Component Description
Sera from residual specimens from NHANES 2003-2004 participants were tested for IgG antibodies against seven norovirus strains: GI.1 Norwalk, GI.4, GII.3, GII.4 US95/96, GII.4 Farmington Hills, GII.4 New Orleans, and GIV.1. A quantitative enzyme immunoassay that uses norovirus virus-like particles (VLPs) to detect anti-norovirus IgG was used. Samples were diluted 1:50 for analysis. The results are reported in optical density (OD) and ng IgG/ml. Samples with OD >3 (i.e. above the measurable range of the assay, strongly positive) are reported as 3.50. Concentrations greater than the highest standard (2000 ng IgG/ml) are reported as 100,000 ng IgG/ml, the upper limit of detection. Samples with OD less than the plate blank are coded 0.00. Missing observations represent samples that could not be tested due to insufficient volume or technical errors.
Eligible Sample
These tests were conducted on residual serum specimens stored at NCHS. Specimens were collected during the 2003-2004 study cycle. A one-third subsample of all subjects aged 16-49, maintaining population representativeness, was tested for anti-norovirus IgG antibody.
Description of Laboratory Methodology
Anti-norovirus IgG was measured using a previously published enzyme immunoassay (EIA) and virus-like particles (VLPs) as antigen. VLPs were provided by Dr. Robert Atmar at Baylor University. The EIA test utilizes VLP-coated wells. The serum samples were diluted 1:50 and incubated in the antigen-coated wells. If present, anti-norovirus antibodies are immobilized in the wells. Residual sample is eliminated by washing and conjugate (enzyme labeled antibodies to human IgG) is added and incubated. If IgG antibodies to norovirus are present, the conjugate will be immobilized in the wells. Residual conjugate is eliminated by washing and the enzyme substrate (p-nitrophenyl phosphate) is added. In the presence of the enzyme, the substrate is converted to a yellow end product which is read photometrically. Each sample was tested in duplicate.
Each plate had an IgG standard curve with known concentrations of human IgG ranging from 2000 ng/ml to 31.25 ng/ml. The standards were run in duplicate on each plate and the average of the duplicates was used to generate the standard curve. The curve was fit using a 4-point parametric fit in the Gen5 software and this curve was used to calculate concentrations for the samples on the same plate. Concentrations in ng IgG/ml were calculated for each well and averaged across the duplicates. Extrapolation beyond the standard curve was not allowed.
Optical density was determined on a BioTek ELx800 microplate reader and computer equipped with Gen5 software (BioTek, Winooski, VT).
Laboratory Quality Assurance and Monitoring
Quality control samples specific to each VLP antigen were included on every plate. Human serum from subjects with norovirus infections strain-matched to the assay VLPs was used as a positive control for all VLPs except GIV.1. For GIV.1, serum with high reactivity to GIV.1 (kind gift from B. di Martino) was used as a positive control because serum from subjects with known GIV.1 infections was not available. Negative control serum was selected from a panel of sera from uninfected subjects in norovirus human challenge studies. The serum sample with the lowest reactivity across all seven VLPs was used as the assay negative control.
Data Processing and Editing
Data was compiled after all the antibody testing was completed. Values above the upper limit of detection (OD = 3.0 or concentration = 100,000 ng IgG/ml) were replaced with 3.5 or 100,000, respectively. Values that were less than the plate blank were replaced with 0. Mean OD and concentration values were calculated using the replacement values. Data Access: All data are publicly available.
Analytic Notes
Due to cross-reactivity between strains and declining antibody levels after infection, there is no standard cutpoint value to determine seropositivity to norovirus in a single serum sample. Based on results of previous human challenge studies, a cutpoint of OD=1.5 is suggested to distinguish seropositive from seronegative samples. Additionally, an OD ≥3 (represented by a value of 3.50) is suggested to identify samples with recent (within the last year) infections.
Parallel analysis using the same selection and assay criteria was performed on serum samples from the 1999-2000 study cycle.
Variable |
Definition |
SEQN |
Respondent Sequence Number |
SSGI1C1 |
antibody concentration in duplicate 1 for GI.1 antigen |
SSGI1C2 |
antibody concentration in duplicate 2 for GI.1 antigen |
SSGI1CM |
mean antibody concentration for GI.1 antigen |
SSGI1OD1 |
optical density in duplicate 1 for GI.1 antigen |
SSGI1OD2 |
optical density in duplicate 2 for GI.1 antigen |
SSGI1ODM |
mean optical density for GI.1 antigen |
SSGI4C1 |
antibody concentration in duplicate 1 for GI.4 antigen |
SSGI4C2 |
antibody concentration in duplicate 2 for GI.4 antigen |
SSGI4CM |
mean antibody concentration for GI.4 antigen |
SSGI4OD1 |
optical density in duplicate 1 for GI.4 antigen |
SSGI4OD2 |
optical density in duplicate 2 for GI.4 antigen |
SSGI4ODM |
mean optical density for GI.4 antigen |
SSGII3C1 |
antibody concentration in duplicate 1 for GII.3 antigen |
SSGII3C2 |
antibody concentration in duplicate 2 for GII.3 antigen |
SSGII3CM |
mean antibody concentration for GII.3 antigen |
SSGII3O1 |
optical density in duplicate 1 for GII.3 antigen |
SSGII3O2 |
optical density in duplicate 2 for GII.3 antigen |
SSGII3OM |
mean optical density for GII.3 antigen |
SSGII4FC1 |
antibody concentration in duplicate 1 for GII.4 Farmington Hills antigen |
SSGII4FC2 |
antibody concentration in duplicate 2 for GII.4 Farmington Hills antigen |
SSGII4FCM |
mean antibody concentration for GII.4 Farmington Hills antigen |
SSII4FO1 |
optical density in duplicate 1 for GII.4 Farmington Hills antigen |
SSII4FO2 |
optical density in duplicate 2 for GII.4 Farmington Hills antigen |
SSII4FOM |
mean optical density for GII.4 Farmington Hills antigen |
SSII4UC1 |
antibody concentration in duplicate 1 for GII.4 US95/96 antigen |
SSII4UC2 |
antibody concentration in duplicate 2 for GII.4 US95/96 antigen |
SSII4UCM |
mean antibody concentration for GII.4 US95/96 antigen |
SSII4UO1 |
optical density in duplicate 1 for GII.4 US95/96 antigen |
SSII4UO2 |
optical density in duplicate 2 for GII.4 US95/96 antigen |
SSII4UOM |
mean optical density for GII.4 US95/96 antigen |
SSII4NC1 |
antibody concentration in duplicate 1 for GII.4 New Orleans antigen |
SSII4NC2 |
antibody concentration in duplicate 2 for GII.4 New Orleans antigen |
SSII4NCM |
mean antibody concentration for GII.4 New Orleans antigen |
SSII4NO1 |
optical density in duplicate 1 for GII.4 New Orleans antigen |
SSII4NO2 |
optical density in duplicate 2 for GII.4 New Orleans antigen |
SSII4NOM |
mean optical density for GII.4 New Orleans antigen |
SSGIV1C1 |
antibody concentration in duplicate 1 for GIV.1 antigen |
SSGIV1C2 |
antibody concentration in duplicate 2 for GIV.1 antigen |
SSGIV1CM |
mean antibody concentration for GIV.1 antigen |
SSGIV1O1 |
optical density in duplicate 1 for GIV.1 antigen |
SSGIV1O2 |
optical density in duplicate 2 for GIV.1 antigen |
SSGIV1OM |
mean optical density for GIV.1 antigen |