Chicken sera rarely give nonspecific positive agglutination reactions in this test and any pretreatment of
the sera is unnecessary. Sera from species other than chickens may sometimes cause agglutination of
chicken RBCs resulting in nonspecific agglutination. Therefore, each serum should first be tested for
this idiosyncrasy and, if present, it should be inhibited by adsorption of the serum with chicken RBCs.
This is done by adding 0.025 ml of packed chicken RBCs to each 0.5 ml of antisera, mixing gently and
leaving for at least 30 minutes; the RBCs are then pelleted by centrifugation at 800 g for 2–5 minutes
and the adsorbed sera are decanted. Alternatively, RBCs of the avian species under investigation
could be used. Nonspecific inhibition of agglutination can be caused by steric inhibition when the H
antigen and serum in the HI test have the same N subtype. The steric inhibition reaction can result in
RBC buttoning in the bottom of the plate or streaming at the same rate as the control. To prevent steric
nonspecific inhibition the H antigen used to test unknown serum must be of a different N subtype than
the unknown sera, or the H antigen use can be recombinant or purified H protein that lacks N protein.
The HI test is based on antigenic binding between the H antigen and antisera and thus other factors
may cause nonspecific binding of the H antigen and sera leading to a nonspecific inhibition reaction. At
this time there are no documented cross reactions or nonspecific inhibition reactions between the
different haemagglutinin subtypes of influenza A.
The neuraminidase-inhibition test has been used to identify the influenza A neuraminidase type of
isolates as well as to characterise the antibody in infected birds. The procedure requires specialised
expertise and reagents; consequently this testing is usually done in an OIE Reference Laboratory. The
DIVA (differentiating infected from vaccinated animals) strategy used in Italy also relies on a serological
test to detect specific anti-N antibodies; the test procedure has been described (Capua et al., 2003).
Chicken sera rarely give nonspecific positive agglutination reactions in this test and any pretreatment of
the sera is unnecessary. Sera from species other than chickens may sometimes cause agglutination of
chicken RBCs resulting in nonspecific agglutination. Therefore, each serum should first be tested for
this idiosyncrasy and, if present, it should be inhibited by adsorption of the serum with chicken RBCs.
This is done by adding 0.025 ml of packed chicken RBCs to each 0.5 ml of antisera, mixing gently and
leaving for at least 30 minutes; the RBCs are then pelleted by centrifugation at 800 g for 2–5 minutes
and the adsorbed sera are decanted. Alternatively, RBCs of the avian species under investigation
could be used. Nonspecific inhibition of agglutination can be caused by steric inhibition when the H
antigen and serum in the HI test have the same N subtype. The steric inhibition reaction can result in
RBC buttoning in the bottom of the plate or streaming at the same rate as the control. To prevent steric
nonspecific inhibition the H antigen used to test unknown serum must be of a different N subtype than
the unknown sera, or the H antigen use can be recombinant or purified H protein that lacks N protein.
The HI test is based on antigenic binding between the H antigen and antisera and thus other factors
may cause nonspecific binding of the H antigen and sera leading to a nonspecific inhibition reaction. At
this time there are no documented cross reactions or nonspecific inhibition reactions between the
different haemagglutinin subtypes of influenza A.
The neuraminidase-inhibition test has been used to identify the influenza A neuraminidase type of
isolates as well as to characterise the antibody in infected birds. The procedure requires specialised
expertise and reagents; consequently this testing is usually done in an OIE Reference Laboratory. The
DIVA (differentiating infected from vaccinated animals) strategy used in Italy also relies on a serological
test to detect specific anti-N antibodies; the test procedure has been described (Capua et al., 2003).
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