Serology

AGGLUTINATION TESTS and IMMUNOASSYS

BASICS Dr.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 particles
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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)
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Steps in Agglutination
Primary phenomenon (SENSITIZATION)
First reaction involving Ag-Ab combination Single antigenic determinant on the surface particle 1) Initial reaction: rapid and reversible 2) 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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Direct agglutination
Principle combination of an insoluble particulate antigen with its soluble antibody

used for antigen detection

forms antigen-antibody complex particles clump/agglutinate bacterial agglutination tests for sero-typing and serogrouping e.g., Vibrio cholerae,
Salmonella spp

Ag-Ab complex

Examples

Positive

Negative

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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Direct agglutination
Principle combination of an insoluble particulate antigen with its soluble antibody

used for antigen detection

forms antigen-antibody complex particles clump/agglutinate bacterial agglutination tests for sero-typing and serogrouping e.g., Vibrio cholerae,
Salmonella spp

Ag-Ab complex

Examples

Positive

Negative

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 @37C Particular antigen clumps at the bottom of the test tube Test is read at 50% agglutination Quantitative Confirmatory test for ELISA reactors 1/11/2013 Dr.T.V.Rao MD 20 Example: Brucellosis screening , Widal Testing

Tube Agglutination Test

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Tube Agglutination Test

Agglutination No agglutination

1/10

1/20

1/40

1/80

1/160

1/320

Neg. ctrl

In this case, the titre is 1/40

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 LATEX e.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 PASSIVE
Agglutination 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

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Quantitative Micro Hemagglutination Test (HA)

Haemagglutination Test (HA)

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Haemagglutination

RBC

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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 1/11/2013 Dr.T.V.Rao agglutination becomes visible MD

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Readings The results

2

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
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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Hemagglutination test: method

1:8 1:2 1:2 1:2 1:2 1:2

virus

serial dilution 8 mix with red blood cells side view 16 32 64 128 256

top view

Titer = 32 HA units/ml

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

In the absence of anti-virus antibodies

Erythrocytes

Virus

Virus agglutination of erythrocytes

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In the presence of anti-virus antibodies

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.

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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
Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes

+
Patients RBCs Coombs Reagent (Antiglobulin)
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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
Step 1 Patients Serum Step 2

+
Target RBCs

+
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Coombs Reagent (Ant globulin) Dr.T.V.Rao MD

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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 Inhibition Tests

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
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

NO AGGLUTINATION of carriers: AGGLUTINATION of carriers: Positive test for hCG Negative test for hCG PREGNANT NOT PREGNANT 1/11/2013 Dr.T.V.Rao MD 48

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 heatkilled 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, nonspore forming, highly pleomorphic bacteria that can present as Cocci (0.1 m in diameter), rods (14 m long) or thread-like (10 m 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 OX19 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 Qfever. False positive reaction may occur in urinary or other Proteus infections Test may be negative in 50 percent scrub typhus

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Weil-Felix test indicated 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 56C for 20 30 minutes. Complement binds to Ag-Ab complex When the Ag is an RBC it causes lysis of RBCs.

Principle
Complement takes part in many of the immunological reactions. It gets absorbed during the combination of antigens and antibody. This property of antigenantibody 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 CFT
Test 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 37C Antigen + Antibody + Complement (from serum) Complement gets fixed 1 Hour

Step 2:
Fixed Complement complex + Hemolytic system At 37C No Hemolysis 1 Hour (Test Positive)

Negative Test

Step 1:
At 37C

Antigen + Antibody absent + Complement

Complement not fixed 1 Hour

Step 2:

Free Complement + Hemolytic system 1 Hour

At 37C Hemolysis (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

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

Radio-immunoassays
Principle
Radioactively labelled-antibody (or antigen) competes with the patients 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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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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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

Labeling technique

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

ELISA

Micro-plate reader

Positive result

96-well micro-plate
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Types 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

Labeling technique Types of ELISA used in the detection of antigens and antibodies
Non-competitive
must remove excess/unbound Ag or 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:
Advantages

Performance, applications

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 day

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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

Labeling technique

Cell infected with Dengue virus

V. Cholerae

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.
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Labeling technique

Types of immuno-fluorescence
Steps
1st

Direct FA

Indirect FA

Sandwich FA

Direct immunofluorescence
Used to detect antigen

2nd

3rd

Legend
Ag= Ab= =FITC-conjugated Ab =FITC-conjAnti-Ig

Indirect and sandwich immuno-fluorescence

Antigen detection Antibody detection
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4th

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 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 crossreacting antibodies identified more specifically Examples T. pallidum, B.burgdorferi, Herpes simplex virus types 1 and 2
Anti HIV-1
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Western-blot analysis

Western-blot analysis
Serum, saliva, urine can be tested Kits are commercially available Recombinant immunoblotting 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
Bound AB Free labled AB Nitrocellulose strip 1/11/2013 Dr.T.V.Rao MD 82 Lysing agend Labled AB. Test band Control band (bound AB) (bound 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
Captured Ag-labelled Ab-complex Captured labelled Ab

Labelled AB-AGcomplex Captured by bound AB of test band

Labelled AB-AGcomplex Captured by bound AB of control band

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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 hour

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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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Email

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