Haemagglutination Assay

Topics: Polymerase chain reaction, DNA, Reverse transcriptase Pages: 6 (876 words) Published: August 14, 2011

■ 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 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

(4 HA).

■ 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.




■ 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



■ 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.



■ 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...
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