TY - JOUR
T1 - Stool desorbing activity
T2 - A possible cause of false-positive reactions in competitive enzyme immunoassays
AU - Hanvanich, M.
AU - Viscidi, R.
AU - Laughon, B. E.
AU - Bartlett, John
AU - Yolken, R. H.
PY - 1985
Y1 - 1985
N2 - We have developed a competitive enzyme immunoassay for the measurement of purified toxin A of Clostridium difficile. However, when we applied this assay to the detection of C. difficile toxin in stool specimens, we noted a high rate of nonspecific activity in fecal specimens which did not contain toxin. We found that the low specificity (26%) of the assay was due to the presence in stool specimens of interfering factors which desorbed the antigen coated on the solid-phase surface. These factors could be detected by measurement of the desorption of biotin-labeled proteins attached to the solid-phase surface. In addition, these interfering factors were partially inactivated by heating at 56° C for 10 min and partially inhibited by phenylmethylsulfonyl fluoride (2 mM) or soybean trypsin inhibitor (10 mg/ml). These data suggested that the desorbing activity was due to proteolytic activity in the fecal specimens. Fetal calf serum (50%) was found to be the most effective measure in preventing the interfering effect. By using 50% fetal calf serum as a diluent, we increased the specificity of the antibody inhibition enzyme immunoassay to 93%. Interfering factors in stool specimens could be a cause of false-positive results in other competitive immunoassay systems. The use of diluents which neutralize protease activity can result in a marked improvement in the specificity of competitive immunoassay systems.
AB - We have developed a competitive enzyme immunoassay for the measurement of purified toxin A of Clostridium difficile. However, when we applied this assay to the detection of C. difficile toxin in stool specimens, we noted a high rate of nonspecific activity in fecal specimens which did not contain toxin. We found that the low specificity (26%) of the assay was due to the presence in stool specimens of interfering factors which desorbed the antigen coated on the solid-phase surface. These factors could be detected by measurement of the desorption of biotin-labeled proteins attached to the solid-phase surface. In addition, these interfering factors were partially inactivated by heating at 56° C for 10 min and partially inhibited by phenylmethylsulfonyl fluoride (2 mM) or soybean trypsin inhibitor (10 mg/ml). These data suggested that the desorbing activity was due to proteolytic activity in the fecal specimens. Fetal calf serum (50%) was found to be the most effective measure in preventing the interfering effect. By using 50% fetal calf serum as a diluent, we increased the specificity of the antibody inhibition enzyme immunoassay to 93%. Interfering factors in stool specimens could be a cause of false-positive results in other competitive immunoassay systems. The use of diluents which neutralize protease activity can result in a marked improvement in the specificity of competitive immunoassay systems.
UR - http://www.scopus.com/inward/record.url?scp=0021922121&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0021922121&partnerID=8YFLogxK
U2 - 10.1128/jcm.21.2.184-188.1985
DO - 10.1128/jcm.21.2.184-188.1985
M3 - Article
C2 - 3882746
AN - SCOPUS:0021922121
SN - 0095-1137
VL - 21
SP - 184
EP - 188
JO - Journal of clinical microbiology
JF - Journal of clinical microbiology
IS - 2
ER -