■ Haemagglutinin proteins (H-16) has the ability to agglutinate the Red Blood Cell which is an indicator of the presence of virus.
■ Magnitude of the agglutination depends on quantity of virus.
■ Two fold serial dilution of the suspected virus material in P.B.S is treated with 1% washed R.B.C.s.
■ Virus agglutination can be seen in micro titration plates.
■ Reciprocal of the last virus dilution giving agglutination is Haemagglutination titer (HA titer).
HAEMAGGLUTINATION INHIBITION TEST
■ Haemagglutination activity of the virus can be inhibited by the addition of the specific antibodies present in serum or specific antisera.
■ Field serum samples are serially diluted in P.B.S & allowed to react with standard antigens
■ Half an hour is given to the reaction so that antigen and antibody may react.
■ 1% R.B.Cs are added to evaluate the Haemagglutination inhibition.
■ Serum samples containing specific antibodies against the antigen used will inhibit the haemagglutination activity of the virus.
■ Reciprocal of the last serum dilution causing Haemagglutination inhibition will consider as (HI titer).
■ Positive serum control, negative serum control, antigen control, R.B.Cs control are run to validate the test result.
ENZYME LINKED IMMUNOSORBANT ASSAY
■ Antigen and antibody reaction are highly specific.
■ Antibody’s FC portion can be attached with enzyme.
■ Specific antigen & antibody reactions with enzyme can be detected by the addition of the substrate specific to the enzyme.
■ Magnitude of antigen and antibody present in the reaction seen with the intensity of colors cab be read by ELISA reader.
Types of ELISA
■ Direct ELISA
■ Indirect ELISA
■ Sandwich ELISA
■ Competitive ELISA
■ Multiplex ELISA
AGAR GEL PRECIPITATION TEST
■ As a result of interaction of antibody and antigen in semisolid phase complexes of two type of the molecule will form precipitation band depending upon the relative concentration of two reactants.
■ Agar gel plays a semisolid phase medium for the reaction and precipitation of specific antigen and antibody present in the serum.
■ Test is helpful in primary screening and qualitative expressions, can be used as semi quantitative test.
■ 1% Noble agar gel containing .005% sodium azide in P.B.S boil for 10 min & and allow it to cool in Petri dish, about 30 ml of gel is sufficient to get 4 mm thickness. Allow it to cool in refrigerator till use
■ Cut the wells by the cylinders 6 wells out side having a central well.
■ Put the standard antigen or antibody in central well and test antigen or antibody in outer 6 wells
■ Allow it to react at 37oC in humid environment for 24, 48 or 72 hours.
■ A line of precipitation show the positive reaction.
POLYMERASE CHAIN REACTION (P.C.R)
■ It is a reaction by which D.N.A is amplified in billions of copies by using polymerase enzyme with the help of specific primer by the use of thermocycler machine.
■ This is a technique from which a small amount of genetic material in a test sample can be identified in short time.
■ Highly sensitive, reproducible, and reliable test.
■ Recently PCR is very useful technique in the diagnosis of poultry disease and research.
■ For influenza reverse transcription PCR is used.
■ R.N.A is converted in to DNA by reverse transcription.
■ Reverse transcriptase enzyme, for conversion of R.N.A to D.N.A.
■ d.N.T.Ps, building blocks for the synthesis of D.N.A.
■ Genomic R.N.A (for R.N.A viruses only).
■ c D.N.A for R.N.A viruses, genomic D.N.A for others.
■ Taq. Polymerase enzyme, for the synthesis of D.N.A.
■ MgCl2 to provide proper...