Understanding

Immunoassays

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Assays and Analytes

Assay: A test run on the Access

Analyte: The specific substance
measured (analyzed) during an
assay.
Diagnostic groupings: Assays that
measure analytes from similar
biological funtional areas

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Access Diagnostic
Groupings

Thyroid

Anemia

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Folate, Vitamin B12, Ferritin,

Intrinsic Factor Aby, EPO

Reproductive

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hTSH ,T3, T4, Free T3, Free

T4, TU, Tg, Tg Aby, TPO Aby

hCG, hFSH, hLH, Prolactin,

Progesterone, Estradiol,
Unconjugated Estriol,
Testosterone, DHEA-S

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Access Diagnostic Groupings

Cardiac

Tumor markers

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Insulin

Inflammation

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PSA, Free PSA, AFP, CEA

OV, GI and BR Monitors

Diabetes

AccuTnI, BNP (BioSite),

CKMB,
Myoglobin, Digoxin,

IL-6

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Access Diagnostic Groupings

Adrenal pituitary

Cortisol

Bone Metabolism

Ostase

hGH
Intact PTH

Infectious disease
Rubella IgG, Toxo IgG,
Toxo IgM II
OUS - Rubella IgM

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Access Diagnostic Groupings

Blood Virus - OUS only,

certain countries

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HBV
HBsAg, HBsAg
Confirmatory,
HBsAb, HBcAb,
HBc IgM
HAV
HAV Ab, HAV IgM
HCV - BioRad only
HIV - BioRad only
HIV 1/2, HIV 1/2 Ab
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What is Immunoassay?

Immunoassay is a test method using a specific antibody

to bind with a substance not readily detected by simple
chemical methods

Y
Antigens

Antibodies

Antibodies are formed as an immune response

to a challenge from an antigen
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Lock and key

Specific binding to
one type of analyte only

Nonspecific binding to any solid phase surface

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What happens in an Assay?

Specific analyte from the sample is captured

by magnetic particles from the pack
The analyte is labelled with an enzyme
from the pack
The enzyme reacts with substrate to produce
light which is measured by a luminometer
The light (RLU) is compared to a stored
calibration
The concentration of the analyte is reported

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Immunoassay Process
Sample

Washing

Capture
Separate

Particle

Conjugate

Label

Detect
Substrate

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Capture Technology

The magnetic particles in the reagent pack are bound

to specific antibodies.

The sample contains analytes that can be captured

by these specific antibodies.

Particles and samples are added to a reaction vessel

where capture occurs

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Label Technology

The reagent pack contains an enzyme (alkaline

phosphatase)
*

*
*

The enzyme alkaline phosphatase is coupled to a

molecule that can form a complex with the magnetic
particle in the reaction vessel

*
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Separation Technology
Side Dispense Probes

Rings In RV

Wash Buffer Dispense

*
Magnet

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*
*

Detection Technology

Chemiluminescent Enzymatic Immunoassay

Alkaline phosphatase on the magnetic particle complex

reacts with chemiluminescent molecules added to the
reaction vessel from the Substrate Bottle

*
Light

Read via a simple photometer (luminometer)

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Reaction in Action

Paramagnetic
particles

Goat antimouse
IgG

Mouse
monoclonal
anti-BhCG

Washing removes
Substrate
Light is material
is
emitted
added
unbound

Sample
containing BhCG

37 C
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Rabbit anti BhCG

- alkaline phosphatase conjugate

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What makes RLUs?

RLU = Relative Light Unit

Measure of light emitted by a
sample
Substrate
Alkaline Phosphatase

Reagent (conjugate)
Environment (contamination bacteria)

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Washing Process

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Calibration Types

Two basic formats of calibration

curve:

Sandwich
Competitive

Determined by APF Assay Protocol File

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Calibration

Calibration: Measurement of the response of a

test system to a set of special samples called
calibrators whose concentrations are known

Calibrator: sample of known concentration at

given dose which the laboratory uses to:
Generate concentration curves
Determine cutoff values

Calibrators typically span the useful analytical

range of values, from low to very high

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Why do we need
calibrators?

Plot a curve used to determine analyte

amounts present in patient samples

Ensure that current lots of reagents are

performing appropriately

Ensure that instrument is performing within

limits of operation

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Generating a calibration
curve

Each calibrator is tested, usually in duplicate, by

the instrument

Results for each replicate are generated

Results are plotted on a graph (each test is

plotted separately)

Points are then connected by a curve fit routine

Calibration curve is valid for about 4 weeks

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Sandwich Assay

+
Antibody coated
Paramagnetic
Particle

+
Sample

*
*

*
*

*
*

Incubation
at 37 C

Antibody
specific
conjugate

3 wash cycles

Formation of
a Sandwich

Dioxetane
+

Dioxetane-P

Energy

Substrate

Fluorescein

*
*
*
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Alkaline
Phosphatase

The signal generated is

directly proportional to the
concentration in the sample

Stable
Glow
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Sandwich Calibration
6 levels of increasing concentration
Response directly proportional to concentration

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Competitive Assay
+
Sample

*
* *

Antibody
specific
conjugate

Incubation
at 37C

Antibody
coated
paramagnetic
particle

*
*

3 wash cycles

Dioxetane
+

Dioxetane-P
Substrate
*

Energy

The signal generated is

inversely proportional to
the concentration in the
sample

Fluorescein

Stable
*

Glow

Alkaline Phosphatase
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Competitive
Calibration
6 levels of increasing concentration
Response inversely proportional to concentration

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Calibration vs. Quality

Control

Calibration

Relates RLUs to known concentrations

Run about once a month

Quality Control (QC):

Relates concentrations to historical

performance
Run every day or shift

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Quality Control

Quality Control (QC):

process which monitors the accuracy and

precision of results through the use of control
specimens

Quality Assurance (QA):

Monitoring of:

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Instruments
Reagents
Technologist performance
Accuracy of reported results

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Why run QC?

QC is used:

To determine if the assay is working

To monitor over time for drifting/trending catch a
variety of instrument or reagent or other problems

Laboratory may use same lot of QC over a long

time frame

Engineers run QC to verify that the instrument

still reports results in the expected range after
service
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QC Terms

Accuracy: An instruments ability to

correctly determine the concentration of
the analyte in question

Precision: An instruments ability to

reproduce a result

Reliability: An instruments ability to

maintain accuracy and precision over time

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Bullseye
Precision

Accuracy

Reliability
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Basic Statistics

Mean: the arithmetic mean value; a

measurement of the central tendency of the data
points within a distribution of values. THE
AVERAGE

Standard Deviation: Measure of the dispersion

or variation in results about the mean; how close a
data point is to the mean, or correct value THE
SPREAD

Coefficient of variation (%CV): a description of

standard deviation as a percentage of the mean

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Use Excel for these

calculations

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%CV and Standard

Deviations

%CV is more useful

when comparing
precision of different
QC levels

Concentrations differ
Precision (%cv) is same

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Distribution and Spread

Normal distribution: Spread of data

points collected over time from the analysis of
replicates of the same sample

Replicates of a sample will give results

scattered about a mean value for that
sample

Inherent random error within the system

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F req u ency of occurren ce

Normal Distribution

68%

95%

99%
-3SD

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-2SD

-1SD

MEAN

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+1SD

+2SD

+3SD

Standard Deviation =
Spread

What does 1SD, or 2SD, or even 3SD

tell us?

Values we would fall somewhere in these

ranges

68% would fall within 1SD of the mean

95% would fall within 2SD of the mean

99% would fall within 3SD of the mean

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Normal Distribution =
Levey-Jennings Chart
Frequency of occurrence

-3SD
-2SD
-1SD

68%

95%

99%

MEAN
+1SD
+2SD
+3SD

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Levey-Jennings Chart

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QC Terms

Shift: Abrupt change in QC results is seen;

successive results are similar

Parts are replaced

After a major PM

Trend: Successive values slowly rising or

falling

Instrument part wearing out

Reagent gradually deteriorating

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Shift and Trend

g
n
i
d
n
e
Tr

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)
p
(u

Shift

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Relating QC to Instrument
Performance

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Relating QC to Instrument
Performance

Customer complains about concentration

My QC is out low!

Determine which assays affected (sandwich

or competitive)

Engineer translates concentration into RLUs

Low concentration = High RLU for
competitive assays

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Competitive
Calibration
Response inversely proportional to
concentration

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Sandwich Calibration
Response directly proportional to
concentration

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Another complaint!

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Is this a different
problem?

NO! Both complaints are because of

high RLUs

Translate concentration to RLUs

High concentration = High RLUs for

sandwich assay

So we have only one problem to

troubleshoot

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