Reagents and methods for detecting influenza virus proteins
Two universally conserved sequences from influenza type A neuraminidases were identified by large scale sequence analysis then chemically modified and conjugated to carrier proteins to generate mono-specific and monoclonal antibodies. The two antibodies, one targeting the N-terminus of the type A neuraminidase and the other sequence close to enzymatic active site, were capable of binding to all 9 subtypes of neuraminidase while demonstrating remarkable specificity against the viral neuraminidase sequences since no cross-reactivity against allantoic proteins was observed. Quantitative analyses of NA using slot blot suggest that the antibodies can be used for NA antigen quantitation in vaccines. These represent the first time the antibody-based immunoassay can be used for NA quantitative determination.
1. A method of preparing antibodies against influenza virus neuraminidase comprising inoculating an animal with an effective amount of a composition comprising an influenza neuraminidase peptide consisting of the amino acid sequence as set forth in SEQ ID No. 1 attached to a first end of a spacer, and a carrier protein attached to a second end of the spacer; and, after the animal has produced antibodies against said composition, recovering said antibodies from said animal.
2. The method according to claim 1 wherein the spacer is selected from the group consisting of amino acids, peptides, phosphoramidite, ε-aminohexanoic acid and 6-aminocaproic acid.
3. The method according to claim 1 wherein the carrier protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), thyroglobulin (THY) and human gamma globulin (HOG).
4. The method according to claim 1 wherein the spacer is 6-aminocaproic acid.
5. The method according to claim 1 wherein the carrier protein is attached to the spacer by a linker.
6. The method according to claim 5 wherein the linker is selected from the group consisting of an amino acid, a peptide of 2-10 amino acids and KKC.
7. The method according to claim 5 wherein the linker is KKC and the spacer is 6-aminocaproic acid.
8. A method of preparing monoclonal antibodies against influenza virus neuraminidase comprising inoculating an animal with an effective amount of a composition comprising an influenza neuraminidase peptide consisting of the amino acid sequence as set forth in SEQ ID No. 1 attached to a first end of a spacer, and a carrier protein attached to a second end of the spacer; and, after the animal has produced antibodies against said composition, removing antibody-producing cells from said animal, fusing a respective one of said antibody-producing cells with a respective one of an immortal cell line, thereby producing a respective one hybridoma cell, and selecting for hybridoma cells producing antibodies against said composition.
9. The method according to claim 8 wherein the spacer is selected from the group consisting of amino acids, peptides, phosphoramidite, ε-aminohexanoic acid and 6-aminocaproic acid.
10. The method according to claim 8 wherein the carrier protein is selected from the group consisting of keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), rabbit serum albumin (RSA), ovalbumin (OVA), thyroglobulin (THY) and human gamma globulin (HGG).
11. The method according to claim 8 wherein the spacer is 6-aminocaproic acid.
12. The method according to claim 8 wherein the carrier protein is attached to the spacer by a linker.
13. The method according to claim 8 wherein the linker is selected from the group consisting of an amino acid, a peptide of 2-10 amino acids and KKC.
14. The method according to claim 8 wherein the linker is KKC and the spacer is 6-aminocaproic acid.