Quantitation of immunoglobulin to hepatitis E virus by enzyme immunoassay.
We developed a quantitative enzyme immunoassay (EIA) for antibody to hepatitis E virus (HEV) by using truncated HEV capsid protein expressed in the baculovirus system to improve seroepidemiology, to contribute to hepatitis E diagnosis, and to enable vaccine evaluations. Five antigen lots were characterized; we used a reference antiserum to standardize antigen potency. We defined Walter Reed antibody units (WR U) with a reference antiserum by using the four-parameter logistic model, established other reference pools as assay standards, and determined the conversion factor: 1 WR U/ml = 0.125 World Health Organization unit (WHO U) per ml. The EIA performed consistently; median intra- and inter-test coefficients of variation were 9 and 12%, respectively. The accurate minimum detection limit with serum diluted 1:1,000 was 5.6 WR U/ml; the test could detect reliably a fourfold antibody change. In six people followed from health to onset of hepatitis E, the geometric mean antibody level rose from 7.1 WR U/ml to 1,924.6 WR U/ml. We used the presence of 56- and 180-kDa bands by Western blotting as a confirmatory test and to define true-negative and -positive serum specimens. A receiver-operating characteristics plot identified 30 WR U/ml as an optimum cut-point (sensitivity, 86%; specificity, 89%). The EIA detected antibody more sensitively than a commercially available test. The EIA was transferred to another laboratory, where four operators matched reference laboratory results for a panel of unknowns. Quantitation of antibody to HEV and confirmation of its specificity by Western blotting make HEV serology more meaningful.
Innis
Bruce L
BL
Department of Virus Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910, USA. bruce.2.innis@gsk.com
Seriwatana
Jitvimol
J
Robinson
Robin A
RA
Shrestha
Mrigendra P
MP
Yarbough
Patrice O
PO
Longer
Charles F
CF
Scott
Robert M
RM
Vaughn
David W
DW
Myint
Khin Saw Aye
KS
D002213:Capsid / Q000276:immunology / Q000302:isolation & purification; D006508:Hepatitis Antibodies / Q000032:analysis*; D018963:Hepatitis Antigens / Q000276:immunology / Q000302:isolation & purification; D016751:Hepatitis E / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D016752:Hepatitis E virus / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D015203:Reproducibility of Results
D006508:Hepatitis Antibodies; D018963:Hepatitis Antigens; D011933:Reagent Kits, Diagnostic; D011993:Recombinant Fusion Proteins