All participants aged 2 years and older are eligible to be tested.
The AUSAB EIA for anti-HBs uses the “sandwich principle” a solid phase enzyme-linked immunoassay technique (1, 2) to detect anti-HBs levels in serum or plasma. Polystyrene beads coated with human Hepatitis B Surface Antigen (HBsAg) are incubated with either the patient specimen or the appropriate controls.
During incubation, antibody, if present, is immunologically coupled to the solid phase antigen. After removal of the unbound material and washing of the bead, human HBsAg tagged with biotin (B-HBsAg) and rabbit anti-biotin, conjugated with horseradish peroxidase (anti-H- HRPO), are incubated with the antibody-antigen complex on the beads. The biotinylated surface antigen binds to this complex crating an antigen-antibody-antigen “sandwich”. The anti-biotin horseradish peroxidase binds to the biotin component of the “sandwich”, forming a sold phase network. Unbound conjugates are removed and the beads are washed. Next, o-Phenylenediamine (OPD) solution containing hydrogen peroxide is added to the bead, and after incubation, a yellow color develops in proportion to the amount of anti-HBs which is bound to the bead. Within limits, the greater the amount of antibody in the sample, the higher the absorbance. The enzyme reaction is stopped by the addition of acid. The absorbance of controls and specimens is determined using a spectrophotometer with wavelength set at 492 nm. Testing for anti-HBs can be useful for:
The detection of anti-HBs is indicative of a prior immunologic exposure to the antigen or vaccine.
The anti-HBs standards contained in the AUSAB quantitation panel kit are assayed with the AUSAB EIA for the quantitative determination of anti-HBs in human serum or plasma. The concentration of anti-HBs expressed in milli-international units per mL (mIU/mL) is determined by comparison with a standard curve generated from measurement of the standards run in duplicate with the AUSAB EIA kit. A curve is obtained by plotting the anti-HBs concentration of the standard vs. the absorbance. The anti-HBs concentration of specimens run concurrently with the standards can then be read from the curve. Specimens with values above the highest standard can be diluted with the specimen dilution buffer and retested. For thepurposes of this study, samples with an absorbance above the highest standard curve will not be diluted, but reported out as > 150 mIU/mL.
Blood specimens are processed, stored, and shipped to the Division of Viral Hepatitis, National Center for Infectious Diseases, National Centers for Disease Control and Prevention. Detailed specimen collection and processing instructions are discussed in the NHANES LPM. Read the LABDOC file for detailed data processing and editing protocols. The analytical methods are described in the Description of Laboratory Methodology section above. Detailed instructions on specimen collection and processing can be found on the NHANES website.
The NHANES quality assurance and quality control (QA/QC) protocols meet the 1988 Clinical Laboratory Improvement Act mandates. Detailed quality control and quality assurance instructions are discussed in the NHANES Laboratory/Medical Technologists Procedures Manual (LPM). Read the LABDOC file for detailed QA/QC protocols. A detailed description of the quality assurance and quality control procedures can be found on the NHANES website.
The analysis of NHANES laboratory data must be conducted with the key survey design and basic demographic variables. The NHANES Household Questionnaire Data Files contain demographic data, health indicators, and other related information collected during household interviews. They also contain all survey design variables and sample weights for these age groups. The phlebotomy file includes auxiliary information such as the conditions precluding venipuncture. The household questionnaire and phlebotomy files may be linked to the laboratory data file using the unique survey participant identifier SEQN.
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