IMMUNOLOGICAL

LABORATORY Dr. Wisanu Wanasawaeng

Ph.D. (Pathobiology), M.Sc. (Avian Medicine), DVM.

Veterinary Laboratory Utilization (VMCL3231)

Contents

Antigens &Antibodies

Precipitations

Agglutinations

Enzyme immunoassay

Immunochromatography

Test for cell-associated antigen

Fluorescent activated cell sorters

Immunoblotting

Antibody microarray

Antigens: • Antigen is a substance that stimulate the production of specific antibodies and

combine specifically with the antibodies produced.

• Most antigens are foreign to the blood and other bodily fluids.

Antibodies:

• Antibody proteins (immunoglobulins) are found in the gamma globulin class of plasma proteins.

• There are five main subclasses : IgG, IgA, IgM, IgD, and IgE.

• Most antibodies in serum are from the class IgG.

3

Antigens

Antibody Structure

Antibody consists of four

interconnected polypeptide chains

Four interconnected polypeptide chains.

Two heavy chains (H-chains) and

joined to two shorter chains (L-chains).

These four chains are arranged in the

form of a ‘Y’ ; with the stalk of the Y is

called the “crystallizable fragment”

The top of the Y is known as the

“antigen-binding fragment”. 4

antigenicity

The ability to combine specifically with the

final products of immune response e.g.

secreted antibodies

Immunogenicity

The ability to induce a humoral and/or cell-

mediated immune response

Epitope or antigenic determinant

The distinct molecular surface features of

an antigen capable to being bound by an

antibody

5

Antigenicity

Immunogenicity

Epitope or antigenic determinant

Ag-ab interaction

The bonds holding Ags and Abs

combining site are all non-

covalent in nature

Hydrogen bonds, electrostatic

bonds, Hydrophobic bonds and

Van der Walls forces

Multiple bonding ensures that

Ags will be bound tightly to Abs

6

Non-covalent bonds

Lock and key concept

The combining site of Abs is located

in the Fab portion of the molecule and

is constructed from Hypervariable

regions of the heavy and light chains

X-ray crystallography studies of Ag-

Ab interactions show the antigenic

determinant nestles in a cleft formed

by the combining site of the antibody

7

Lock and Key concept

affinity

The strength of the reaction between a single antigenic

determinant and a single Ab combining site on Abs

The sum of the attractive and repulsive forces operating

between the antigenic determinant and the combining

site of the antibody

Affinity: the equilibrium constant of the Ag-Ab reactions

Apply the law of mass action

Ka (K1/K2) = [Ag-Ab]

8

Affinity = ∑ attractive + repulsive forces

[Ag] [Ab]

Affinity constant determination

Equilibrium dialysis

9

A measurement of the overall strength of binding of Ag with many antigenic

determinants and multivalent Ab

Avidity is influenced by both the valence of the Ab and the valence of Ag

Avidity is more than the sum of the individual affinities

Avidity

Affinity is the strength between single antigenic

determinant and individual antibody combining site

Avidity is the overall strength of binding between

multivalent Ags and Abs

10

Cross Reactivity

The ability of an individual antibody combining site to react with more than one

antigenic determinant.

The ability of a population of Ab molecules to react with more than one Ag.

Cross reactions arise because the cross reacting Ag shares epitope in common

with immunizing Ag.

Anti-

A Ab

Ag A

Anti-

A Ab

Ag B

Shared epitope

Anti-

A Ab

Ag C

Similar epitope

Cross reactions

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Cross reactions

12

Classical precipitin curve

Antigen-antibody reaction:

Precipitin reactions in gels

Ab is incorporated into the agar gel &different

dilution of Ag are placed in holes punched into

the agar

Ag diffuses into gel &reacts with Ab

Equivalence point: a ring of precipitation is

formed

The positioning of Ab &Ag solution within

wells cut into an agar matrix to allow

visualization of precipitin bands formed by

reaction of components from the two solutions.

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Radial immunodiffusion

Double immunodiffusion

Radial Immunodiffusion

Interpretation:

Diameter of ring is

proportional to the

concentration

Quantitative:

Ig levels

• Method

– Ab in gel

– Ag in a well

Ag Concentration

Dia

me

ter2

Ag Ag Ag Ag

Ab in gel Mancini method

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Double immunodiffusion

1. Reaction of identity

2. Reaction of partial identity

3. Reaction of non-identity

“Spur”

Ouchterlony method

Application:

Anti-Nuclear Antibody

(ANA) e.g. SLE, Influenza

Immunoelectrophoresis

Incorporation of electrophoresis with

double diffusion

1. Ag mixture is separated by charge

2. Troughs are cut to direction of electrophoresis field

3. Anti-sera is added to trough

4. Ag and Ab diffusion towards each other to produce

precipitin bands

Application:

1. Presence/Absence of specific proteins or Ig classes

2. Immunodeficiency or immunoproliferative

disorders

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Countercurrent electrophoresis

Method

Ag and Ab migrate toward each other by electrophoresis

Used only when Ag and Ab have opposite charges

A rapid version of Ouchterlony double diffusion technique

Qualitative: rapid

Ag Ab

- +

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Agglutination tests

When Ag is particulate, the

reaction of Ab with Ag can

be detected by agglutination

(clumping) of Ag.

Simple, inexpensive,

but sensitive

Agglutinin is used when Abs

agglutinate particulate Ag.

Haemaggluinin is used when

Ag is erythrocyte.

Several types exist:

• 1. Haemagglutination

• 2. Bacterial agglutination

• 3. Passive agglutination

• 4. Agglutination

inhibition e.g. HI

+

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Direct agglutination

Direct agglutination:

Reactions test patient serum against large, cellular antigens to screen for the

presence of antibodies

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Blood agglutination

Agglutination (clumping) of type A red blood cells (RBCs) by anti-A Ab.

The Abs have two combining sites and are able to attach to the A Ag on adjacent

RBCs, thus causing the RBCs to bond together.

RBC + anti-A Ab ----- Haemagglutination = Blood group A

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Blood agglutination

Group O: No clumping in blood drops A or B

Group A: Clumping in blood drop A with anti-A Ab

Group B: Clumping in blood drop B with anti-B Ab

Group AB: Clumping in both blood drops A and B

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Blood agglutination

Serogrouping of bacteria

Place 1 drop of reagent from each bottle in

agglutination site on the test

plate.

Use a conventional 10µl loop to pick some

fresh colonies from the plate

where the bacteria have been

isolated, and transfer adjacent to the test plate.

Place the loop in the drop and

return this small volume to mix with bacterial

material.

Then mix &spread with the rest of the reagent

with in agglutination site.

Using a fresh loop, repeat the

procedure above for each serotype and agglutination site on the test

plate.

Agitate the test plate gently for

1 minute.

Read agglutination pattern while

agitating.

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indirect agglutination

Indirect agglutination

• To test patient serum for the presence of Abs against soluble Ags, serum is mixed

with latex spheres with the soluble Ags attached.

• Abs will then cause visible agglutination of the latex spheres with the soluble

antigens attached.

• Abs may be attached to the latex spheres to test for the presence of soluble antigens

in patient serum.

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Hae

mag

glu

tin

atio

n

Agglutination reactions

using red blood cells.

Using in blood typing,

the diagnosis of certain

diseases, &identification

of viruses.

Viral HA occurs when

spikes on the virus cause

agglutination of red blood

cells - there is no antigen-

antibody interaction.

Haemagglutination test

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HA

Influenza virus

Influenza/Rubella/Mump Ag

(Haemagglutinin) + RBC

Haemagglutination

Haemagglutination test

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HA titration of antigens

A B

C D E F G H

1 2 3 4 5 6 7 8 9 10 11 12

HA titer 2 4096

PBS 50μl

50l 50l 50l 50l 50l 50l 50l 50l 50l 50l 50l 50l

Add 50l of antigen

Add 50l of antigen

Add 50l of antigen

Add 50l of antigen

Add 50l of antigen

Add 50l of antigen

Determination of HA titers

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Haemagglutination Inhibition

RBC

Haemagglutination Inhibition (HI) RBC

Virus Haemagglutination (HA)

Virus

Ab - Virus

Ab Ab - Virus

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Dilution of anti sera for HI test

A B

C D E F G H

1 2 3 4 5 6 7 8 9 10 11 12

Serum

titer

50 l 50 l 50 l 50 l 50 l 50 l

50l 50 l 50 l 50 l 50 l 50 l

Add 50l of serum

Add 50l of serum

Add 50l of serum

Add 50l of serum

Add 50l of serum

Add 50l of serum

Add 50l of serum

Add 50l of PBS (control)

+ 50 l

8HA antigen

to A-G wells

Then, incubate

30min.

Add 50 l

CRBC

Incubate 30min

PBS 50μl

2 4096

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Haemagglutination Inhibition

Enzyme-linked immunosorbent

The most sensitive of tests for Ag/Ab

ELISA tests depend on enzyme conjugated to antibody reacting with a specific substrate to produce a color reaction

Allows for qualitative or quantitative testing of Ag or Ab

• Valuable tools for use in clinical labs, can measure Abs or Ags

• Inexpensive, rapid, quantitative, specific, sensitive (pg/ml)

• Expensive equipment not required (but helps!)

• Can be automated

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Types of ELISA

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Indirect ELISA

Antigen

Primary antibody

Secondary antibody HRP conjugated Species specific

anti-IgG antibody

Substrate Uncolored/Colored substrate

Positive Negative

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Positive Negative

Antigen

Conjugate HRP conjugated Species specific

Anti-IgG Monoclonal antibody

Substrate Uncolored/Colored substrate

Capture Antibodies

Sandwich ELISA

Detector Antibodies

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Add TMB

Competitive ELISA

Sheep anti-rabbit IgG

Rabbit anti-CAP IgG Sheep anti-rabbit IgG CAP Substrate

Rabbit anti-CAP + CAP-E + CAP

E E E E

E CAP+E

Positive Negative

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• The most sensitive method

to measure antigen-specific T

cell function

eLISPOT

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Blue-black colored precipitate forms at the sites of cytokine localization and

appears as spots

Each individual spot representing an individual cytokine-secreting cell.

The spots can be counted with automated ELISpot reader systems or

manually, using a stereomicroscope

eLISPOT

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ELISPOT

New technology to detect individual activated

effector T cells

T-SPOT.TB is suitable for use with all patients at

risk of latent tuberculosis infection (LTBI) or

suspected of having TB disease, whether

immunocompetent or immunocompromised.

T-SPOT.tb

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Immunochromatography

The need of on-site examination at the point-of-care system ex. farms, hospital

Not require any expert knowledge and complex procedure

Simple to use but quick responses

Principle

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Positive semples with

Ag Influenza A

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Influenza a test kit

Application

Pregnancy test kit: HCG pregnancy test

Drugs of abuse test kit: amphetamine, methamphetamine etc.

Steroid test kit in herbal medicine drug: dexamethazone, prednisolone etc.

Human infectious diseases test kit: influenza, HIV (1+2), HBsAb, HBsAg,

Dengue, Adeno/Rota, H. pylori etc.

Pet animal diseases test kit: CPV, FELV, FIV, LSH, RTV, Giardia, Crypto

Food borne pathogen test kit: salmonella, listeria etc.

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Immunochromato Reader

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Immunochromato Reader

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Fluorescence microscope necessary for analysis of specimens

High structure resolution possible

Advanced image reconstruction (3D) and signal quantification

Multiple labeling easy

Limit shelf life of labeled specimens

Method of choice for labeling of live cells

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Immunofluorescence

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A.H. Coons (1941): fluorochrome-labeled antigens or antibodies

FITC (Fluorescein isothiocyanate): green

Absorption at wavelength 490-495 nm

Emission at wavelength 517 nm

TRITC (Tetramethyl rhodamine isothiocyante): red

Absorption at wavelength 550 nm

Emission at wavelength 580 nm

Fluorescence microscopy

1. Direct immunofluorescence

2. Indirect immunofluorescence

Immunofluorescence

Multiple immunofluorescence

IHC uses Abs to detect

&visualize Ags in cells

&tissues

Abs can be raised against almost any type

of Ags (protein, carbohydrate, lipid etc.)

Abs bind to Ag in a specific

manner

Bound Abs can be detected in several ways

54

Immunohistochemistry

A very important diagnostic &research tools

55

Two strategies used for the immmunohistochemical detection

of antigens in tissue

1. Direct method

2. Indirect method

Modified avidin-biotin complex (ABC) method

> Biotinylated secondary antibody

> Strept (avidin)-horseradish peroxidase

> 3, 3’-Diaminobenzidine (DAB)> brown

Immunohistochemistry

Fix

• Fix embed & section tissue

• Wash section in physiological buffer e.g. PBS

Block

• Incubate with protein solution (BSA) to reduce non-specific binding

of Abs to specimen

1 Ab

• Incubate with primary Ab + (Pos. &Neg. Control)

• Wash section in physiological buffer e.g. PBS

Detect

• Apply suitable detection system

• Mount specimens and analyze microscopically

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Outline of procedures

Antigen expressing Cell

Primary Antibody (species X)

Biotinylated

Secondary Antibody

(anti species X)

Enzyme-conjugated

Avidin-Biotin Complex

Substrate-chromogen

solution

ABC (Avidin Biotin Complex)

Biotin (Vitamin B) binds with high affinity to Avidin-thus good linker system

Extremely high sensitivity

Endogenous biotin may be present in tissue-risk of background

59

Flow cytometry

Flow

Tip

Laser

FL

Detector

Light

Scatter

Detector

Flow cytometry

Cells in suspension labeled with fluorescent-tag

Cells analyzed on flow cytometer

FCM is a technique for

counting and examining

microscopic particles, such as

cells and chromosomes, by

suspending them in a stream of

fluid and passing them by an

electronic detection apparatus.

Simultaneous multiparametric

analysis of the physical and/or

chemical characteristics of up to

thousands of particles per second.

Data analysis

Data displayed

Gating: Histogram

Computational analysis

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Fluorescent activated cell sorters

Cell sorting Monitor for fluorescence Forward angle light scatter (FALS or FSC) > cell size 90o light scatter > cytoplasmic granularity Deflection plates

Ultrasonic nozzle vibrator FACS

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immunoblotting

• Immunoblotting or Western blotting is an extremely powerful technique for identifying a single protein (or epitope) in a complex mixture following separation based on its MW by SDS-PAGE, size and charge (non denaturing gel electrophoresis) or isoelectric point (isoelectric focusing)

• Three steps: 1. SDS-PAGE 2. Electro transfer 3. Antibody detection • Limit of Detection: 10 pg with HRP or AP labeling

A detergent that can dissolve

hydrophobic molecules with a negative

charge attached to it.

To denature all proteins to the same

linear shape.

Two important features:

1. All proteins retain only primary structure

2. All proteins have a large negative charge

and migrate toward the positive pole in

electric field

SDS-PAGE

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SDS (Sodium dodecyl sulfate)

To put the proteins into an environment

that will allow different sized proteins to

move at different rates.

The environment of choice is

polyacrylamide, which is a polymer of

acrylamide monomers.

A polyacrylamide gel is made of a

labyrinth of tunnels through a meshwork

of fibers.

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SDS-PAGE

PAGE (Polyacrylamide electrophoresis)

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• Protein separation on SDS-polyacrylamide gel

• Electrophoretic transfer of protein blot into nitrocellulose membrane

SDS-PAGE

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1. Analysis of the protein purity

2. Determination of protein molecular weight

3. Verification of protein concentration

4. Detection of proteolysis

5. Identification of immunoprecipitated proteins

6. First stage of immunoblotting

7. Detection of protein modification

8. Separation and concentration of protein antigens for antibody production

9. Separation of radioactively labeled proteins

applications

To identify specific proteins in mixtures

Proteins are separated on SDS-PAGE

Proteins are transferred to membrane

Membrane flooded with enzyme-linked

polyclonal Abs specific for protein

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Western blotting

Western Blot

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• Immunoblotting can be divided into two steps

• 1. Transfer of protein from the gel to the matrix

• 2. Decoration of the epitope with the specific antibody

Western Blot

Western blotting

70

• Block membrane with 3 % BSA/TBS for 1 hr • Wash membrane wit TBS • Add primary antibody at appropriate dilution in 0.5 % BSA/TBS for 1 hr • Wash membrane with TBS • Add secondary antibody at appropriate dilution in 0.5 %

BSA/TBS for 1 hr • Wash membrane with TBS • Add the adequate substrate solution: DAB

Western blotting

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Lane 1: SF-9 cell lysate

2: Baculovirus wild type

3. H9N2 ELISA antigen

4: Molecular weight marker (kDa)

5: Blank

6: rH9N2 NP

~61kDa: Hemagglutinin

~56kDa: Nucleoprotein

~50kDa: Neuraminidase

~27kDa: Matrix Protein

Western blotting

Protein Microarray

Application of Protein arrays

Protein binding microarray, Biochip,

Protein chip

A multiplex approach to identify

protein-protein interactions, to

identify the targets of biologically

active small molecules

An important tool for proteomic

researches

Related microarray technology

including DNA microarrays, cellular

microarrays, tissue microarrays,

chemical compound microarrays and

antibody microarrays

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Antibody (Ab) Microarray

A specific from of protein microarrays.

A collection of capture Abs are spotted and fixed on a solid surface such glass, plastic and silicon chip for the

purpose of detecting Ags.

Antibody microarray is often used for detecting protein expressions from cell lysates.

It is used for general research such as toxicity testing and drug discovery, and special markers from serum or

urine for diagnostic application

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Procedure

Scanning of the array and the analysis of the results.

Incubation of labeled protein with the array.

Removal of unbound dye.

Labeling of extracted protein with fluorescent dyes Cy5 and Cy3.

Extraction of total cellular protein from biological samples of interest (e.g. Serum samples).

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Biochip Array Technology

Biochip Array Technology for Multi-Analyte Testing

Simultaneous determination of multiple analystes

from a single sample.

Biochip is a solid substrate containing an array of

discrete test regions of immobilized Abs specific to

different antimicrobials

Competitive chemiluminescent immunoassay

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Antimicrobial array 12 S

ulp

honam

ides

Oth

ers

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A few ways to create microarrays

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Comparison of Immunoassay

Method Western’s

Blot ELISA ELISPOT

Immono

precipitation

Immono

cytochemistry

Immono

histochemistry Flow cytometry

Target

(Analyte)

Proteins or

polypeptide

Peptide,

proteins

Cells Protein or complex Cells Tissue section Cells

Primary

antibody

Binds

antigen

Binds

antigen

Can be linked to

organic dye or

quantum dot

Can be linked to

beads

Can be linked to

organic dye or

quantum dot

Can be linked to

organic dye or

quantum dot

Can be linked to

organic dye or

quantum dot

Secondary

antibody

Used for

signal

generation

Enzyme

linked for

generating

signal

Enzyme or dye or

dot linked

May be omitted Enzyme or dye or

dot linked

Enzyme or dye or

dot linked

Enzyme or dye or

dot linked

Data forms Protein

binding

image

Numerical

and curve

Image or

numerical

Protein binding

image

Image Image Numerical or

curve

Sensitivity ng/ml pg/ml pg/ml ng/ml ng/ml ng/ml 1/105 cells

Reproducibility Good High Good Variable Variable Variable Good

Quantification Semi Yes Semi Semi Semi Semi Yes

Applications Protein

analysis and

ident.

Antigen

analysis

Antigen analysis

Protein analysis

and ident.

Protein analysis

and ident.

Protein analysis and

ident.

Antigen analysis

Methods

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Method Western’s

Blot ELISA ELISPOT

Immono

precipitation

Immono

cytochemistry

Immono

histochemistry Flow cytometry

Learn and

operation

Easy Easy Less easy Less easy Difficult Difficult More difficult

Assay

development

Simple Take time

(Days)

Take time

(Days)

Longer time

(Weeks)

Longer time

(Weeks)

Longer time

(Weeks)

Longer time

(Months)

Validation Simple Complicate Difficult Difficult Difficult Difficult Complicate

Standardization Straight

forward

Need

definition

Control variables Control variables More variables More variables

Need definition

High through

put

No Possible

with

automation

No No No Possible with

automation

Yes

Cost for set up Low ($1-

2K)

Low ($5-

10K)

Low ($5-10K) Low ($2-5K) Middle ($20-40K) Middle ($30-50K) High ($50-

100K)

Cost per assay Low Middle High High

High

Middle High

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Comparison of Immunoassay

Methods