A radioactive antigen-binding assay for the measurement of antibody to Haemophilus influenzae type b capsular polysaccharide
Abstract
A new polyethylene glycol (PEG) radioimmunoprecipitation assay was developed for the detection of antibody to Haemophilus influenzae b capsular polysaccharide, polyribosylribitol phosphate (PRP). The radioactive antigen, [3H]PRP, with a high specific activity, was produced by growing the organism in the presence of [3H]ribose and was purified by hydroxylapatite and Sepharose 4B column chromatography. In the assay, PEG (12.5%) was used to separate antibody-bound [3H]PRP from free [3H]PRP. The assay covered the range of 0.5 and 20 ng antibody/assay at a maximum sensitivity of 0.5 approximately 1.0 ng antibody/assay. With various dilutions (1-20 ng antibody/assay) of S. Klein reference antiserum, the within-run coefficient of variation (CV) of 10 replicates ranged from 3.5 to 8.5%. Average CVs of 8.9% and 11.0% were obtained in the between-run and day-to-day reproducibility studies. The binding of [3H]PRP to S. Klein reference antiserum was severely inhibited by a minute amount of non-radioactive PRP; however, no significant interference was found in the presence of high concentrations of polysaccharides from Escherichia coli K100 and Streptococcus pneumoniae indicating that the RIA was highly specific for antibody to H. influenza...Continue Reading
References
Use of polyethylene glycol to separate free and antibody-bound peptide hormones in radioimmunoassays
Citations
Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates
Outer membrane protein subtypes and investigation of recurrent Haemophilus influenzae type b disease
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