Heightened sensitivity of quantitative ELISA for IgM rheumatoid factor with the use of the biotin-streptavidin system.
Rheumatoid factors (RFs) have been traditionally detected by the agglutination of IgG-coated particles such as latex beads or erythrocytes. These agglutination assays are only semiquantitative and do not detect materials other than pentameric IgM RF. In order to optimize a quantitative enzyme-linked immunoassay (ELISA) for IgM RF that could be used for detecting small amounts of IgM RF in materials derived from large numbers of biologic fluids, several steps were investigated. In comparison with an ELISA using a peroxidase-conjugated antibody, substitution of a biotinylated antibody followed by peroxidase-conjugated streptavidin led to a steeper slope of absorbance versus U/mL of RF without increasing the background. By using as a chromogenic substrate tetramethylbenzidine (TMB) instead of o-phenylenediamine (OPD), an even steeper slope and a lower background were achieved. The biotin-streptavidin ELISA proved to be more sensitive than a commercial latex agglutination test and similar in sensitivity to a screening slide-agglutination test using IgG-coated human erythrocytes. When sera that had had positive results in tube-dilution assays both with sensitized human erythrocytes and sensitized sheep erythrocytes were compared with other sera with positive results for only agglutination of sensitized human cells but not sheep, the group with positive results for both tube dilution assays had higher IgM RF levels by ELISA than those in the group with only the positive assay for sensitized human cells.
Moxley
G
G
Medical College of Virginia, Virginia Commonwealth University, Richmond.
D001426:Bacterial Proteins; D001560:Benzidines; D001710:Biotin; D002863:Chromogenic Compounds; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007075:Immunoglobulin M / Q000032:analysis*; D011786:Quality Control; D012016:Reference Values; D012217:Rheumatoid Factor / Q000032:analysis*; D019809:Streptavidin
D001426:Bacterial Proteins; D001560:Benzidines; D002863:Chromogenic Compounds; D007075:Immunoglobulin M; C021758:3,3',5,5'-tetramethylbenzidine; D001710:Biotin; D012217:Rheumatoid Factor; D019809:Streptavidin