HAEMAGGLUTINATION TEST ¦ 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). [pic] 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 (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. [pic] ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA) ¦ PRINCIPLE ¦ 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. [pic] Types of ELISA ¦ Direct ELISA ¦ Indirect ELISA Sandwich ELISA ¦ Competitive ELISA ¦ Multiplex ELISA [pic] 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.
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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. [pic] 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 hermocycler 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. REACTANTS ¦ 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 concentration of ions. ¦ P. C. R buffer, to provide proper pH, and medium for reaction. ¦ Forward and reverse primers, specific for short stretch selected sequence on D. N. A. D3 water to maintain volume of the reactants & products. STEPS IN THE REACTION ¦ 1. Reverse Transcription, cD. N. A is formed from the viral R. N. A by reverse transcriptase enzyme ¦ P. C. R reaction 1. Denaturation, 94oC, double strand D. N. A is melted in single strand and now is open for reaction . Annealing, 54 oC, primers are attached at specific sites to the strand 3. Extension, 72 oC, polymerase enzyme adds dN. T. Ps and make copies of the specific copies of the cites selected by the primers. (30 to 35 cycles are used in a reaction and billions of copies are made of the specific size selected by the primers which can be detected by the electrophoresis) [pic] [pic] ¦ Electrophoresis. P. C. R product is run across the electro potential from negative to positive in the presence of agarose gel and visualize by using Ethidium bromide that reacts with the P.
C. R products and visualize under U. V light. Wight of the P. C. R product is compared with D. N. A ladder. [pic] [pic] INTRODUCTION ¦ Avian influenza is an enveloped virus with surface proteins Haemagglutinatin H (16 type) and Neuraminidse (9 Type), Matrix proteins (M proteins), segmented genome RNA virus. ¦ When the virus enters in the host system initiates both haemoral and cell mediated immune response. ¦ The laboratory diagnosis is designed on the bases of viral structure and immune responses. [pic]