonal (usually an IgM rheumatoid factor) and the other
polyclonal IgG; and type III, mixed polyclonal cryoglobulins, which are
present in autoimmune diseases, viral infections, parasitic diseases,
and other inflammatory conditions. To determine the levels of
cryoglobulins accurately, the blood must be collected and the serum
must be separated at 37C. The serum is then placed in Wintrobe

tubes and the amount of precipitate is established after a period of as

many as 7 days by centrifugation. The result is reported as the
"cryocrit." The immunochemical identification of the cryoglobulin
follows the isolation of the cryoprecipitate and its washing to remove
contaminating serum. The nature and composition of the cryoglobulin
is determined by immunoelectrophoresis or by other immunologic
techniques. Sometimes immune complexes precipitate in the cold
(cryoproteins of SLE), because normal serum proteins, through
intermolecular attractive forces, initiate cryoprecipitation .
[11]

Complement
Common clinically relevant methods include the methods for CH50 or
total hemolytic assay as well as the immunochemical measurements of
individual complement components.
The hemolytic assay is performed in the liquid phase as well as in gels.
A simple technique involves the use of agarose gels impregnated with
antibody-coated erythrocytes. The unknown serum is placed in a small
well and is allowed to diffuse into the gel. Serum complement
hemolyzes the erythrocytes, generating a clear area around the well.
The diameter of the clear zone is proportional to the logarithm of the
number of CH50 units of complement.
Individual complement components, most commonly C3 and C4 (see
Chapter 21 ), can be determined by nephelometry or immunodiffusion.
Applications

The normal range of C3 is between 1.01 and 1.98 g/L. Changes from
normal are seen in association with a number of clinical conditions.
45

In some forms of acute glomerulonephritis, C3 levels are reduced (less

than 0.7 g/L) during the active stage of the disease. The C3 value
returns to normal during recovery. C3 levels can be used to
differentiate between acute nephritis and other kidney diseases, such
as nephrosis, because in the latter, the C3 level is either normal or
slightly elevated.
During the active stage of lupus erythematosus with nephritis, the
levels of C3 globulin are decreased (less than 0.7 g/L). Serial C3
determinations are recommended to assess the disease activity as well
as the effect of therapy. Levels of C3 have been shown to reflect
accurately the activity of the disease in general and, in particular, the
degree of renal involvement.
The synovial fluid obtained from patients with active RA has reduced
levels of C3. A comparison of C3 levels in the synovial fluid and the
total protein levels may be even more helpful.
In patients with acute hepatitis with severe liver necrosis, the level of
C3 is reduced because of a low rate of synthesis of this protein.

Autoantibodies and Autoimmune Diseases

Investigation of Connective Tissue Disease

[12]

Connective tissue diseases include a group of autoimmune diseases

with multisystem involvement. Laboratory investigation helps ( a) to
arrive at a specific diagnosis, and ( b) to evaluate disease activity. To
reach the first objective, the clinician may choose from a number of
assays by which to identify antibodies directed against selfconstituents. Although these autoantibodies are not specific for any
disease, the results of the tests, when used as a profile, provide useful
information for diagnosis (vide infra). The autoantibodies, however, do
not reflect disease activity accurately. Because connective tissue
diseases are associated with tissue inflammation during their active
stage, disease activity is best monitored with changes of serum levels
of certain glycoproteins. The increase of the normal plasma levels of
these proteins is associated with tissue injury or inflammation and is
known as acute-phase response. The proteins used most commonly as
acute-phase reactants are C-reactive protein, alpha-1 glycoprotein and
fibronectin (vide infra).
Rheumatoid Factors

The sera of patients suffering from RA and other diseases contain

antibodies capable of interacting with Ig molecules. Such antibodies
are known collectively as rheumatoid factors (RF). The classic RF is an
antibody with specificity against the Fc fragment of human Ig or Ig of
other species such as rabbits, but other Ig determinants also provide
binding sites for RF.
The sheep cell agglutination test (SCAT) detects RF capable of
interacting with rabbit Ig. Sheep red cells coated with rabbit antibodies
will agglutinate in the presence of RF.
Another test for the detection of RF is the latex fixation test, in which
the indicator system consists of latex particles coated with human Ig.
The serum is heated for 30 minutes at 56C to inactivate complement
that may interfere with the test.
The concentration of RF is determined most commonly by
nephelometry. In this test, the serum is added to a standard quantity of
human Ig in solution. In the presence of RF, the aggregated Ig results
in increased light scattering, the extent of which is proportional to the
RF concentration. The technique has certain advantages over other
assays, in that it is simple, rapid, sensitive, and more objective.
Recently, RIA
and ELISA
have been developed for the specific
detection of IgM and IgG RF. Because IgG RF usually is "missed" with
the use of the classic methods just mentioned, these tests offer
specific advantages. A solid phase assay for the detection of IgG RF
involves the use of polystyrene tubes coated with rabbit IgG. To
prevent detection of IgM RF, the serum of the patient is treated with
[13] [14]

[15] [16]

pepsin . Rabbit F(ab.)2 fragments specific for human IgG also have
been used in ELISA for IgG RF determinations ; however, they are
still cumbersome and have not found wide applications in routine
laboratory investigations.
[17]

[15]

Significance of RF

[18] [19] [20] [21]

Approximately 75 to 80% of sera from patients with RA have a positive

latex test result. The role of RF in the pathogenesis of RA is
controversial as is the usefulness of the RF assay . A recent report
revealed a positive predictive value of only 24 to 34% with limited
clinical value. However, others found the positive and negative
predictive values to be 80% and 96% respectively. These differences
may be due to differences in the populations examined
. Many
people have RF but no disease. A correlation apparently exists,
however, between the presence of RF, especially at high titers, and
some manifestations of RA, such as subcutaneous nodules, symmetric
deforming arthritis, visceral manifestations, vasculitis, and Felty
syndrome. In addition, seropositivity has a statistically significant
association with sustained (as opposed to intermittent) disease,
although some patients are consistently seronegative in spite of
sustained disease. Also, RFs are not specific for RA; they can be
detected in high titers in patients with a variety of conditions including
Sjogren syndrome, bacterial endocarditis, syphilis, pulmonary
tuberculosis, and autoimmune diseases other than RA. Patients with
juvenile RA usually remain seronegative. IgGRF may play an important
pathogenetic role in promoting inflammation within synovial tissue.
This IgGRF forms self-associated IgG complexes
(Fig. 3.10) of high
avidity, which precipitate within the joint tissues, activate complement,
and lead to tissue damage. Levels of IgGRF in the serum correlate with
erythrocyte sedimentation rates but not with disease activity ,
although synovial fluid levels may be a better index of disease activity
. More work is needed to determine whether IgGRF determinations
are of any value diagnostically. RF also can cross-react with antigens
found in cell nuclei
. Possibly, therefore, RF is formed originally
against other antigens that possess structural similarities to part of the
Fc fragment of human Ig. RA sera also contain antibodies that react
with a nuclear antigen present in human lymphoblastoid cell lines
infected with Epstein-Barr virus, known as RA-associated nuclear
antigen (RANA). The significance of these antibodies to the etiology of
RA remains to be established.
The role of T cells and cytokines in rheumatoid arthritis has recently
been thoroughly investigated. A broad range of
[22]

[22] [23]

[24]

[25] [26]

[27]

[28]

[29] [30] [31]

46

Figure 3-10 Self-associating complexes involving rheumatoid factor (RF). IgG RF

binds to the Fc antigenic determinant of other IgG RF molecules, giving rise to soluble

immune complexes.
proinflammatory cytokines has been detected by a variety of methods such as mRNA or protein secretion
from rheumatoid synovia. TNF-alpha and IL-1 drive cytokine production generating a cascade of pro-as
well as anti-inflammatory cytokines. A cytokine disequilibrium in favor of the proinflammatory category
may be important in the perpetuation of synovial inflammation [32] .
Antinuclear Antibodies

The sera of patients suffering from SLE and other collagen diseases
contain antibodies against nuclear material
. These antibodies,
known collectively as antinuclear antibodies (ANA), are directed
against several antigens present in nuclei, as shown in Table 3.1 .
[33] [34]

Antinuclear Antibody Assays.

The detection of ANA by fluorescence requires nuclei from any one of

several sources, such as human white cells, mouse liver cells,
nucleated chicken erythrocytes, or rat kidney, because ANA are not
species specific. Mouse liver cells possess large nuclei and constitute a
useful substrate for ANA detection, as do cells from a human laryngeal
epithelioma (Hep-2), which have been used to some advantage over
other substrates in detecting certain antibody specificities
.
Different patterns of fluorescence have been identified with the use of
antibodies of different specificities. A homogeneous pattern ( Fig. 3.11
A), in which the entire nucleus fluoresces diffusely, indicates the
presence of antibodies against nucleoprotein. A ring pattern ( Fig. 3.11
B), in which only the periphery of the nucleus fluoresces, indicates the
presence of anti-DNA antibodies. A speckled pattern ( Fig. 3.11 C) over
the nucleus indicates the presence of antibodies against a number of
nuclear antigens extractable by saline that are known collectively as
extractable nuclear antigens (ENA).
Twenty different antigens are known to belong to the category of ENA
. Some are known by the initials of the name of the patient in whose
serum the antibody was first detected, (e.g., Sm ), whereas the
designation of other antigens reflects their composition, (e.g.,
ribonucleoprotein [RNP] ), or their association with certain diseases,
(e.g., the sicca syndrome associated antigens SS-A and SS-B ). The
Sm and RNP antigens are RNA-protein complexes
and belong to
the category of nuclear RNA known as small nuclear RNA (snRNA) .
These snRNA are of low molecular weight comparedwith other nuclear
RNA and may be involved in the processing of the heterogeneous
nuclear RNA (hnRNA) to mature mRNA by splicing out sequences that
need to be removed . Other antigens that stimulate antibodies in
patients with autoimmune diseases are histones, particularly, H2A-H2B
.
[35] [36]

[37]

[38]

[39]

[40]

[41] [42]

[43]

[44]

[45]

Anti-DNA Assays.

Anti-DNA antibodies may be detected by RIA

or ELISA techniques
. In RIA, anti-DNA antibodies in the patient's serum are allowed to
[46] [47] [48]

[49]

react with DNA-coated plastic surfaces or with DNA in solution. In the

latter system, the DNA-anti-DNA complexes are separated from the
DNA remaining free in solution by ammonium sulfate at 50%
saturation, which precipitates the DNA bound to the antibody but not
the unbound form (Farr technique). The degree of binding of DNA to
antibody is influenced by the molecular weight of DNA . The amount
of radioactivity bound to the antibody is expressed as a percentage of
the total. Binding of less than 20% of the added radioactivity
[50]

TABLE 3-1 -- Antinuclear Antibodies

Antibody Specificity

Disease

Incidence (%)

Nucleic acids
Double-stranded DNA

SLE

60-70

Single-stranded DNA

SLE

70

Drug-induced SLE

80

Histones

SLE

30-70

RA

15-20

Drug-induced SLE

95

Nuclear proteins
Sm antigen

SLE

25-35

RNP (SnRNA-protein)

SLE

30-40

MCTD
SSA/Ro

SSB/La

SLE

30-40

Sjogren's Syndrome

60-70

SLE
Sjogren's Syndrome

Scl-70 1

95

15
40-60

Scleroderma

70

Centromere

CREST Syndrome

80

JO-1 2

Polymyositis

30

Dermatomyositis

SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; MCTD, mixed connective tissue disease.
Topiosomerase I.
Histidyl-tRNA synthetase. (Data from McCarty GA. Autoantibodies and their relation
to rheumatic diseases. Med Clin North Am 1986; 70:237-261; Bentwich Z et al.
Laboratory investigations in clinical immunology: methods, pitfalls, and clinical
indications. A second IUIS/WHO report. Clin Immunol Immunopathol 1988; 49:478497; Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases
and probes for cell biology. Adv Immunol 1989; 44:93-151.)
1
2

47

Figure 3-11 Antinuclear antibodies (ANA) detected by immunofluorescence in

mouse fresh frozen liver sections (A-C) and Crithidium luciliae (D,E). A,
Homogeneous pattern. B, Ring pattern. ANA binds to the periphery of the nucleus.
This pattern correlates with antibodies to DNA, C, Speckled pattern, associated with
antibodies to RNA-protein complexes present within the nucleus (i.e., Sm, RNP). D,
Kinetoplast fluorescence, indicative of the presence of anti-double-stranded DNA
antibodies. E, Fluorescence of kinetoplast and nucleus.
is considered normal. Sera from patients with active SLE usually bind more than 60% of the antigen.
Intermediate ranges of 20 to 40% are seen in the sera of patients with other diseases, such as RA. The Farr
method has been standardized against the First International Standard (WO/80) and the results are
expressed in International Units per milliliter (IU/ml). A value less than 7 IU/ml is considered normal.

In the ELISA assay, microwells are coated with dsDNA from various
sources, for example, calf thymus. Serum or fluids from patients are
allowed to react with the DNA and after washing, monoclonal anti-IgG
and anti-IgM antibodies (conjugated to the enzyme alkaline
phosphatase) are added. After washing, the substrate is added and the
reaction is stopped at a fixed time interval. The color intensity of the
substrate is measured and the concentration of the anti-dsDNA
antibody is calculated from a standard curve made against the WO/80
WHO Reference Preparation. The results are reported in International
Units (IU)/ml. A value less than 30 IU/ml is considered negative for antidsDNA antibodies.
The Crithidium Luciliae assay
is another anti-DNA assay. C.
luciliae is a trypanosome that contains a structure known as a
kinetoplast. This small round body is located where the flagellum is
attached to the cell and contains double-stranded DNA in the absence
of protein. Fluorescence of the kinetoplast indicates the presence of
anti-double-stranded DNA antibodies, whereas fluorescence of the
nucleus (located in the front end of the microorganism) does not ( Fig.
3.11 D,E).
[51] [52] [53] [54]

Significance of ANA: Their Association with Specific Clinical Diseases or Syndromes

[59] [60]
.

[1] [33] [55] [56] [57] [58]

Although the
48

detection of ANA is not in itself diagnostic of SLE or other autoimmune diseases, these assays, when used
and interpreted properly, provide important help in the diagnosis of disease and the follow-up care of

patients. For diagnostic purposes, it is preferable to determine several antibody specificities rather than a
single, specific antibody. An important step is to measure complement levels, especially of C3 and C4, in
addition to ANA. Several measurements over a period of time should be made, because results from a
single determination may not always reflect the underlying disease process. In some patients, the
autoimmune disorder may not conform to a distinct disease entity, and in such cases the term
undifferentiated connective tissue syndrome has been used [61] . When followed over extended periods, such
patients eventually may develop a distinct disease entity.

DNA binding, as determined by any immunoassay, usually yields high

levels in patients with SLE but low levels in those with RA and other
autoimmune diseases . In contrast to all the other ANA, anti-DNA
antibodies show a good correlation with disease activity and kidney
complications
, especially the anti-native (double-stranded)
DNA antibodies that are rarely, if ever, detected in other collagen
vascular diseases. In only a few patients with RA are anti-DNA
antibodies detected, and the binding activity is usually low. When
binding is borderline, the test should be repeated at a later date,
because only SLE patients demonstrate high DNA binding during the
course of their disease.
Low complement levels may also help to confirm the diagnosis of SLE
when the disease is in relapse. Both tests should be used to monitor
disease activity . Sometimes, complement levels are low or anti-DNA
antibody levels are high in the absence of active disease . Anti-DNA
antibodies have been detected also against left-handed or "Z" DNA
(the predominant configuration of DNA is right-handed or "B"). Patients
with RA show higher binding for Z DNA than do patients with SLE .
Anti-ENA antibodies, giving a speckled fluorescence pattern, are said to
be associated with a syndrome called mixed connective tissue disease
(MCTD)
. MCTD may represent a variant of SLE or a distinct form of
"collagen disease" . Anti-ENA antibodies, however, as detected by
immunofluorescence, are present in typical cases of SLE, but the titer
is usually low relative to the high titers associated with MCTD. Use of
other methods with which to identify specific anti-ENA antibodies, such
as anti-Sm and RNP, reveals that anti-Sm are characteristic of SLE (in
approximately 30% of patients), whereas anti-RNP are often found in
patients with clinical features characteristic of MCTD. Other
investigators have not found any specific association between the Sm
and RNP antigen systems and SLE or MCTD .
Among the other components of ENA, the SS-B antibody shows high
specificity for the sicca syndrome , the antibodies to Scl-70 antigen
(topoisomerase I) for progressive systemic sclerosis , and an
antibody to a determinant of the centromere to the CREST variant of
scleroderma . Antibodies to SS-A (RO) antigen are associated with
neonatal lupus and congenital heart block . Antibodies to singlestranded DNA and histones have been detected in patients under
treatment with procainamide, hydralazine, or quinidine . Antihistone
antibodies are detected in more than 95% of these patients, but
antibodies to double-stranded DNA are absent. Induction of these
[62]

[63] [64] [65] [66]

[67]

[65]

[68]

[59] [69]

[70]

[71]

[40]

[72]

[72]

[73]

[74]

[33]

antibodies is correlated with the capacity to acetylate drugs through

liver acetyltransferase activity. Slow acetylators develop ANA and
clinical symptoms more quickly than rapid acetylators.
The large number of autoantibodies found in patients with SLE may be
the result of polyspecificity or cross-reactivity of a single antibody
population that ultimately is specific for a phosphodiester bond
included in the backbone structure of many substances, such as
nucleic acids, phospholipids, cardiolipin, phosphaditic acid, and others
. This polyspecificity may explain the well-known association of SLE
with false-positive Wassermann reactions and circulating
anticoagulants. Two other intriguing cross-reactivities of the ANA have
been identified. Some ANA cross-react with leukocyte cell membranes
and are specific for the core histones (H2A and H2B) of nucleosomes
. Some human monoclonal anti-DNA antibodies also bind to
lymphocyte membranes and exert a cytotoxic effect . Another group
of ANA express distinct RF activity
. The widespread crossreactivity detected among ANA raises the question of the nature of the
original immunogenic stimulus. Characterization of anti-DNA antibodies
by monoclonal antibodies showed that they share a common idiotype,
suggesting that they are products of a restricted number of germ-line
genes
.
[75] [76]

[77]
[78]

[79]

[29] [30]

[80] [81] [82]

Evaluation of Disease Activity

Rheumatoid Arthritis (RA)

Elevations of the erythrocyte sedimentation rate and the serum levels

of C-reactive protein are useful markers of disease activity; however,
the sedimentation rate may remain elevated during remissions as a
result of persistently high levels of serum Igs. Increases in serum levels
of C-reactive protein correlate better with active joint disease
.
Fibronectin levels are elevated in the synovial fluid but not in
peripheral blood, and the ratio of these levels with synovial C3 levels
(which are decreased) can be used to monitor disease activity in
patients with RA .
[21] [83]

[84]

Systemic Lupus Erythematosus (SLE)

Evaluation of disease activity has advanced recently to reliable clinical

activity indices. The most widely used are SLEDAI, SLAM, BILAG, and
LAI
. Serologic measures of disease activity include an elevation
of sedimentation rate, an increase in acute-phase reactants, and low
levels of serum complement, particularly C3. The C-reactive protein
level is elevated, but not to the same extent as in bacterial infections.
Therefore, this value may help to differentiate infections in SLE from
active disease . The level of alpha-1 acid glycoprotein also is
elevated in patients with SLE, and because its electrophoretic mobility
is altered during infections, these changes may be used to identify
patients with infections . In contrast to RA patients, serum levels of
[85] [86] [87]

[88]

[89]

fibronectin are elevated in persons with active lupus . Anti-dsDNA

may also be a useful predictor of "flares," but it increases only in
approximately 27% of flares .
[90]

[91]

Antiphospholipid Antibodies and Their Significance

It has long been recognized that in some patients with systemic lupus
erythematosus (SLE) there is an abnormality in the phospholipid
dependent coagulation reaction that
49

prevents the conversion of prothrombin to thrombin. This inhibitory activity has been known as "lupus
anticoagulant" and has been shown to be an Ig which is directed against anionic phospholipids.

More recently, sensitive solid phase enzyme-linked immunoassays

have been developed to detect such antibodies using cardiolipincoated microtiter plates. The nature of these antibodies has been
controversial and it has been shown that they are quite heterogeneous
in their specificity. Some "anti-cardiolipin" antibodies actually require a
plasma cofactor for their binding . It is now realized that the
antibodies bind complexes of phospholipids with serum proteins such
as beta2 -glycoprotein I (beta2 GPI, a non-complement member of the
complement control family), prothrombin, protein C, and protein S
. The heterogeneity of these autoantibodies is reflected by the
variety of the names used: the lupus anticoagulants inhibit
phospholipid-dependent coagulation assays in vitro, interfere with the
conversion of prothrombin to thrombin, and are specific not only for
anionic phospholipids but also complexes of phospholipids with beta2
GPI or prothrombin ; the anticardiolipin antibodies are detected by
the ELISA test, which also detects antibodies to phospholipid-protein
complexes. Serum is always present in the test system and is used also
for blocking non-specific binding sites of the microtiter plates. These
two classes of antibodies as they are defined by these two methods
are distinct but probably have overlapping specificities . The term
anti-phospholipid antibodies (APA) is more generic, and it applies to
autoantibodies detected by all assays including syphilis tests. The
detection of APA is clinically important, because they have been
associated with arterial and/or venous thrombosis, recurrent fetal loss,
and thrombocytopenia. These four features are the major clinical
manifestations of what became known as the anti-phospholipid
syndrome (APLS)
.
Several mechanisms have been suggested as causes of thrombosis by
the antiphospholipid antibodies, but none has found wide acceptance.
These mechanisms implicate a direct involvement of the antibodies in
blood clotting such as interference with the regulatory protein C and
protein S
, impairment of fibrinolytic mechanism
, platelet
aggregation
, or interference with the formation of complexes of
[92]

[93] [94]

[95]

[96]

[97]

[98] [99]

[100]

[101]

[102]

antithrombin III and thrombin which in turn permits thrombin to

promote coagulation
.
APA are detected in patients with classical SLE, but also in other
individuals without serological or clinical evidence of autoimmunity. In
non-SLE individuals, clinical APA-associated events define the primary
APS (PAPS)
, while in those with SLE they define the secondary
APS. Comparison between these two groups of patients indicated that
PAPS and APS plus SLE have similar APA-associated symptoms, which
suggests that APA are directly responsible for the symptoms associated
with their presence
. In patients with SLE, presence of APA is an
important risk factor for thrombosis, neurologic disease,
thrombocytopenia, and fetal loss
. The APA are also present in other
disorders such as syphilis and acute infections, but their association
with thrombosis in these conditions is controversial
. Finally, APA
may develop in response to certain drugs. Chlorpromazine,
procainamide, and hydralazine are responsible for almost all instances
of drug-induced APA.
There is some interlaboratory variation with the ELISA test in the
detection of anticardiolipin antibodies, even with the use of standards
and careful adherence to test performance. Some kits use a mixture of
phospholipids to decrease chances of false positive results for syphilis
and still obtain comparable positive results for other diseases. Isotypes
of the antibodies are identified using isotype-specific anti-human Ig
antisera. Antibodies in the three main Ig classes, IgG, IgM, and IgA,
have been detected. Values of IgG and IgM anticardiolipin antibodies
are estimated from calibrated standards that contain known amounts
of antibodies expressed in units separately for IgG (GPL units) or IgM
(MPL units). The unit was defined with the use of affinity-purified IgG
and IgM anticardiolipin antibodies. The binding activity of 1 mug/ml of
IgG or IgM anticardiolipin antibody was called one GPL or MPL unit
respectively
. Healthy donors have less than 23 GPL and less than
11 MPL units, which defines the normal range. Interpretation of results
may be difficult. If the test is positive for IgG at medium to high levels
for several weeks to months, the diagnosis of APS is most likely. IgM
antibodies at high titers may be important but at low levels may be
false positive. IgA antibodies have been found to correlate with
thrombocytopenia better than IgG or IgM
.
[103]

[104] [105]

[106]

[107]

[107] [108]

[109]

[110]

Antineutrophil Cytoplasmic Antibodies (ANCA)

Antibodies to cytoplasmic constituents of neutrophils

were
originally detected in sera from patients with vasculitis or
glomerulonephritis (GMN)
and later in Wegener granulomatosis (WG)
and systemic vasculitis
.
[111] [112]

[113]

[114]

[115]

Several methods are used to detect ANCA:

indirect immunofluorescence (which still remains the standard

method), ELISA, Western blotting, and radioimmunoassay. The

fluorescence assay is reliable, depending on the source of cells,

fixation, incubation, and washing steps. With alcohol-fixed cells, there
are two patterns of fluorescence: the classic "cytoplasmic" pattern
(cANCA), the perinuclear (pANCA), and an atypical pattern, which
consists of a mixture of both.
The cANCA autoantigen has been identified as a serine protease known
as PR3, an elastinolytic enzyme of 29 kD molecular weight
. It is
variously called azurophilic granule protein (AGP7) or myeloblastin. It
has been renamed "Wegener autoantigen"
. This enzyme
degrades elastin, fibronectin, laminin, vitronectin, and collagen IV and
may be responsible for some of the damage seen in vasculitis and
GMN.
The pANCA pattern seen by indirect immunofluorescence is an artifact
of alcohol fixation, which translocates proteins rich in basic amino
acids to the negatively charged nucleus. This pattern is not seen in
formaldehyde-fixed neutrophils. To distinguish this fluorescence from
antinuclear antibodies, the HEP-2 substrate should be used. The
antigen of pANCA is myeloperoxidase. Other antigens may also give
the perinuclear pattern. The pANCA is associated with systemic
vasculitides and some cases of polyarteritis nodosa, necrotizing
crescentic GMN, chronic inflammatory rheumatic disorders, SLE,
Sjogren syndrome, and others. pANCA can also be induced by drugs,
such as hydralazine and L-tryptophan
.
The ANCA-associated vascular lesions consist of focal
[116]

[117] [118]

[119]

50

necrotizing inflammation with granulocyte infiltration, but no evidence of immune complex deposition.
These lesions are similar independent of their localizations in kidney, lungs, muscle, and elsewhere. The
most life-threatening manifestation is pulmonary hemorrhage as a result of necrotizing inflammation of
alveolar capillaries or renal failure. The pathophysiology of ANCA-induced vasculitis is not well
understood. It is hypothesized that a proinflammatory cytokine (such as TNF-alpha) may translocate the
autoantigen from the granule to the surface and make it accessible to the circulating ANCA, causing
granulocyte activation, degranulation, and endothelial injury.

Circulating Immune Complexes

[120] [121]

Immune complexes form when antibodies interact with the

corresponding antigens. The fate of the complexes depends on many
variables, including the nature of the antigen and antibody, the size of
the complex, the rate of production, and the rate of removal, the latter
depending in turn on the functional integrity of the phagocytic system.
Immune complexes are formed as part of the normal immune response
and are released into the circulation. They are then deposited in
tissues, where the resulting complement fixation and
polymorphonuclear leukocyte accumulation leads to tissue damage
through the release of mediators of inflammation. Because of the
pathogenetic role of immune complexes in several human diseases,

their demonstration by specific, sensitive, and reliable assays is

essential. Numerous methods are available, but not all are suitable for
routine clinical use. The assays can be divided into two major groups:
those based on the physical properties of the complexes and those
based on their biologic properties.
Assays based on physical properties are not used commonly in routine
work. Most important are techniques involving ultracentrifugation, gel
filtration, cryoprecipitation, and the precipitation of complexes by
polyethylene glycol. Mixed cryoglobulins (see Chapter 103 ) are often
found in association with connective tissue disease, such as SLE or RA,
and represent an example of complexes demonstrated on the basis of
their physical properties.
Most biologic methods depend on the capacity of immune complexes
to interact with complement. The C1q binding assay can be performed
in the fluid or solid phase. In the fluid phase assay, radiolabeled C1q
added to the serum binds to the complexes and is separated from the
unbound fraction by polyethylene glycol. In the solid phase assay,
complexes interact with C1q bound to plastic surfaces and
subsequently are quantitated with a radiolabeled or enzyme-labeled
anti-Ig antiserum. The fluid phase assay has the advantage of lack of
interference by DNA or endotoxin, because complexes of these
substances are not precipitated by polyethylene glycol. False-positive
results may be obtained, however, in the presence of heparin. The
solid phase assay has the distinct advantage that it is Ig specific, but
RF may interfere with the results. The C1q deviation test is based on
the ability of complexes to inhibit the binding of radiolabeled C1q to
specific substrates, such as antibody-coated sheep erythrocytes. Falsepositive results may be obtained with DNA and endotoxin as well as RF.
Conglutinin binding assays depend on the ability of conglutinin, a high
molecular weight protein in bovine sera, to bind complement avidly. In
one assay, serum is added to plastic tubes coated with conglutinin.
Immune complexes contained in such sera bind to the tubes and are
detected by means of anti-Ig reagents.
Another group of assays for the detection of immune complexes
depends on their reaction with RF. The ability of immune complexes to
interact with monoclonal RF is used in one assay. RF is conjugated to
cellulose and is mixed with serum containing complexes and with
aggregated IgG labeled with radioactive iodine. The degree to which
the binding of aggregated IgG to the RF is inhibited by the serum
constitutes a measure of the quantity of complexes present in the
serum.
The Raji cell assay involves the use of a culture line derived from a
patient with Burkitt lymphoma. The cells have a few receptors for the
Fc fragment and many complement receptors but they have no surface
Ig. Complexes containing complement bind to the surface of the Raji
cells. The cells then react with radiolabeled anti-Ig and the amount of

bound radioactivity is calculated. Antilymphocytic antibodies often

found in SLE may bind to these cells and cause false-positive results.
All techniques available for the detection of immune complexes have
advantages and drawbacks
. All methods are affected by a variety
of substances other than immune complexes, and none differentiate
between aggregated Ig and immune complexes. Thus, all procedures
that generate such aggregates have their own built-in disadvantages.
In addition, the various assays show significantly different response
patterns when all are tested simultaneously against groups of patients
suffering from different kinds of immune complex diseases. The cause
of these differences is not understood, but it has been attributed to a
variety of factors, such as the nature of the antigen, the size of the
complex, and interaction with other plasma components in vivo. Under
certain conditions, single-stranded DNA reacts with C1q and interferes
with most tests involving use of the C1q assay. Some investigators
recommend the use of more than one assay for all patients suspected
of suffering from immune complex diseases.
[122] [123]

Monoclonal Antibodies
In the diagnostic laboratory, immunologic assays routinely involve the
use of antibodies raised in animals against a variety of antigens.
Conventional immunization procedures usually yield heterogeneous
populations of antibodies with varying degrees of cross-reactivity to
structurally related antigens. These problems have made it difficult to
standardize immunoassays and to accumulate large quantities of
antisera that can be used as reference reagents.
In 1975, Kohler and Milstein
fused cultured mouse myeloma cells
with spleen cells from mice immunized with sheep red blood cells, and
showed that some of the resulting hybrids made homogeneous
(monoclonal) populations of antibodies that reacted with the
immunizing antigen. The hybrid, or hybridoma, is the clonal progeny of
the fusion of a single antibody-forming cell and a single myeloma cell.
[124]

51

These hybridoma cells retain the properties of the malignant myeloma cell parent in that they grow
continuously in culture and at the same time synthesize structurally identical antibody molecules, a
property they have inherited from the antibody-forming cell parent. The main steps in the production of
hybridomas are shown in Figure 3.12 . The antibodies produced by these hybridoma cells are known as
monoclonal antibodies, to indicate their origin from the single antibody-forming cell parent. Hybridoma
technology now is used widely in research and in clinical practice, because monoclonal antibodies have
distinct advantages over the antibodies produced by conventional immunization. These advantages include
their homogeneity, their predictable specificity and affinity, and the ability to produce such antibodies in
unlimited quantities.

Because monoclonal antibodies can be produced to any antigen, they

have broad applicability in clinical medicine as potent diagnostic tools.
In oncology, monoclonal antibodies are used in the detection of tumor
antigens
and in attempts to localize tumors by means of
[125]

radioimmunodetection techniques
. Monoclonal antibodies
conjugated with toxic agents are used experimentally for the treatment
of cancer. These conjugates are known as immunotoxins
. In
addition, monoclonal antibodies raised against tumor cells have been
used to treat patients suffering from lymphoma
.
In the diagnosis of infectious diseases, particularly of
[126]

[127] [128]

[129]

Figure 3-12 Production of hybridomas. PEG, polyethylene glycol.

sexually transmitted diseases, monoclonal antibodies have led to the development of rapid and simple
diagnostic procedures for the detection of infections such as those caused by Chlamydia and the herpes
viruses [130] [131] . Monoclonal antibodies can be used directly on clinical specimens, and sometimes antigens
can be detected even when routine cultures reveal no infectious particles.

Monoclonal antibodies now are used widely to identify subgroups of

leukemias and lymphomas that differ in their prognosis and required
approaches to therapy
. The development of human monoclonal
antibodies has not yet reached the same level of development as that
of mouse monoclonal antibodies, principally because of the instability
of human hybridomas and their limited secretion of antibody
.
[132] [133]

[134]

Lymphocyte Function
The tests commonly employed for assessing lymphocyte function
include: ( a) mitogenic stimulation; ( b) mixed lymphocyte cultures;
and ( c) lymphocyte cytotoxicity studies.
Mitogenic Stimulation
Lymphocytes exposed to certain plant lectins undergo divisions
(mitogenesis) and differentiation. Lectins bind to simple sugars, such
as galactose and fucose, or to complex sugars, such as those present
in glycoproteins. Sometimes, the lectin agglutinates the cells. The
lectins used most commonly are phytohemagglutinin (PHA) present in
the kidney bean Phaseolus vulgaris, concanavalin A (con A) derived
from Jack beans ( Canavalia ensiformis), and pokeweed mitogen (PWM)
from Phytolacca americana. PHA is mitogenic for T cells, con A
stimulates T cells in solution and will stimulate B cells only if con A is
attached to solid surfaces, and PWM is mitogenic for B cells in the
presence of T cells. The lectins stimulate the lymphocytes to proliferate
and to differentiate and secrete lymphokines in the case of T cells and
Ig in the case of B cells. Lectins therefore induce changes identical to
those induced by antigen, with the fundamental difference that lectins
activate all lymphocytes (polyclonal activation); antigens activate only
the lymphocyte clones that bear the antigen-specific receptors. The
mitogenic effect of lectins on lymphocytes can be measured by
counting the number of "blast" cells or by measuring the amount of
3H-thymidine incorporated into newly synthesized DNA. The mitogen is
added to lymphocytes purified from the peripheral blood; after

incubation for 48 to 72 hours at 37C in an atmosphere of 5% CO2 , a

small amount of 3H-thymidine is added for a few hours to be
incorporated into DNA (pulse labeling). After washing to remove excess
thymidine, the amount incorporated is determined by scintillation
counting. The data are expressed as counts per minute (cpm). In some
laboratories, the results are expressed as a ratio of stimulated to
control cultures, called the stimulation index. The degree of activation
depends on many factors, both technical (such as cell number, shape
of tube, mitogen dose, and time in culture) and biologic (such as the
presence of suppressor cells or monocytes). To obtain the most
accurate evaluation of the ability of lymphocytes to be activated, it is
important to conduct a complete kinetic study
52

as opposed to measuring DNA synthesis at a single point in time only, when the maximum response in
patients may occur at a different time than in normal controls.

Mixed Lymphocyte Reaction (MLR)

When lymphocyte populations from two individuals with different HLA
antigens are mixed, a potent proliferative reaction takes place, which is
triggered through the recognition of class II molecules by T
lymphocytes. MLR is characterized by a brisk proliferative response
and new DNA synthesis. The MLR can be "two-way" or "one-way." In
the former, the cells from each individual stimulate as well as respond,
and the DNA synthesis is the combination of responses from both cell
populations. In the one-way test, the cells from one individual are
irradiated or treated with mitomycin C so that they cannot divide and
synthesize new DNA. The treated cells then act as stimulators and the
DNA synthesis represents the response of the untreated cells.
MLR is used in histocompatibility assays and as an in vitro test for the
immunocompetence of T cells. In most immune deficiency diseases
involving T cells, the patient's response in the MLR is absent or
severely deficient. The reader should bear in mind that the degree of
response in the MLR varies depending on the HLA differences between
the stimulator and responder cells and the density of class II antigens.
For this reason, viable pooled human cells should be used as
stimulators. The intensity of MLR is measured as the amount of 3Hthymidine incorporated into the DNA of responding cells.
Lymphocyte Cytotoxicity
In response to allogeneic stimulation, T lymphocytes differentiate into
cytotoxic (CTL) or killer cells. This response is described in Chapter 21 .
CTL responses occur in mixed lymphocyte cultures and the cytotoxic
cells react predominantly against class I molecules. The most
convenient way for assaying CTL responses is by the use of targets

that contain an intracellular radioactive label, usually chromium 51.

When the target cell is lysed, chromium 51 is released and the amount
of label released is proportional to the number of cells killed. The result
is expressed as percent cytotoxicity, which can be calculated according
to the formula:

in which control release is the amount of radioactivity released in the

absence of CTL and maximal release is the amount released when the
targets are lysed by freezing and thawing. It is an index of the total of
isotope that can be released from the target cells. The CTL response
usually is measured after 5 days of culture.
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