• Component Description
• Eligible Sample
• Description of Laboratory Methodology
• Laboratory Quality Assurance and Monitoring
• Data Processing and Editing
• Analytic Notes
• References
• Codebook
  • SEQN - Respondent sequence number
  • SSHEWSCR - Wantai anti-HEV assay SCR
  • SSHEW - Anti-HEW IgE
  • SSHEWB - HEV Western blot

National Health and Nutrition Examination Survey

1988 - 1994 Data Documentation, Codebook, and Frequencies

IgG Antibody to Hepatitis E Virus and anti-HEV Western Blot (SSNH3HEW)

Data File: SSNH3HEW.xpt

First Published: September 2016

Last Revised: NA

Component Description

IgG hepatitis E virus (HEV) antibody testing of stored sera specimens from NHANES III using the Wantai HEV-IgG ELISA assay was conducted to evaluate correlation of results with those of other IgG anti-HEV assays. Testing was also performed using an in-house anti-HEV Western Blot assay.

Eligible Sample

A stratified random sample of 6000 participants aged ≥6 years from NHANES III with anti-HEV IgG test results from testing performed by Kuniholm and colleagues was selected for testing by the CDC Hepatitis Laboratory with the DSI IgG and IgM anti-HEV assays (data previously reported). Further testing with the Wantai HEV-IgG ELISA assay and an in-house anti-HEV Western Blot assay was performed on a subset of 1803 of the 6000 samples.

Description of Laboratory Methodology

IgG anti-HEV
WANTAI HEV-IgG ELISA is an enzyme-linked immunosorbent assay for the qualitative detection of IgG-class antibodies to hepatitis E virus in human serum or plasma. WANTAI HEV-IgG ELISA employs solid phase, indirect ELISA method for detection of IgG-class antibodies to HEV (anti-HEV) in two-step incubation procedure. Polystyrene microwell strips are pre-coated with HEV recombinant. During the first incubation step, anti-HEV specific antibodies, if present, will be bound to the solid phase pre-coated HEV antigens. The wells are washed to remove unbound serum proteins and then, rabbit anti-human IgG antibodies (anti-IgG) conjugated to horseradish peroxidase (HRP-Conjugate) is added. During the second incubation step, these HRP-conjugated antibodies will be bound to any antigen-antibody (IgG) complexes previously formed and the unbound HRP-conjugate is then removed by washing. Chromogen solutions containing Tetramethylbenzidine (TMB) and urea peroxide are added to the wells and in presence of the antigen-antibody-anti-IgG (HRP) immunocomplex, the colorless Chromogens are hydrolyzed by the bound HRP conjugate to a blue colored product. The blue color turns yellow after stopping the reaction with sulfuric acid. The amount of color intensity can be measured and is proportional to the amount of antibody captured in the wells, and to the sample respectively. Wells containing samples negative for anti-HEV-IgG remain colorless. Samples with a signal to cutoff ratio (SCR) ≥ 1.0 are considered positive for anti-HEV; those with SCR < 1.0 are considered anti-HEV negative.

Anti-HEV Western Blot
Sixty (60) µl of recombinant HEV protein is mixed with the sample buffer and run on 12 % preparative gels. The protein is transferred to a nitrocellulose membrane using the iBlot apparatus (Invitrogen) and evaluated for proper transfer by Ponceau S staining. The membranes are blocked in 5 % non-fat dry milk in TBS buffer for 1 hour. During this time, 60 µl of each clinical sample is mixed with 5 % milk in TBS. Blocked membranes are placed in a multiscreen apparatus, and the samples are added to the wells, followed by an overnight incubation at room temperature with gentle rocking. The next day, the membranes are washed at room temperature with agitation three times for 10 minutes in 5 % non-fat milk in TBST and incubated with a secondary antibody for two hours at room temperature with gentle rocking. Two additional washes are performed for 10 min each at room temperature in 5 % non-fat milk in TBST, followed by one 10 min wash in TBST. The signal is developed using a colorimetric assay, DAB with metal enhancer.

Laboratory Quality Assurance and Monitoring

The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act mandates.

Data Processing and Editing

Data were received after all the laboratory testing was complete. The data were not edited.

Data Access: All data are publicly available.

Analytic Notes

There are four variables:
SEQN: Respondent sequence number
SSHEWSCR: Wantai anti-HEV assay signal to cutoff ratio
SSHEW: IgG Anti-HEV (1=Positive, 2=Negative)
SSHEWB: Anti-HEV Western Blot (1=Positive, 2=Negative)

Western blot and Wantai IgG anti-HEV testing was performed to determine correlation with the DSI and Kuniholm NIH in-house IgG anti-HEV assays. These test results were not intended to produce population estimates, thus no subsampling weights to generate such estimates are provided in this data file.

References

• Kuniholm MH, Purcell RH, McQuillan GM, et al. Epidemiology of hepatitis E virus in the United States: results from the Third National Health and Nutrition Examination Survey, 1988-1994. J Infect Dis 2009; 200:48-56.

Codebook and Frequencies