Antigenic drift leads to hemagglutinin variants within each HA subtype from different globe

regions at different times. Certain Mabs that specifically target a given HA epitope of one type AIV, may not be able to recognize other AIV strains with a click here mutated antigenic epitope even if such a mutation is slight. Therefore, using one single Mab for H5 AIV antigen detection, in most cases, will not cover all the H5 subtype AIV circulating world around. Here we report the development of an antigen-capture dot ELISA based on a pair of Mabs targeting the same epitope on H5, however, by two different and dominant amino acids respectively, in an attempt to make a universal H5 AIV rapid detection test. Results Identification of monoclonal antibodies recognizing complementary epitopes on H5 hemagglutinin A panel of Mabs against influenza hemagglutinin was screened for efficient detection of different strains of H5N1 viruses. Based on PCI-32765 research buy the results of the HI assay, Mabs 6B8 and 4C2 CH5183284 order were chosen for further studies due to their high HI activity (Table 1) against a wide range of rescued reassortant viruses from different clades. Both Mabs were found to be of the IgM isotype. After the virus neutralizing activity has been confirmed (data not shown), the amino acids involved in forming the epitopes of the Mabs were analyzed using escape mutant analysis. All

HA amino acid numbering in this work uses H5 numbering excluding the signal peptide. Upon sequencing escape mutants from 3 different parental strains (Table 2), a few mutant clones of Mab 6B8 showed mutations at either Lys189 or Asn155, while clones of Mab 4C2 presented

mutations at Arg189, Ser155 or Asn155. The results indicated that the 189th and 155th amino acids were involved in the epitopes of both Mabs, but in different forms. Mab 6B8 is able to bind to H5 with either Lys189 or Asn155 independently. Mab 4C2 binds 5-Fluoracil to Arginine on 189th amino acid of H5, and it recognizes both Serine and Asparagine at position 155. Table 1 Hemagglutination Inhibition (HI) titers of the Mab 6B8 and 4C2 (1 mg/ml) against H5N1 influenza viruses. Virus Clade 6B8 4C2 A/Indonesia/CDC669/06 2.1 <8 512 A/Indonesia/CDC594/06 2.1 256 128 A/Vietnam/1203/04 1 512 <8 A/Hongkong/156/97 0 256 128 A/turkey/Turkey1/05 2.2 256 128 A/Anhui/1/05 2.3 256 <8 A/goose/Guiyang/337/06 4 128 128 A/chicken/Shanxi/2/06 7 256 256 A/chicken/Henan/12/04 8 256 128 Table 2 Amino acids on HA of H5N1 influenza viruses recognized by Mab 6B8 and 4C2, which identified in the comparison between parental virus and cloned escape mutants. Virus 6B8 4C2 A/Indonesia/CDC669/06 (155Ser, 189Arg) — 189Arg, 189Arg155Ser A/Indonesia/CDC594/06 (155Asn, 189Arg) 155Asn 155Asn A/Vietnam/1203/04 (155Ser, 189Lys) 189Lys — The number indicated the amino acid position in H5 HA. The amino acid type on the position was shown after the number. –: The Mab does not react with the parental virus.