METHOD AND KIT FOR DETECTION OF HEPATITIS A VIRUS NEUTRALIZING ANTIBODIES
A rapid immunoassay method for the detection of anti-Hepatitis A Virus (HAV) neutralizing antibodies is described herein. This microplate-based enzymatic assay may be applicable in virological diagnostics, in evaluating the immunogenicity of candidate immunogenic compositions, such as HAV vaccines, or in quantifying functional neutralizing antibodies during the course of HAV infection.
1 . A rapid in vitro method for detecting hepatitis A virus (HAV) neutralizing antibodies in sera comprising evaluating HAV neutralizing antibody response after exposure of HAV-permissive cells to the sera in combination with a rapidly replicating HAV.
2 . The method of claim 1 wherein the rapidly replicating HAV comprises HM175/18f-HAV.
3 . The method of claim 1 wherein sera is derived from a subject suspected of exposure to HAV infection, an HAV vaccine, or an immunogenic composition based on HAV used to elicit an HAV neutralizing antibody response.
4 . The method of claim 1 , wherein no more than one replication cycle of HAV is required to evaluate HAV neutralizing antibody response.
5 . The method of claim 1 , wherein the HAV-permissive cells are fetal rhesus monkey kidney FRhK-4 cells.
6 . The method of claim 1 , wherein evaluating HAV neutralizing antibody response comprises detecting HAV growth in said HAV-permissive cells with an antibody to a HAV-VP3 structural protein, wherein suppressed HAV growth is indicative of HAV neutralizing antibodies.
7 . The method of claim 1 , which is a microplate-based method.
8 . The method of claim 1 , wherein evaluating HAV neutralizing antibody response comprises comparing the ability of sera to neutralize HAV relative to a virus control value.
9 . A rapid in vitro method for detecting hepatitis A virus (HAV) neutralizing antibodies in sera, the method comprising:
incubating the sera with a rapidly replicating HAV to form a sera-HAV mixture;
incubating the sera-HAV mixture with pre-seeded HAV permissive cells;
fixing the cells; and
detecting HAV-VP3 in cells by incubating the cells with an anti-HAV VP3 antibody;
wherein HAV-VP3 in cells is indicative of HAV growth in said cells; suppressed HAV growth being indicative of HAV neutralizing antibodies in sera.
10 . The method of claim 9 , wherein the cells are fetal rhesus monkey kidney FRhK-4 cells.
11 . The method of claim 9 , wherein the rapidly replicating HAV is a cytopathic variant of HAV.
12 . The method of claim 11 , wherein the cytopathic variant of HAV is HM175/18f-HAV.
13 . The method of claim 9 , wherein a detectable antibody to the anti-HAV VP3 antibody is used to detect HAV-VP3.
14 . The method of claim 13 , wherein the detectable antibody is horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody.
15 . A kit for detecting hepatitis A virus (HAV) neutralizing antibodies in sera comprising:
a rapidly replicating cytopathic variant of HAV for incubating with sera;
HAV permissive cells comprising fetal rhesus monkey kidney FRhK-4 cells for incubating with a mixture of sera and said cytopathic variant of HAV; and
an anti-HAV VP3 antibody.
16 . The kit of claim 15 , wherein the rapidly replicating cytopathic variant of HAV is HM175/18f-HAV.
17 . The kit of claim 15 , additionally comprising a cell fixative.
18 . The kit of claim 15 , additionally comprising a detectable antibody to the anti-HAV VP3 antibody.
19 . The kit of claim 18 , wherein the detectable antibody comprises HRP-conjugated goat anti-mouse antibody.
20 . The kit of claim 15 , additionally comprising instructions for use in detecting HAV neutralizing antibodies in sera.