Agglutination tests - جامعة الملك سعود

Serology AGGLUTINATION TESTS and IMMUNOASSYS BASICS Saud AbdUllah Almasri 434104889 Methods for Ag-Ab detection Precipitation Agglutination Hemagglutination and Hemagglutination inhibition

Viral neutralization test Radio-immunoassays ELISA Immunofluorescence Immmunoblotting Immunochromatography 2 Agglutination Agglutinins Antibodies that produce such

reactions Involves twostep process: Sensitization or initial binding Lattice formation or formation of large aggregates 3 Agglutination - Types of particles that participate in such

reactions: Erythrocytes Bacterial cells Inert carriers such as latex particles 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 5 Examples Slide Agglutination Test Plate Agglutination Test

Tube Agglutination Test Passive Agglutination Test Microscopic Agglutination Test Haemagglutination test (HAT) 6 Steps in Agglutination Primary phenomenon (SENSITIZATION) First reaction involving Ag-Ab

combination Single antigenic determinant on the surface particle I. Initial reaction: rapid and reversible II. Cross link formation visible aggregates (stabilization) 7 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 8 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 9 DIRECT AGGLUTINATION - Test patient serum against large, cellular antigens to screen for the presence of antibodies. o Antigen is naturally present on the surface of the cells.

o In this case, the Ag-Ab reaction forms an agglutination, which is directly visible. 10 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 11 12 DIRECT AGGLUTINATION These reactions

can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required. 13 Direct agglutination Principle combination of an insoluble

particulate antigen with its soluble antibody - forms antigen-antibody complex. - particles clump/agglutinate used for antigen detection Ag-Ab complex Examples

- bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp 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 16 17 Direct agglutination Principle

combination of an insoluble particulate antigen with its soluble antibody forms antigen-antibody complex particles clump/agglutinate Ag-Ab complex used for antigen detection

Examples bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp Positive Negative Slide Agglutination Test

02/26/2020 Dr.T.V.Rao MD 19 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 Example: Brucellosis screening , Widal Testing 20 Tube Agglutination Test

02/26/2020 Dr.T.V.Rao MD 21 Tube Agglutination Test Agglutination 1/10

1/20 No agglutination 1/40 1/80 1/160 In this case, the titre is 1/40 1/320

Neg. ctrl 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 23 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 24 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. 25 Reverse passive agglutination Principle - antigen binds to soluble antibody coated on carrier particles and results in agglutination

- detects antigens Example - detecting cholera toxin 26 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 27

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