A method for the rapid purification of serum IgM for the diagnosis of recent viral infections of chickens.
The rapid purification of chicken IgM from serum was achieved by affinity chromatography. IgM immunoadsorbent gels were prepared using monoclonal antibodies specific to chicken IgM. Five different eluting agents were compared for the dissociation of the adsorbed IgM; the most convenient for routine purposes was 2 M NaCl, Tris-HCl, EDTA (NTE), as this enabled direct assay of eluents by ELISA without requiring the intermediate step of dialysis, which the other eluting agents did. Eluents prepared from sera obtained from infectious bronchitis virus (IBV) and infectious laryngotracheitis (ILTV)-infected chickens, together with samples of the same serum fractionated by gel chromatography, were tested by ELISA for virus-specific antibodies and to confirm the identity of the antibody class. In the case of both IBV and ILTV, similar results were obtained using immunoaffinity and gel chromatography. IBV-specific IgM, as determined by both methods in the ELISA, reached its highest concentration at the 8th day after inoculation and was virtually absent by the 24th day, whilst the highest concentration of ILT-specific IgM was detected at 6 days and no or little IgM was present at 16 days after inoculation. Purification of serum IgM by affinity chromatography followed by ELISA was considered suitable for routine serological diagnosis of IB and ILT, since the time required to complete the assay (3 hours) was considerably less than that for gel chromatography and many samples could be assayed simultaneously.
Martins
N R
NR
Instituto de Pesquisas Veterinarias Desiderio Finamor, Porto Alegre, Brasil.
Mockett
A P
AP
Cook
J K
JK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003333:Coronaviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D004797:Enzyme-Linked Immunosorbent Assay; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D001352:Herpesvirus 1, Gallid / Q000276:immunology*; D007075:Immunoglobulin M / Q000302:isolation & purification*; D001351:Infectious bronchitis virus / Q000276:immunology*; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology; D013047:Specific Pathogen-Free Organisms
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M