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SerologyAGGLUTINATION TESTS and    IMMUNOASSYS          BASICS      Dr.T.V.Rao MD
Methods for Ag-Ab detection•   Precipitation•   Agglutination•   Hemagglutination and Hemagglutination inhibition•   Viral...
Agglutination• Agglutinins      – Antibodies that        produce such        reactions• Involves two-step  process:      –...
Agglutination• Types of particles that  participate in such reactions:  –Erythrocytes  –Bacterial cells  –Inert carriers s...
Agglutination tests• Antibodies can agglutinate multivalent  particulate antigens, such as Red Blood Cells  (RBCs) or bact...
Examples• Slide Agglutination Test• Plate Agglutination Test• Tube Agglutination Test• Passive Agglutination Test• Microsc...
Steps in Agglutination• Primary phenomenon  (SENSITIZATION)      First reaction involving Ag-Ab combination      Single ...
Secondary phenomenon:LATTICE FORMATION   – Ab + multivalent Ag  stable network (visible     reaction)   – conc. of Ag and...
Secondary Phenomenon      • Lattice Formation      • The Fab portion of the Ig molecule attaches to antigens on        2 a...
DIRECT AGGLUTINATION- Test patient serum against large, cellular  antigens to screen for the presence of  antibodies.• Ant...
DIRECT AGGLUTINATION• The particle antigen may be a bacterium. e.g.: Serotyping of E. coli, Salmonella using a  specific a...
1/11/2013   Dr.T.V.Rao MD   12
DIRECT AGGLUTINATION• These reactions  can be performed  on slides (rapid  tests) or on  microliter plates  or tubes for  ...
Direct agglutinationPrinciple• combination of an insoluble  particulate antigen with its soluble  antibody   – forms antig...
Slide Agglutination Test• Used for serotyping (e.g. Salmonella)• Antigen: isolated Salmonella in suspension• Antibody: spe...
1/11/2013   Dr.T.V.Rao MD   17
Direct agglutinationPrinciple• combination of an insoluble  particulate antigen with its soluble  antibody   – forms antig...
Slide Agglutination Test1/11/2013     Dr.T.V.Rao MD    19
Tube Agglutination Test• Also known as the standard agglutination test or     serum agglutination test (SAT)• Test serum i...
Tube Agglutination Test1/11/2013    Dr.T.V.Rao MD   21
Tube Agglutination Test    Agglutination                   No agglutination 1/10       1/20    1/40     1/80      1/160   ...
Passive Agglutination• An agglutination reaction that employs  particles that are coated with antigens not  normally found...
Passive Agglutination• Passive agglutination has been used  in the detection of :      –Rheumatoid factor      –Antinuclea...
Passive Agglutination Test • Converting a precipitating test to an   agglutinating test • Chemically link soluble antigen ...
Reverse passive agglutination            Principle      – antigen binds to        soluble antibody        coated on carrie...
REVERSE PASSIVE                Agglutination Tests • Antibody rather than antigen is attached to a carrier particle • For ...
The latex particles are coated with IgG and        mixed with the patients serum1/11/2013           Dr.T.V.Rao MD         ...
Quantitative Micro       Hemagglutination Test (HA)     Haemagglutination Test (HA)1/11/2013         Dr.T.V.Rao MD     29
Haemagglutination                     RBC1/11/2013           Dr.T.V.Rao MD   30
Viral Haemagglutination• Some viruses and microbes contain proteins which  bind to erythrocytes (red blood cells) causing ...
Viral Hemagglutination• the attachment of viral particles by their receptor sites to     more than 1 cell.• As more and mo...
Readings The results•         Titer: The maximum dilution that gives visible          agglutination.•         The end poin...
Hemagglutination test: method                        1:8                          1:2         1:2        1:2        1:2   ...
In the absence of anti-virus                     antibodies                  Erythrocytes              Virus              ...
In the presence of anti-virus                     antibodies                   Erythrocytes               Virus       Anti...
1/11/2013   Dr.T.V.Rao MD   37
What is Antibody Titer• Is the lowest concentration of antibodies against a particular antigen.  1/11/2013           Dr.T....
1/11/2013   Dr.T.V.Rao MD   39
Readings•      The end point is the well with the lowest    concentration of the serum where a clear button is seen.    2 ...
Coombs Test an Agglutination Test• The Coombs test is  actually two  separate tests: the  "direct" and  "indirect" Coombs ...
Coombs (Antiglobulin)Tests                                          • Incomplete Ab                                     • ...
Coombs Test     Direct ant globulin test (also called the Coombs’                           test,1/11/2013                ...
Coombs (Antiglobulin)Tests   • Indirect Coombs Test            – Detects anti-erythrocyte antibodies in serumStep 1       ...
Application of Coombs (Antiglobulin)Tests• Applications      –Detection of       anti-Rh Ab      –Autoimmune       hemolyt...
Agglutination Inhibition• Based on the competition between  particulate and soluble antigens for  limited antibody combini...
Agglutination Inhibition                         Tests• Pregnancy Testing      -classic example of            agglutinatio...
Agglutination Inhibition          Urine                                        Antiserum    No hCG in urine:              ...
Co-agglutination• Co agglutination is similar to the latex  agglutination technique for detecting antigen  (described abov...
Coagglutination• Name given to systems  using inert bacteria as  the inert particles to  which the antibody is  attached• ...
Highly specific but not very sensitive in detecting               small quantities of antigen1/11/2013                 Dr....
Co agglutination Test  Agglutination test in   which inert particles   (latex beads or heat-   killed S aureus Cowan   1 s...
Rickettsia and Serology• Rickettsia is a genus of motile, Gram-negative, non-  spore forming, highly pleomorphic bacteria ...
Weil and Felix contribute for testing• In 1915, Weil and Felix showed that serum of  patients infected with any member of ...
Weil-Felix reaction – A Heterophile                     agglutination Test• A Weil-Felix reaction is a type of  agglutinat...
Weil-Felix a Heterophile agglutination test• The agglutination reactions,  based on antigens common to  both organisms, de...
Reading/Grading Agglutination Reactions• Done by gently shaking the tubes containing  the serum and cells, and observing t...
Interpretations in Weil-Felix reaction• Sera from endemic typhus agglutinate OX19, OX2.  Tick borne spotted fever agglutin...
Weil-Felix test indicated in when patients present with rashes• Test for diagnosis of  typhus and certain  other Rickettsi...
Latex Agglutination • Antibody molecules can be bound to each   latex beads • It will increase the potential number of   e...
Complement fixation Test• The complement fixation test (CFT) was extensively  used in syphilis serology after being introd...
Principle• Complement takes part in many of the immunological reactions.  It gets absorbed during the combination of antig...
Components of CFTTest System• Antigen: It may be soluble or particulate.• Antibody: Human serum (May or may not contain An...
Positive Test• Step 1:                                  At 37°CAntigen + Antibody + Complement      Complement gets fixed ...
Negative Test                                                         Step 1:                                            ...
Results and Interpretations:•   No hemolysis is considered as a positive test.•   hemolysis of erythrocytes indicative of ...
Radio-immunoassays• Principle     – Radioactively labelled-antibody (or antigen) competes with the patient’s       unlabel...
Radio Immuno Assay
Radio-immunoassays:       Performance, applicationsAdvantages     – highly sensitive     – can be used for detection of sm...
Enzyme-linked                    Labeling                        Immuno-Sorbant                                technique  ...
ELISA                               Micro-plate reader                    Positive result                                 ...
Labeling       Types of ELISA (Ag Abs           tests)                                         technique         Competiti...
Labeling     Types of ELISA used in the detection of antigens and                          antibodies            technique...
ELISA:            Performance, applications• Advantages     – Automated, inexpensive     – Objective     – Small quantitie...
Labeling           Immuno-fluorescence                             technique•Principle    – Use fluorescein isothiocyanate...
Immunofluorescence Helps inDiagnosis of Various Diseases
Immuno-fluorescence:       Performance, applications• Advantages     – Sensitive and specific     – Can be used for discre...
Labeling            Types of                                            technique                                         ...
• Principle    Western-blot analysis   – Antigens are separated by Poly Acrylomide Gel     Electrophoresis (PAGE) and tran...
Western-blot analysis• Serum, saliva, urine can  be tested• Kits are commercially  available• Recombinant immuno-  blottin...
Immuno-chromatography:                  Principle• Dye-labelled antibody, specific for target antigen, is present on  the ...
Immuno-chromatography:                   Principle• If antigen is present, some labelled antibody will  be trapped on the ...
Immuno-chromatography:             Performance, applications• Advantages     – Commercially available     – Single use, ra...
Chemiluminescent Immunoenzymatic                   Assay• Process for the quantitative and qualitative  determination of a...
• Programme Created by Dr.T.V.Rao MD    for Medical Students in the Developing                    World                   ...
AGGLUTINATION TESTS and IMMUNOASSYS
AGGLUTINATION TESTS and IMMUNOASSYS
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  1. 1. SerologyAGGLUTINATION TESTS and IMMUNOASSYS BASICS Dr.T.V.Rao MD
  2. 2. Methods for Ag-Ab detection• Precipitation• Agglutination• Hemagglutination and Hemagglutination inhibition• Viral neutralization test• Radio-immunoassays• ELISA• Immunofluorescence• Immmunoblotting• Immunochromatography 1/11/2013 Dr.T.V.Rao MD 2
  3. 3. Agglutination• Agglutinins – Antibodies that produce such reactions• Involves two-step process: – Sensitization or initial binding – Lattice formation or formation of large aggregates1/11/2013 Dr.T.V.Rao MD 3
  4. 4. Agglutination• Types of particles that participate in such reactions: –Erythrocytes –Bacterial cells –Inert carriers such as latex particles1/11/2013 Dr.T.V.Rao MD 4
  5. 5. 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 precipitation1/11/2013 Dr.T.V.Rao MD 5
  6. 6. Examples• Slide Agglutination Test• Plate Agglutination Test• Tube Agglutination Test• Passive Agglutination Test• Microscopic Agglutination Test• Haemagglutination test (HAT)1/11/2013 Dr.T.V.Rao MD 6
  7. 7. 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)1/11/2013 Dr.T.V.Rao MD 7
  8. 8. 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 • temperature1/11/2013 Dr.T.V.Rao MD 8
  9. 9. 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, precipitation1/11/2013 Dr.T.V.Rao MD 9
  10. 10. 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.1/11/2013 Dr.T.V.Rao MD 10
  11. 11. 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 groups1/11/2013 Dr.T.V.Rao MD 11
  12. 12. 1/11/2013 Dr.T.V.Rao MD 12
  13. 13. DIRECT AGGLUTINATION• These reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required.1/11/2013 Dr.T.V.Rao MD 13
  14. 14. Direct agglutinationPrinciple• combination of an insoluble particulate antigen with its soluble antibody – forms antigen-antibody complex – particles clump/agglutinate• used for antigen detection Ag-Ab complexExamples – bacterial agglutination tests for sero-typing and sero- grouping e.g., Vibrio cholerae, Salmonella spp Positive Negative
  15. 15. 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 agglutination1/11/2013 Dr.T.V.Rao MD 16
  16. 16. 1/11/2013 Dr.T.V.Rao MD 17
  17. 17. Direct agglutinationPrinciple• combination of an insoluble particulate antigen with its soluble antibody – forms antigen-antibody complex – particles clump/agglutinate Ag-Ab complex• used for antigen detectionExamples – bacterial agglutination tests for sero-typing and sero- grouping e.g., Vibrio cholerae, Salmonella spp Positive Negative
  18. 18. Slide Agglutination Test1/11/2013 Dr.T.V.Rao MD 19
  19. 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 @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 Testing1/11/2013 Dr.T.V.Rao MD 20
  20. 20. Tube Agglutination Test1/11/2013 Dr.T.V.Rao MD 21
  21. 21. 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
  22. 22. 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 – charcoal1/11/2013 Dr.T.V.Rao MD 23
  23. 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 spiralis1/11/2013 Dr.T.V.Rao MD 24
  24. 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.1/11/2013 Dr.T.V.Rao MD 25
  25. 25. Reverse passive agglutination Principle – antigen binds to soluble antibody coated on carrier particles and results in agglutination – detects antigensExample – detecting cholera toxin1/11/2013 Dr.T.V.Rao MD 26
  26. 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 albicans1/11/2013 Dr.T.V.Rao MD 27
  27. 27. The latex particles are coated with IgG and mixed with the patients serum1/11/2013 Dr.T.V.Rao MD 28
  28. 28. Quantitative Micro Hemagglutination Test (HA) Haemagglutination Test (HA)1/11/2013 Dr.T.V.Rao MD 29
  29. 29. Haemagglutination RBC1/11/2013 Dr.T.V.Rao MD 30
  30. 30. 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 1/11/2013 Dr.T.V.Rao MD 31
  31. 31. 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 MD1/11/2013 Dr.T.V.Rao 32
  32. 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 4096The HA titer of this virus in this row is 256 or 28(1:256 dilution contains (1 HA unit) (one haemagglutinating unit) 1/11/2013 Dr.T.V.Rao MD 33
  33. 33. Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 virusserial dilution 8 16 32 64 128 256mix with redblood cells side view top view Titer = 32 HA units/ml One HA unit :minimum amount of virus that causes complete agglutination of RBCs
  34. 34. In the absence of anti-virus antibodies Erythrocytes Virus Virus agglutination of erythrocytes1/11/2013 Dr.T.V.Rao MD 35
  35. 35. In the presence of anti-virus antibodies Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes1/11/2013 Dr.T.V.Rao MD 36
  36. 36. 1/11/2013 Dr.T.V.Rao MD 37
  37. 37. What is Antibody Titer• Is the lowest concentration of antibodies against a particular antigen. 1/11/2013 Dr.T.V.Rao MD 38 Figure 18.6
  38. 38. 1/11/2013 Dr.T.V.Rao MD 39
  39. 39. 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 ) 1/11/2013 Dr.T.V.Rao MD 40
  40. 40. 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).1/11/2013 Dr.T.V.Rao MD 41
  41. 41. Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test – Detects antibodies on erythrocytes + ↔Patient’s RBCs Coombs Reagent (Antiglobulin)1/11/2013 Dr.T.V.Rao MD 42
  42. 42. Coombs Test Direct ant globulin test (also called the Coombs’ test,1/11/2013 Dr.T.V.Rao MD 43
  43. 43. Coombs (Antiglobulin)Tests • Indirect Coombs Test – Detects anti-erythrocyte antibodies in serumStep 1 + ↔ Patient’s Target Serum RBCsStep 2 + ↔ Coombs Reagent1/11/2013 (Ant globulin) Dr.T.V.Rao MD 44
  44. 44. Application of Coombs (Antiglobulin)Tests• Applications –Detection of anti-Rh Ab –Autoimmune hemolytic anemia1/11/2013 Dr.T.V.Rao MD 45
  45. 45. 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 proteins1/11/2013 Dr.T.V.Rao MD 46
  46. 46. Agglutination Inhibition Tests• Pregnancy Testing -classic example of agglutination inhibition – Human chorionic gonadotropin (hCG) • Appears in serum and urine early in pregnancy1/11/2013 Dr.T.V.Rao MD 47
  47. 47. Agglutination Inhibition Urine Antiserum No hCG in urine: hCG in urine: Anti-hCG free Anti-hCG neutralized Carriers coated with hCG added Carriers coated with hCG added AGGLUTINATION of carriers: NO AGGLUTINATION of carriers: Negative test for hCG Positive test for hCG NOT PREGNANT PREGNANT1/11/2013 Dr.T.V.Rao MD 48
  48. 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 reactions1/11/2013 Dr.T.V.Rao MD 49
  49. 49. 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 antibody1/11/2013 Dr.T.V.Rao MD 50
  50. 50. Highly specific but not very sensitive in detecting small quantities of antigen1/11/2013 Dr.T.V.Rao MD 51
  51. 51. 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.1/11/2013 Dr.T.V.Rao MD 52
  52. 52. 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 test1/11/2013 Dr.T.V.Rao MD 53
  53. 53. 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.1/11/2013 Dr.T.V.Rao MD 54
  54. 54. 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.1/11/2013 Dr.T.V.Rao MD 55
  55. 55. 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.1/11/2013 Dr.T.V.Rao MD 56
  56. 56. 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).1/11/2013 Dr.T.V.Rao MD 57
  57. 57. 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 infections Test may be negative in 50 percent scrub typhus1/11/2013 Dr.T.V.Rao MD 58
  58. 58. 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)..1/11/2013 Dr.T.V.Rao MD 59
  59. 59. 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)1/11/2013 Dr.T.V.Rao MD 60
  60. 60. 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.
  61. 61. 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.
  62. 62. 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.
  63. 63. Positive Test• Step 1: At 37°CAntigen + Antibody + Complement Complement gets fixed (from serum) 1 Hour• Step 2: At 37°CFixed Complement complex + Hemolytic system No Hemolysis 1 Hour (Test Positive)
  64. 64. Negative Test  Step 1: At 37 CAntigen + Antibody absent + Complement Complement not fixed 1 Hour  Step 2: At 37 C Free Complement + Hemolytic system Hemolysis 1 Hour (Test Negative)
  65. 65. 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
  66. 66. 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 test1/11/2013 Dr.T.V.Rao MD 68
  67. 67. Radio Immuno Assay
  68. 68. Radio-immunoassays: Performance, applicationsAdvantages – highly sensitive – can be used for detection of small quantities – quantification possibleLimitations – expensive – requires isotopesTime taken – 1 day1/11/2013 Dr.T.V.Rao MD 70
  69. 69. Enzyme-linked Labeling Immuno-Sorbant technique 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 readerExamples – Hepatitis A (Anti-HAV-IgM, anti-HAV Iggy)
  70. 70. ELISA Micro-plate reader Positive result 96-well micro-plate1/11/2013 Dr.T.V.Rao MD 72
  71. 71. Labeling Types of ELISA (Ag Abs tests) technique Competitive•Antigen or antibody are labeled withenzyme and allowed to compete withunlabeled ones (in patient serum) forbinding to the same target•Hydrolysis signal from Ag-Abcomplex (enzyme-labeled) ismeasured•Antigen or antibody in serum is thencalculated•No need to remove theexcess/unbound Ag or Ab from thereaction plate or tubes)
  72. 72. Labeling Types of ELISA used in the detection of antigens and antibodies technique •Non-competitive•must remove excess/unbound Agor Ab before every step of reactions•Direct ELISA•Indirect ELISA•Sandwich ELISA•Ab Capture ELISA (similar tosandwich ELISA but in 1st step, anti-Ig (M or G) is coatedon the plate•Then antibodies in patient serumare allowed to capture in next step
  73. 73. 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 day1/11/2013 Dr.T.V.Rao MD 75
  74. 74. Labeling Immuno-fluorescence technique•Principle – Use fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systems – Requires a fluorescent microscope Cell infected with Dengue virus•Examples – Herpes virus IgM – Dengue virus – Rabies virus – Scrub and murine typhus V. Cholerae
  75. 75. Immunofluorescence Helps inDiagnosis of Various Diseases
  76. 76. 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.1/11/2013 Dr.T.V.Rao MD 78
  77. 77. Labeling Types of technique immuno-fluorescenceSteps Direct FA Indirect FA Sandwich FA •Direct immuno- 1st fluorescence – Used to detect 2nd antigen •Indirect and sandwich 3rd Legend immuno-fluorescence Ag= Ab= =FITC-conjAnti-Ig – Antigen detection – Antibody detection =FITC-conjugated Ab 4th 1/11/2013 Dr.T.V.Rao MD 79
  78. 78. • Principle Western-blot analysis – 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 Anti HIV-1 1/11/2013 Dr.T.V.Rao MD 80
  79. 79. Western-blot analysis• Serum, saliva, urine can be tested• Kits are commercially available• Recombinant immuno- blotting assays (RIBA) uses recombinant proteins
  80. 80. 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 Lysing agend Test band Control band Bound Labled AB. (bound AB) (bound AB) AB Free labled AB Nitrocellulose strip1/11/2013 Dr.T.V.Rao MD 82
  81. 81. 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 Captured labelled Ab-complex Ab Labelled AB-AG- Labelled AB-AG- complex complex Captured by Captured by bound AB of bound AB of test band control band1/11/2013 Dr.T.V.Rao MD 83
  82. 82. 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 hour1/11/2013 Dr.T.V.Rao MD 84
  83. 83. 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 Chemiluminescences 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.1/11/2013 Dr.T.V.Rao MD 85
  84. 84. • Programme Created by Dr.T.V.Rao MD for Medical Students in the Developing World • Email • doctortvrao@gmail.com1/11/2013 Dr.T.V.Rao MD 86
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