Improved enzyme immunoassays for the detection of antigens in fecal specimens. Investigation and correction of interfering factors.
Solid phase enzyme immunoassays (EIA) are widely used for the detection of infectious agents in body fluids such as stool specimens. However, we found that stool specimens contained substances which desorb from 50% to 68% of the immunoreactant from solid phase surfaces. This desorbing activity decreased the sensitivity of EIA systems for toxin A of C. difficile, rotavirus and adenovirus. The desorbing activity in stool specimens was partially heat labile at 56 degrees C for 30 min, was present in stool fractions corresponding to an estimated molecular weight of 25,000 and was shown to degrade solid phase protein. In addition, the desorbing activity was partially reversed by specific and nonspecific protease inhibitors. Thus, the desorption may reflect the enzymatic activity of stool proteases. The desorption was markedly reduced by diluting specimens in 50% fetal calf serum or an acid-protein buffer such as 0.25 M citrate buffer, pH 4.7, containing 5% bovine serum albumin. These diluents were shown to improve the recovery of toxin A of C. difficile, rotavirus and adenovirus in EIA systems for these antigens.
D000469:Alkaline Phosphatase; D000818:Animals; D000942:Antigens, Bacterial / Q000032:analysis*; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003015:Clostridium Infections / Q000276:immunology; D006224:Cricetinae; D005188:False Negative Reactions; D005243:Feces / Q000032:analysis* / Q000201:enzymology; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000378:metabolism; D008297:Male; D008647:Mesocricetus; D010447:Peptide Hydrolases / Q000378:metabolism
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007074:Immunoglobulin G; C031815:toxB protein, Clostridium difficile; D000469:Alkaline Phosphatase; D010447:Peptide Hydrolases