AGGLUTINATION TESTS and IMMUNOASSYS

SerologyAGGLUTINATION TESTS and IMMUNOASSYS

BASICSDr.T.V.Rao MD

Methods for Ag-Ab detection• Precipitation• Agglutination• Hemagglutination and Hemagglutination inhibition• Viral neutralization test• Radio-immunoassays• ELISA• Immunofluorescence• Immmunoblotting• Immunochromatography

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Agglutination• Agglutinins

– Antibodies that produce such reactions

• Involves two-step process:– Sensitization or initial

binding– Lattice formation or

formation of large aggregates

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Agglutination• Types of particles that

participate in such reactions:–Erythrocytes–Bacterial cells–Inert carriers such as latex

particles04/11/2023 Dr.T.V.Rao MD 4

Agglutination tests• Antibodies can agglutinate multivalent

particulate antigens, such as Red Blood Cells (RBCs) or bacteria

• Some viruses also have the ability to agglutinate with RBCs.

• This behavior is called agglutination.• Serological tests based on agglutination are

usually more sensitive than those based on precipitation

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Examples• Slide Agglutination Test• Plate Agglutination Test• Tube Agglutination Test• Passive Agglutination Test• Microscopic Agglutination Test• Haemagglutination test (HAT)04/11/2023 Dr.T.V.Rao MD 6

Steps in Agglutination•Primary phenomenon

(SENSITIZATION) First reaction involving Ag-Ab combination Single antigenic determinant on the surface

particle1) Initial reaction: rapid and reversible2) Cross link formation visible aggregates

(stabilization)

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Secondary phenomenon:LATTICE FORMATION

– Ab + multivalent Ag stable network (visible reaction)

– conc. of Ag and Ab– Governed by physiochemical factors:

• Ionic strength of milieu• pH• temperature

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Secondary Phenomenon • Lattice Formation• The Fab portion of the Ig molecule attaches to antigens on

2 adjacent cells-visible results in agglutination• If both antigen and antibody are SOLUBLE reaction will

become visible over time, ie, precipitation

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DIRECT AGGLUTINATION- Test patient serum against large, cellular

antigens to screen for the presence of antibodies.

• Antigen is naturally present on the surface of the cells.

• In this case, the Ag-Ab reaction forms an agglutination, which is directly visible.

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DIRECT AGGLUTINATION• The particle antigen may be a bacterium. e.g.: Serotyping of E. coli, Salmonella using a

specific antiserum• The particle antigen may be a parasite. e.g.: Serodiagnosis of Toxoplasmosis• The particle antigen may be a red blood cell. e.g.: Determination of blood groups

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DIRECT AGGLUTINATION

• These reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required.

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Positive Negative

Ag-Ab complex

Direct agglutination Principle• combination of an insoluble

particulate antigen with its soluble antibody – forms antigen-antibody

complex – particles clump/agglutinate

• used for antigen detection

Examples– bacterial agglutination tests

for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp

Slide Agglutination Test• Used for serotyping (e.g. Salmonella)• Antigen: isolated Salmonella in suspension• Antibody: specific antisera against Salmonella• Place test Salmonella in a drop of saline on a

slide • Add a drop of antiserum, mix and rock slide

for approx. 1 minute• Examine for agglutination

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Positive Negative

Ag-Ab complex

Direct agglutination

Principle• combination of an insoluble

particulate antigen with its soluble antibody – forms antigen-antibody

complex – particles clump/agglutinate

• used for antigen detectionExamples

– bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp

Slide Agglutination Test

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Tube Agglutination Test• Also known as the standard agglutination test or

serum agglutination test (SAT)• Test serum is diluted in a series of tubes (doubling

dilutions)• Constant defined amount of antigen is then added to

each tube and tubes incubated for ~20h @37°C• Particular antigen clumps at the bottom of the test

tube• Test is read at 50% agglutination• Quantitative• Confirmatory test for ELISA reactors• Example: Brucellosis screening , Widal Testing04/11/2023 Dr.T.V.Rao MD 20

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Tube Agglutination Test

No agglutinationAgglutination

1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl

In this case, the titre is 1/40

Tube Agglutination Test

Passive Agglutination

• An agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfaces

• Particle carriers include:– Red blood cells– Polystyrene latex– Bentonite– charcoal

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Passive Agglutination

• Passive agglutination has been used in the detection of :–Rheumatoid factor–Antinuclear antibody in LE–Ab to group A streptococcus antigens–Ab to Trichinella spiralis

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Passive Agglutination Test• Converting a precipitating test to an

agglutinating test• Chemically link soluble antigen to inert

particles such as LATEX or RBC• Addition of specific antibody will cause the

particles to agglutinate• Reverse PAT: antibody linked to LATEXe.g. Lancefield grouping in Streptococci.

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Reverse passive agglutination

Principle– antigen binds to

soluble antibody coated on carrier particles and results in agglutination

– detects antigens

Example– detecting cholera toxin

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REVERSE PASSIVEAgglutination Tests

• Antibody rather than antigen is attached to a carrier particle• For the detection of microbial antigens such as:

▫ Group A and B streptococcus▫ Staphylococcus aureus▫ Neisseria meningitides▫ Haemophilus influenza▫ Rotavirus▫ Cryptococcus neoformans▫ Mycoplasma pneumoniae▫ Candida albicans

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The latex particles are coated with IgG and mixed with the patient's serum

Quantitative Micro Hemagglutination Test (HA)

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Haemagglutination Test (HA)

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Haemagglutination

RBC

Viral Haemagglutination• Some viruses and microbes contain proteins which

bind to erythrocytes (red blood cells) causing them to clump together

• NDV• Adenovirus III• AIV• IBV• Mycoplasma

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Viral Hemagglutination

• the attachment of viral particles by their receptor sites to more than 1 cell.

• As more and more cells become attached in this manner agglutination becomes visible 04/11/2023 Dr.T.V.Rao MD 32

Readings The results • Titer: The maximum dilution that gives visible

agglutination.• The end point: is the well with the lowest

concentration of the virus where there is haemagglutination

2 4 8 16 32 64 128 256 512 1024 2048 4096

The HA titer of this virus in this row is 256 or 28

(1:256 dilution contains (1 HA unit) (one haemagglutinating unit)

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Titer = 32 HA units/ml

Hemagglutination test: method1:8

1:2 1:21:21:21:2

8 16 32 64 128 256

virus

serial dilution

mix with red blood cells

side view

top view

One HA unit :minimum amount of virus that causes complete agglutination of RBCs

In the absence of anti-virus antibodies

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Erythrocytes

Virus

Virus agglutination of erythrocytes

In the presence of anti-virus antibodies

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Erythrocytes

Virus Anti-virus antibodies

Viruses unable to bind to

the erythrocytes

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What is Antibody Titer• Is the lowest

concentration of antibodies against a particular antigen.

04/11/2023 Dr.T.V.Rao MD 38Figure 18.6

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Readings • The end point is the well with the lowest concentration of the serum where a clear button is seen. 2 4 8 16 32 64 128 256 512 1024 2048 4096

The antibody titer in this row will be 512 (29).(the lowest concentration of Abs which inhibit HA caused by the virus )

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Coombs Test an Agglutination Test

• The Coombs test is actually two separate tests: the "direct" and "indirect" Coombs tests. Both aim to identify autoimmune haemolysis of red blood cells (erythrocytes).

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Coombs (Antiglobulin)Tests

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• Incomplete Ab• Direct Coombs Test

– Detects antibodies on erythrocytes

+ ↔

Patient’s RBCs Coombs Reagent(Antiglobulin)

Coombs Test Direct ant globulin test (also called the Coombs’ test,

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Coombs (Antiglobulin)Tests

• Indirect Coombs Test– Detects anti-erythrocyte antibodies in serum

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Patient’s Serum

TargetRBCs

+ ↔Step 1

+ ↔

Coombs Reagent(Ant globulin)

Step 2

Application of Coombs (Antiglobulin)Tests

• Applications–Detection of

anti-Rh Ab–Autoimmune

hemolytic anemia

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Agglutination Inhibition• Based on the competition between

particulate and soluble antigens for limited antibody combining site

• Lack of agglutination is indicator of a positive reaction

• Usually involves haptens complexed with proteins

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Agglutination InhibitionTests

• Pregnancy Testing-classic example of

agglutination inhibition – Human chorionic

gonadotropin (hCG)• Appears in serum

and urine early in pregnancy

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Agglutination Inhibition

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Urine Antiserum

No hCG in urine:

Anti-hCG free

hCG in urine:Anti-hCG neutralized

Carriers coated with hCG added Carriers coated with hCG added

AGGLUTINATION of carriers:Negative test for hCGNOT PREGNANT

NO AGGLUTINATION of carriers:

Positive test for hCGPREGNANT

Co-agglutination• Co agglutination is similar to the latex

agglutination technique for detecting antigen (described above). Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions

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Coagglutination• Name given to systems

using inert bacteria as the inert particles to which the antibody is attached

• S.aureus: most frequently used because it has protein A in its outer surface that naturally adsorbs the Fc portion of the antibody

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Highly specific but not very sensitive in detecting small quantities of antigen

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Co agglutination Test Agglutination test in

which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.

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Rickettsia and Serology• Rickettsia is a genus of motile, Gram-negative, non-

spore forming, highly pleomorphic bacteria that can present as Cocci (0.1 μm in diameter), rods (1–4 μm long) or thread-like (10 μm long). Obligate intracellular parasites

• Because of this, Rickettsia cannot live in artificial nutrient environments and are grown either in tissue or embryo cultures (typically, chicken embryos are used).

• Still we have to dependent on Weil Felix test

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Weil and Felix contribute for testing

• In 1915, Weil and Felix showed that serum of patients infected with any member of the typhus group of diseases contains agglutinins for one or more strains of O X Proteus. In cases of typhus fever the reaction usually appears before the sixth day and reaches its height in the second week.

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Weil-Felix reaction – A Heterophile agglutination Test

• A Weil-Felix reaction is a type of agglutination test in which patients serum is tested for agglutinins to O antigen of certain non-motile Proteus and Rickettsial strains(OX19, OX2, OXk)

• OX19, OX2 are strains of Proteus vulgaris.OXk is the strain of Proteus mirabilis.

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Weil-Felix a Heterophile agglutination test

• The agglutination reactions, based on antigens common to both organisms, determine the presence and type of Rickettsial infection

• Because Rickettsia are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification

• Weil-Felix test that is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris.

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Reading/Grading Agglutination Reactions

• Done by gently shaking the tubes containing the serum and cells, and observing the cell button as it is dispersed

• Hard shaking must be avoided because this may yield to false result

• Attention should also be given to whether discoloration of the supernatant is present (Hemolysis).

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Interpretations in Weil-Felix reaction

• Sera from endemic typhus agglutinate OX19, OX2.Tick borne spotted fever agglutinate OX19, OX2.

• Scrub Typhus agglutinate OXk strain • Test is negative in rickettsia pox, trench fever and Q-

fever.False positive reaction may occur in urinary or other Proteus infectionsTest may be negative in 50 percent scrub typhus

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Weil-Felix testindicated in when patients present with rashes

• Test for diagnosis of typhus and certain other Rickettsial diseases. The blood serum of a patient with suspected Rickettsial disease is tested against certain strains of (OX-2, OX-19, OX-K)..

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Latex Agglutination

•Antibody molecules can be bound to each latex beads

•It will increase the potential number of exposed antigen-binding sites.

•When an antigen is present in test specimen, it may bind to the latex bead thus forming visible cross-linked aggregates.

•Latex particles can be coated with antigen (pregnancy testing, rubella antibody testing)

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Complement fixation Test• The complement fixation test (CFT) was extensively

used in syphilis serology after being introduced by Wasserman in 1909.

• Complement is a protein (globulin) present in normal serum.

• Whole complement system is made up of nine components: C1 to C9

• Complement proteins are heat labile and are destroyed by heating at 56°C for 20 – 30 minutes.

• Complement binds to Ag-Ab complex• When the Ag is an RBC it causes lysis of RBC’s.

Principle• Complement takes part in many of the immunological reactions.

It gets absorbed during the combination of antigens and antibody.

• This property of antigen–antibody complex to fix the complement is used in complement fixation test for the identification of specific antibodies.

• The hemolytic system containing sheep erythrocytes (RBC) and its corresponding antibody (Amboceptor) is used as an indicator which shows the utilization or availability of the complement.

• If the complement is fixed then there will be no lysis of sheep erythrocytes, thus denoting a positive test.

• If the complement is available then there will be hemolysis which is a property of complement, denoting a negative test.

Components of CFTTest System• Antigen: It may be soluble or particulate.

• Antibody: Human serum (May or may not contain Antibody towards specific Antigen)

• Complement: It is pooled serum obtained from 4 to 5 guinea pigs. It should be fresh or specially preserved as the complement activity is heat labile (stored at -30 °C in small fractions). The complement activity should be initially standardized before using in the test.

Indicator System (Hemolytic system)• Erythrocytes: Sheep RBC

• Amboceptor (Hemolysins): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.

Positive Test• Step 1:

At 37°CAntigen + Antibody + Complement Complement gets fixed

(from serum) 1 Hour

• Step 2: At 37°C

Fixed Complement complex + Hemolytic system No Hemolysis 1 Hour (Test Positive)

Negative Test Step 1:

At 37°C

Antigen + Antibody absent + Complement Complement not fixed

1 Hour

Step 2:

At 37°C

Free Complement + Hemolytic system Hemolysis

1 Hour (Test Negative)

Results and Interpretations:• No hemolysis is considered as a positive test. • hemolysis of erythrocytes indicative of a negative test.

1 2 3 4

A

B

• Microtiter plate showing Hemolysis (Well A3, A3 and B4) and No Hemolysis (Well

Radio-immunoassays• Principle

– Radioactively labelled-antibody (or antigen) competes with the patient’s unlabeled antibody (or antigen) for binding sites on a known amount of antigen (or antibody)

– Reduction in radioactivity of the antigen-patient antibody complex compared with control test is used to quantify the amount of patient antibody / antibody bound

– Limited use due to the problems with handling radioisotope

• Example– HBsAg– Thyroid function test

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Res

pons

e

Antibody

Radio Immuno Assay

Radio-immunoassays:Performance, applications

Advantages– highly sensitive– can be used for detection of small quantities– quantification possible

Limitations– expensive – requires isotopes

Time taken– 1 day

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Labeling technique

Enzyme-linked Immuno-Sorbant assay (ELISA)

Principle– use of enzyme-labeled immunoglobulin to

detect antigens or antibodies – signals are developed by the action of

hydrolyzing enzyme on chromogenic substrate– optical density measured by micro-plate reader

Examples– Hepatitis A (Anti-HAV-IgM, anti-HAV Iggy)

ELISA

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Antibody

Res

pons

e

Micro-plate reader

96-well micro-plate

Positive result

Labeling techniqueTypes of ELISA (Ag Abs tests)

Competitive •Antigen or antibody are labeled with enzyme and allowed to compete with unlabeled ones (in patient serum) for binding to the same target•Hydrolysis signal from Ag-Ab complex (enzyme-labeled) is measured•Antigen or antibody in serum is then calculated•No need to remove the excess/unbound Ag or Ab from the reaction plate or tubes)

Labeling technique

Types of ELISA used in the detection of antigens and antibodies

•Non-competitive •must remove excess/unbound Agor Ab before every step of reactions

•Direct ELISA •Indirect ELISA•Sandwich ELISA •Ab Capture ELISA (similar to sandwich ELISA but in 1st step, anti-Ig (M or G) is coated on the plate•Then antibodies in patient serum are allowed to capture in next step

ELISA:Performance, applications

• Advantages– Automated, inexpensive– Objective– Small quantities required– Class specific antibodies measurable

• Limitations– Expensive initial investment – Variable sensitivity / specificity of variable tests– Cross contamination

• Time taken - 1 day04/11/2023 Dr.T.V.Rao MD 75

Labeling technique

Cell infected with Dengue virus

V. Cholerae

Immuno-fluorescence•Principle

– Use fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systems

– Requires a fluorescent microscope

•Examples– Herpes virus IgM– Dengue virus– Rabies virus– Scrub and murine typhus

Immunofluorescence Helps in Diagnosis of Various Diseases

Immuno-fluorescence:Performance, applications

• Advantages– Sensitive and specific– Can be used for discrepant analysis

• Limitations– Expensive (Reagents and equipment)– Subjective– Cross reactivity– Non-specific immuno-fluorescence

• Time taken– few minutes to few hours.04/11/2023 Dr.T.V.Rao MD 78

Labeling techniqueTypes of immuno-fluorescence

Direct FA Indirect FA Sandwich FA

1st

2nd

3rd

4th

Ag=

Ab==FITC-conjugated Ab

Steps

Legend

=FITC-conjAnti-Ig

•Direct immuno-fluorescence

– Used to detect antigen

•Indirect and sandwich

immuno-fluorescence– Antigen detection– Antibody detection

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Western-blot analysis• Principle– Antigens are separated by Poly Acrylomide Gel

Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranes

– Antibodies in serum react with specific antigens – Signals are detected according to the principles of test

systems– Antibodies against microbes with numerous cross-

reacting antibodies identified more specifically• Examples

– T. pallidum, B.burgdorferi, – Herpes simplex virus types 1 and 2

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Anti HIV-1

Western-blot analysis • Serum, saliva, urine can

be tested• Kits are commercially

available• Recombinant immuno-

blotting assays (RIBA) uses recombinant proteins

Immuno-chromatography: Principle

• Dye-labelled antibody, specific for target antigen, is present on the lower end of nitrocellulose strip or in a plastic well provided with the strip.

• Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line

• Either antibody specific for the labelled antibody, or antigen, is bound at the control line

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Lysing agendLabled AB.

Test band(bound AB)

Control band(bound AB)

Nitrocellulose strip

BoundAB

Free labled AB

Immuno-chromatography: Principle

• If antigen is present, some labelled antibody will be trapped on the test line

• Excess-labelled antibody is trapped on the control line

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Captured Ag-labelled Ab-complex

Captured labelled Ab

Labelled AB-AG-complex

Captured by bound AB of

test band

Labelled AB-AG-complex

Captured by bound AB ofcontrol band

Immuno-chromatography: Performance, applications

• Advantages– Commercially available– Single use, rapid test– Easy to perform– Can detect antigen or antibody– Can be used in the field

• Limitations– Cost– Concern validated data

• Time taken - 1 hour04/11/2023 Dr.T.V.Rao MD 84

Chemiluminescent Immunoenzymatic Assay

• Process for the quantitative and qualitative determination of antigens, antibodies and their complexes by means of a chemiluminescing labelling substance activated or excited to Chemiluminescence's by an analytical reagent. By means of a serological reaction, initially an antigen/antibody complex is formed which is treated with a chemiluminescing conjugate containing chemiluminescing triphenylmethane dyes and the chemiluminescence of the chemiluminescing complex formed is measured.

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• Programme Created by Dr.T.V.Rao MD for Medical Students in the Developing

World• Email

• [email protected]

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