Western blot

- Uses :
a- To separate different proteins from a solution depending on weight
b- Measure the amount and weight of antigen

- Steps

a- The mixture is subjected to analytical separation (typically by SDS-PAGE) the final positions of
different proteins in the gel are a function of their molecular size.
b- The proteins transferred from gel to membrane
c- The position of the protein antigen on the membrane can then be detected by binding of an
unlabeled antibody specific for that protein (the primary antibody)
d- Followed by a labeled second antibody that binds to the primary antibody.

- Second antibody probes are labeled with

a- enzymes that generate chemiluminescent signals and leave images on photographic film.
b- Near-infrared fluorophores can also be used to label antibodies

Note :

- Southern blot : DNA detection developed earlier by Edwin Southern.

- Northern blot : RNA detection
Radio- immune assay (RIA) and ELISA

Radioimmunoassay (RIA) :

a- the antigen or antibody is labeled with a radioisotope

b- it may be quantified by instruments that detect radioactive decay events

Enzyme-linked immunosorbent assay (ELISA) :

a- the Ag or Ab is covalently coupled to an enzyme

b- it may be quantified by determining (with a spectrophotometer) the rate at which the enzyme
converts a clear substrate to a colored product

Several variations of RIA and ELISA exist, but the most commonly used version is the sandwich assay.

The sandwich assay uses two different antibodies reactive with different epitopes on the antigen
whose concentration needs to be determined.

Measures amount quantities of antigens or antibody (protein) using radiolabelled sign:

a- Hormones
b- Drugs
c- Tumor markers
d- IgE
e- presence of antibodies that are specific for a microbial antigen (e.g., antibodies reactive with
proteins from human immunodeficiency virus [HIV] or hepatitis B virus) as indicators of infection
f- Viral antigens

ELISA

- Enzyme linked immunosorbent assay (1971)

- The antibody is enzyme linked or radiolabeled
- Done in 96 well micro-titer plates
- To detect the presence of a protein and its quantity
- Ag or Ab coated on wells

Indirect ELISA

- For Ab detection
- Ag coated wells used
- Add patient serum then wash
- If Ab present, it binds to Ag
- Add goat anti-human Ig antibody (enzyme linked or radiolabeled)
 Sandwich ELISA

- For Ag detection
- wells coated with specific Ab.
- add patient specimen then wash
- Ag in specimen binds to coated Ab.
- Add antibody enzyme linked or radiolabeled
- Color produced

Competitive ELISA

- Ag coated wells
- Two specific Abs (enzyme linked or radiolabeled antibody)
- Ab & patient serum added simultaneously then wash
- More specific tested Abs attach to Ag
- Positive test- no colour

Tests for Cell Associated Antigens (Immuno-cyto/histo-chemistry)

a- For cell associated Ags

b- Specimen is tissue not serum
c- Determine the anatomic distribution of antigen in tissues

1- Immunofluorescence
- An antibody labeled with a fluorescent molecule (fluorescein or rhodamine)
- Determine the anatomic distribution of antigen in tissues using the fluorescence emitted
- Used in
a- Tumor antigen detection
b- SLE
c- Rheumatoid arthritis.
A- Direct Immunofluorescence
- Prepare a specific mouse antibody directed against the antigen of interest
- attaching horseradish peroxidase to a specific mouse antibody
- add this to specimen
- observe the result using immune-fluoresce

B- Indirect Immunofluorescence
- Prepare a specific mouse antibody directed against the antigen of interest
- Prepare a 2nd specific mouse or rabbit antibody directed against the 1st antibody
- attaching horseradish peroxidase to a specific 2nd antibody
- add the 1st antibody to the specimen then add the 2nd
- observe the result using immune-fluoresce
- more sensitive than direct immunofluorescence

Note : Although sensitive, the fluorescence microscope is not an ideal tool to identify the detailed
structure of the cell or tissue because of a low structural details

2- confocal microscopy : uses optical sectioning technology to filter out unfocused fluorescent light
3- A conventional light microscope
- antibodies may be coupled to enzymes that convert colorless substrates to colored insoluble
substances that precipitate at the position of the enzyme.
- may then be used to localize the antibody in a stained cell or tissue.
- The most common variant of this method uses the enzyme horseradish peroxidase
- the method is commonly referred to as the immunoperoxidase technique.

4- Electron microscope
- Antibody can be coupled to an electron-dense probe such as colloidal gold
- the location of antibody can be determined subcellularly by means of an electron microscope

5- Flow cytometry
- done by staining the cell with fluorescently labeled probes (fluorochrome-labeled antibodies )that are
specific for some molecules and measuring the quantity of fluorescence emitted by the cell
- specialized instrument that can detect fluorescence on individual cells in a suspension and thereby
determine the number of cells expressing the molecule to which a fluorescent probe binds

- Uses
a- The tissue lineage
b- maturation stage
c- cytokine secretion (inside cells and bound)
 d- activation status of a cell
e- numerating B and T cells, apoptotic cells
f- cell cycle study
g- measure the forward light-scattering properties of cells, which reflect cell size
h- measure the side light-scattering properties of cells, which reflect internal complexity
i- used to distinguish different cell types

Note : lipophilic Fluorescent dyes can be used to study proliferation of T and B cells in vivo The dyes enter
cells, form covalent bonds with cytoplasmic proteins, and then cannot leave the cells One commonly used
dye of this type is (CFSE), which can be detected in cells by standard flow cytometric techniques. Every time
a T cell divides, its dye content is halved

Immunohematology

- serologic, genetic, biochemical, and molecular study of antigens associated blood, as well as the
immunologic properties and reactions of blood components and constituents.

Transfusion medicine :

a multidisciplinary specialty encompassing all aspects of:

blood donation.

blood component preparation.

blood cell serology.

blood transfusion therapy.

divided between blood centers and transfusion services.

1- Blood centers
a- recruit and collect blood from donors
b- manufacture and distribute blood components.
c- Units of WBC are kept refrigerated at 33.8 to 42.8 F (1.0 to 6.0 C), with maximum permitted
storage periods of 35 days
d- Units of RBC are kept refrigerated at 33.8 to 42.8 F (1.0 to 6.0 C), with maximum permitted
storage of 42 days
e- The layer between the red cells and the plasma is referred to as the buffy coat and is sometimes
removed to make platelets for transfusion
 f- Platelets are typically pooled before transfusion and have a shelf life of 5 to 7 days.
g- Platelets are stored at room temperature (72 F or 22 C) and must be rocked/agitated
h- If the plasma is frozen promptly and is intended for transfusion, it is typically labeled as fresh
frozen plasma
i- If it is intended to be made into other products, it is typically labeled as recovered plasma or
plasma for fractionation.

Transfusion services

a- perform pretransfusion compatibility testing (blood grouping and cross match)

b- select and issue blood components for patients
c- provide medical support for blood transfusion.

pretransfusion compatibility testing

Evaluation of the donor includes

Testing of the donor unit for infectious diseases

ABO/Rh typing and antibody screen

Evaluation of the recipient includes

ABO/ Rh typing

Antibody screen. Perform antibody identification if antibody screen is positive to determine

the identity of the antibody,

Crossmatch Tests the compatibility of the recipients serum with RBCs from potential donor unit

The ABO antigens

- carbohydrates linked to cell surface proteins and lipids

- synthesized by polymorphic glycosyl-transferase enzymes
- Most individuals possess a fucosyl-transferase that adds a fucose moiety to a nonterminal sugar
residue of the core glycan
- the fucosylated glycan is called the H antigen
- A single gene on chromosome 9 encodes a glycosyl-transferase enzyme that may further modify the H
antigen
There are three allelic variants

1- O allele gene product is devoid of enzymatic activity.

2- The A allele encoded enzyme transfers a terminal N-acetylgalactosamine moiety onto the H antigen.
3- B allele gene product transfers a terminal galactose moiety.

Note :

Individuals lacking a particular blood group antigen produce natural IgM antibodies against that
antigen.

Alloantibodies: are antibodies directed against the antigens not possessed by the individual

If such individuals are given blood cells expressing the target antigen, the preexisting antibodies bind to
the transfused cells, activate complement, and cause transfusion reactions

ABO incompatibility reactions (can be life-threatening)

1- immediate hemolytic reaction & intravascular lysis of red blood cells : mediated by the complement
system, and phagocytosis of antibody- and complement-coated erythrocytes by macrophages in the
liver and spleen

2- acute renal tubular cell necrosis and kidney failure.

3- High fever, shock, and disseminated intravascular coagulation may also develop, suggestive of release
of massive amounts of cytokines (e.g., TNF or IL-1)

4- delayed hemolytic reactions

a- result from incompatibilities of minor blood group antigens
b- progressive loss of the transfused red blood cells, leading to anemia and jaundice
c- 1-14 days post-transfusion

5- Transfusion associated lung injury (TRALI) : Donor anti-leukocyte antibodies attack

6- Transfusion associated graft-versus-host disease (TA-GVHD):

a- Donor T cells attack immunocompromised patients
b- one week after the transfusion.
c- Signs include a fever, characteristic skin lesions, and diarrhea.
d- Blood tests reveal signs of bone marrow failure and liver malfunction.
e- Prevented by receiving blood products that have been irradiated.
f- Fatal
 7- Allergic reactions
8- Post transfusion purpura (PTP):
a- Platelet incompatibility
b- low number of platelet
c- occurs 5 to 10 days
d- treated by plasmapheresis

9- Febrile non-hemolytic transfusion reaction (FNHTR):

a- White blood cell incompatibility
b- mild leukodepletion
c- treated by antipyretic

d- Anaphylaxis:
a- IgA anti-plasma protein antibodies
b- In case of patients with IgA deficiency
c- life threatening
d- treated with epinephrine

Note :

Mutations in the gene encoding the fucosyltransferase that produces the H antigen are rare; people
who are homozygous for such a mutation are said to have the Bombay blood group

And cannot produce H, A, or B antigens. and cannot receive type O, A, B, or AB blood.

Lewis antigens

- minor antigens have recently received much attention from immunologists because these
carbohydrate groups serve as ligands for E-selectin and P-selectin and thus play a role in leukocyte
migration

Rh antigens and antibodies

- Ag : non-glycosylated, hydrophobic cell surface proteins found in red blood cell membranes
- the RH locus is on chromosome 1 and comprises two highly homologous, very closely linked genes,
RHD and RHCE.
- 15% of the population has a deletion or other alteration of the RhD allele
- The RHCE gene has four main alleles; CE, Ce, ce and cE.
- This concept of D and CcEe genes linked closely and transmitted together is consistent with the Fisher
nomenclature.
 - antibodies directed against all Rh antigens, except d, have been described: anti-D, anti-C, anti-c, anti-E
and anti-e.
- Rh antigens are restricted to red cells
- Rh antibodies result from previous alloimmunization by previous pregnancy or transfusion.
- Immune Rh antibodies are predominantly IgG (IgG1 and/or IgG3).
- Anti-D is clinically the most important antibody.
- It may cause hemolytic transfusion reactions and was a common cause of fetal death resulting from
hemolytic disease of the newborn before the introduction of anti-D prophylaxis.

hemolytic disease of the newborn

- Rh-negative mothers carrying an Rh-positive fetus can be sensitized by fetal red blood cells that enter
the maternal circulation, usually during childbirth.
- IgG antibodies are generated in Rh-negative mothers.
- Subsequent pregnancies in which the fetus is Rh positive are at risk because the maternal anti-Rh IgG
antibodies can cross the placenta and mediate the destruction of the fetal red blood cells.
- This causes erythroblastosis fetalis
- Prevention
a- Rh immune globulin (RhIg) or Rhogam is used to prevent Rh-negative mothers from becoming
sensitized to the Rh antigen of their newborn child
b- Shortly after each birth within 72 hours of an Rh+ baby, the mother is given an injection of anti-Rh
antibodies
c- The preparation is called Rh immune globulin (RhIG) or Rhogam
d- These passively acquired antibodies destroy any fetal cells that got into her circulation before they
can elicit an active immune response in her.