Antigen antibody interaction/Reaction

1. Antigen- Antibody Reaction Prabin Shah BScMLT , MSc(Biochemistry)

2. When a soluble Ag combine with its Abs in the presence of electrolytes at a suitable temperature, pH, the Ag- Ab complex forms an insoluble precipitate. Soluble Ags interact with Abs to form lattice. Occurs best when Ag and Ab are present in optimal proportions. Precipitation Tests

3. Polyclonal antibodies can form lattices or large aggregates however monoclonal antibody can link only two molecules of antigen and no precipitate is formed. The amount of precipitate formed is greatly influenced by the relative proportions of Ags and Abs. Precipitation curve: Zone of antibody excess: precipitation is inhibited and ANTIBODY NOT BOUND TO Ag can be detected in the supernatant. This is also called as Prozone Precipitation reaction

4. Zone of Equivalence: maximal precipitation in which Ab and Ag form large insoluble complexes and neither antibody nor AG can be detected in the supernatant. Zone of Ag Excess: precipitation is inhibited and Ag not bound the Ab can be detected in the supernatant. This is also known as P ost zone .

5. Multivalent Ag combines with bivalent Ab in varying proportions, depending on the Ag and Ab ratio in the reacting mixture. Precipitation results when a large lattice is formed consisting of alternating antigen and Ab molecule. This is possible only in the zone of equivalence. In the zone of Ag- Ab excess, the lattice does not enlarge as the valencies of the Ab and the Ag are fully satisfied. MECHANISM

6. Ring test Slide test Tube test Immunodiffusion E lectroimmnunodiffusion Types of Precipitation Test

7. A) Ring Test Consist of layering the Ag solution over a column of antiserum in narrow tube. A ppt is form at the junction of two liquids. Eg : Ascoli’s thermo precipitin test, Lancefield grouping of streptococci. B) Slide test A drop of Ag and the antiserum are placed on a slide and mixes by shaking, floccules appear. Eg : VDRL test for syphilis is an example of slide flocculation.

8. C) Tube test A quantitative tube flocculation test is used for the standardization of toxins and toxoids. Serial dilution of the toxin/toxoid are added to the tubes containing a fixed quantity of the antitoxin The amount of toxin or toxoid that flocculates optimally with one unit of the antitoxin is defined as an Lf dose Example: Kahn Test for syphilis is an example of tube flocculation test

9. D). Immunodiffusion Test Precipitation test is done in gel. Immunodiffusion procedure are carried out in an agar or agarose gel The precipitate is easily seen in gel yield precipitin lines

10. i ) Single diffusion in one dimension The Ab is incorporated in agar gel in a test tube and the Ag solution is layer over it. The Ag diffuses downward through the agar gel, forming a line of precipitation that appears to move downwards. This is due to the precipitation formed at the advancing front of the Ag and is dissolved as the concentration of Ag at the site increases due to diffusion. The no of bands indicate the no of different Ag present.

11. ii). Double diffusion in One dimension (Oakley- fulthorpe procedure) Ab is incorporated in a gel above which is placed a column of plain agar. The Ag is layered on top of this. The Ag and Ab move towards each other through the intervening column of plain agar and form a band of precipitate where they meet at optimum proportion.

12. iii). Single diffusion in two dimension( Radial immunodiffusion) The antiserum is incorporated in agar gel poured on a flat surface. The Ag is added to the wells cut on the surface of the gel. It diffuses radially from the well and forms ring- shaped bands of precipitation concentrically around the well. This method has been employed for the estimation of the immunoglobulin classes in sera.

13. Radial immunodiffusion

14. iv) Double diffusion in two dimensions Agar gel is poured on a slide and wells are cut using a template . The antiserum is placed in the central well and different Ags in the surrounding well. If two adjacent Ags are identical. The lines of precipitate form by them will fuse. If they are unrelated, the lines will cross each other.

15. v) Immunoelectrophoresis: This technique involves the electrophoretic separation of a composite Ag (such as serum) into its constituent proteins followed by immunodiffusion against its antiserum resulting in separate precipitin lines, indicating reaction between each individual protein with its antibody. It is performed on agar or agarose gel on a slide with an Ag well and Ab trough cut on it. The test serum is placed in the antigen well and electrophoresed for about an hour. Ab against human serum is then placed in the trough and diffusion allowed to proceed for 18-24 hrs. The resulting precipitin lines can be photographed and the slides dried, stained and preserved for record. This is used for testing normal and abnormal proteins in serum and urine

16. Plasma (mixture of antigens) Electrophoresis Antiserum (mixture of antibodies) Immunodiffusion

17. E) Electroimmunodiffusion The development of precipitin can be speeded up by electrically driving the antigen and Ab. Two most frequently technique used in the lab are: Counterimmunoelectrophoresis Rocket electrophoresis

18. Counterimmunoelectrophoresis It involves simultaneous electrophoresis of the Ab in gel in opposite directions resulting in precipitation at a point between them Produces visible precipitation line within 30 mins and is ten times more sensitive than the standard double diffusion techniques Used for the detection of alpha- fetoprotein in serum and specific Ags of Cryptococcus and N. m eningitidis in the CSF.

19. ii) Rocket- electrophoresis The antiserum to the Ag to be quantified is incorporated in agarose and gelled on the glass slide. The Ag in increase concentration, is placed in wells in the set gel. The Ag is then electrophoresed into the Ab containing agarose . The pattern of immunoprecipitation resembles a rocket and hence the name. Application- it is used for quantitative estimation of Ags

20. Particulate Ag + its Antibody Agglutination Reaction Electrolytes and suitable temp. And pH Particles clumped / agglutinated

21. Same principle govern agglutination and precipitation reaction Zone phenomenon is also seen in agglutination. Incomplete or monovalent Abs do not cause agglutination, though they combine with Ag. They act as blocking Abs. More sensitive than precipitation for detection of Abs.

22. A) Slide Agglutination when a drop of the appropriate anti serum is added to a smooth uniform suspension of a particulate Ag in a drop of saline on a slide, agglutination take place. Clumping of Ag and clearing of suspension Uses: Blood grouping and cross matching Confirmation of cultural isolates Application of Agglutination test

23. B) Tube Agglutination Standard quantitative method for measuring Abs Titre estimated by: A fixed volume of a particulate Ag suspension is added to an equal volume of serial dilutions of antiserum in test tubes. Uses: Diagnosis of : Typhoid ( W idal test) Brucellosis

24. Heterophile Agglutination Based on sharing of a common Ag between different species. e.g. Weil- F elix reaction: common Ag between typhus R ickettsiae and strains of P roteus bacilli. Saifudheen etal . Ann Indian Acad Neurol 2012;15:141-4

25. C) The Antiglobulin test It was devised by Coombs, Mourant and Race (1945) Detection of anti- Rh antibodies that do not agglutinate Rh- positive red cells in saline. Used for the detection of incomplete Abs. PRINCIPLE: When sera containing incomplete anti- Rh Abs are mixed with Rh- positive red cells , the Ab globulin coats the surface of the RBCs , though they are not agglutinated.

26. When such erythrocytes coated with the Ab globulin are washed off all unattached protein and treated with a rabbit antiserum against human gammaglobulin (antiglobulin or coombs serum) the cells are agglutinated. The coombs test may be direct or indirect.

27. D) Passive Agglutination Test Soluble Ag when attached to carrier particles, precipitation is converted to agglutination. More sensitive and convenient for Ab detection. Commonly used carrier are: Red Blood Cells Latex particles Carbon particles Bentonite

28. Coagglutination Carrier molecule: Protein A producing Staphylococcus aureus (Cowan I strain)

29. In the presence of the appropriate Abs complement Lyses erythrocytes Kills and in some cases lyses bacteria Immobilises motile organism Promotes phagocytosis and immune adherence. Contributes to tissue damage in certain types of hypersensitivity. The ability of antigen- antibody complexes to fix complement is the basis of CFT. Complement Fixation Test

30. It is a very versatile and sensitive test. Types of CFT- Wasserman reaction: used for serodiagnosis of syphilis Indirect complement fixation test Conglutinating complement adsorption Other complement- dependant serological tests.

31. 1) Wasserman Reaction ANTIGEN + T EST SERUM (Contains antibody) + Complement + Haemolytic system ANTIGEN + TEST SERUM (contains no Ab) + complement + Hemolytic system Complement fixed Result- no haemolysis Positive CF test Complement not fixed Result- Hemolysis Negative of CF test

32. 2) Indirect complement fixation test It is employed in cases of certain avian and mammalian sera which do not fix guinea pig complement. The test is set up in duplicate and after the first step the standard serum known to fix the complement is added to one set. If the test serum contained Ab the Ag would have been used up in the first step and therefore the standard antiserum added would not be fixed to the complement. Therefore haemolysis indicates a positive result.

33. ELISA INTRODUCTION: Enzyme Linked Immuno -Sorbent Assay Term Was Coined By Engvall and Pearlmann in 1971 Similar To RIA, Except No Radiolabel Can Be Used To Detect Both Antibody and Antigen

34. Principle: Works on the principle of antigen – antibody reaction where an enzyme conjugated with an antibody reacts with a colorless substrate to give a colored reaction product.

35. Types of ELISA Indirect ELISA Sandwich ELISA Competitive ELISA

36. ELISA READER ELISA KIT

37. Substrate used is known as chromogenic substrate. Enzymes used are ALP (p-nitro phenyl phosphate) Horse radish peroxidase (o- phenylene diammine dihydrochloride ) β galactosidase

38. INDIRECT ELISA This method can determine the antibody quantitatively. Procedure S erum containing primary Ab is added to an antigen coated microtiter well and is allowed to react with the antigen attached to the well. The specific enzyme- conjugated secondary Ab is added which will then detect the primary Ab that was bound to the antigen. Then a substrate is added which forms a colored reaction product.

39. SANDWICH ELISA This method can detect the antigen present. Procedure: Serum containing antigen is added and allowed to react with the immobilized Ab bound on the microwell . Enzyme conjugated secondary antibody specific for the antigen is added and is allowed to react with the bound antigen. Substrate is added and the colored reaction product formed is measured.

40. COMPETITIVE ELISA This method can detect the antigen present. Procedure : Ab incubated with the sample containing antigen. Antigen – antibody mixture is then added to an antigen coated microtiter well. Enzyme conjugated secondary antibody specific for the primary antibody can be used to determine the primary antibody bound to the well. Substrate is added and the color reaction product is formed which can be measured. Higher the con. of Ag in serum, lower will be the absorbance.

41. source: kuby 6 th edition

42. C alculation of the cut-off value Cut off value is the value which checks whether the sample has to be considered as positive or not. It is calculated as =mean value + 3 x (S.D) + 10% Cut off value in ELISA is

43. Micro titre plate

44. Advantages Sensitive Highly specific Readings can be documented Economic Easy to perform

45. applications Indirect ELISA HIV antibodies HCV antibodies Leptospira antibodies Dengue antibodies TORCH antibodies Sandwich ELISA HbsAg antigen P 24 antigen

46. CLIA Chemiluminescence Immuno Assay Principle: It is based on the principle by which the light emmission from the excited singlet state occurs when the electron returns to the ground state. It involves the oxidation of an organic compound such as luciferin , acridinium esters, isoluminol etc. Chemiluminescence is the measurement of this energy generated from the chemical reaction (an oxidation reaction ).

47. P rocedure Dispense the stds , samples and controls into the appropriate wells. Mix for 10 secs Dispense the enzyme conjugate reagent into each well. Mix gently for 30 secs . Incubate for 1 hour at RT. Remove the incubation mixture. Rinse the microtitre wells 5 times with wash buffer. Add the chemiluminscence substrate solution into each well. Gently mix and read the wells on the chemiluminscence microwell reader.

48. Chem Luminometer Source: google images

49. Enzymes used: ALP Horse radish peroxidase Metal ions Cu(II) Fe(III) hemin

50. Advantage of CLIA over ELISA CLIA detection using microplate luminometers provides A more sensitive , higher throughput and economical alternative to conventional calorimetric methods like ELISA. In CLIA, unlike ELISA the chromogenic substrate is replaced by the use of a luxogenic substrate. Eg . Oxidation of a compound luminol by H 2 O 2 a nd the enzyme horse radish peroxidase (HRP) when used will produce a relatively long lived light emission. It can be measured using a luminometer . Miniminal sensitivity of test is 0.5ng/ml.

51. Applications Chemiluminescence in clinical immunology for the study of; autoimmune diseases , inflammatory responses, endocrine disorders , immunodeficiency's states, It has been applied in a wide variety of techniques including: immunoassays , protein blotting, toxicological and pharmacological tests

52. IMMUNOFLUoRESCENCE History: This technique was implemented by a scientist by name, Albert Coons in the year 1944. Principle: Based on the principle that fluroscent molecules can absorb light of one wavelength( excitation ) and emit light of another wavelength( emission ). Fluorescence is a property where light is absorbed and remitted within a few nanoseconds (approx. 10ns) at a lower energy.

53. It is a type of antigen–antibody reaction using fluroscent dyes observed using a fluroscent microscope. Fluroscent dyes are special dyes that can absorb the rays of lower wavelength and emit the rays of higher. Commonly used dyes are; Fluroscene isothiocyanate Lissamine rodamine

54. Types of immunofluorescence i ) Primary (direct) It uses a single antibody that is chemically linked to a fluorophore . The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected via microscopy. This technique has several advantages over the secondary (or indirect) because of the direct conjugation of the antibody to the fluorophore . This reduces the number of steps in the staining procedure making the process faster and can reduce background signal. However , since the number of fluorescent molecules that can be bound to the primary antibody is limited, direct immunofluorescence is less sensitive than indirect immunofluorescence .

55. Direct IF

56. ( ii)Secondary( indirect): It uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore , recognises the primary antibody and binds to it . Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen. This protocol is more complex and time consuming than the primary (or direct), but it allows more flexibility because a variety of different secondary antibodies and detection techniques can be used for a given primary antibody .

57. TYPE OF IMMUNOFLUROSCENCE ADVANTAGES DISADVANTAGES DIRECT SHORTER SAMPLE STAINING TIME LOWER SIGNAL SIMPLER LABELLING PROCEDURE HIGHER COST LESS FLEXIBILITY INDIRECT MORE SENSITIVE POTENTIAL FOR CROSS REACTIVITY AMPLIFICATION OF SIGNAL SAMPLE WITH ENDOGENOUS Ig MAY EXIHIBIT HIGH BACKGROUND

58. Immunofluroscence microscopic view Source: google images

59. APPLICATIONS Detection of rabies antigen (direct IF) Fluroscent T reponema Antibody test for syphilis- (indirect IF) Analysis of antigen in fresh, frozen or fixed tissues. Detection of presence or absence of specific DNA sequences on chromosomes. Defines the patterns of gene expression within the cells/tissues .

60. LIMITATIONS Photo bleaching Autofluroscence Fluroscence overlap

61. REFERENCES www.wikipedia.com Google images www.pubmed.com Text book on Medical L aboratory T echnology Dr. Praful B Godkar Annual review of Medicine Introduction to chemiluminscence Text book of immunology Kuby