Reassessment of the two-site enzyme immunoassay for human epidermal growth factor (hEGF) and measurement of immunoreactive hEGF in human sera and urine.
We reassessed the enzyme immunoassay (EIA) system for hEGF previously developed by our laboratory (Clin Chim Acta 156:51-60, 1985), since it appeared that the reported EIA system detected not only hEGF but also pS2 protein owing to minor contamination by pS2 protein in the hEGF sample used for immunization. In this study, we purified the hEGF sample using Benzamidine-Sepharose 6B column chromatography as a critical step for purification and newly developed an EIA system with specificity for hEGF. We also measured the hEGF level in serum, plasma, and urine from normal subjects by our new EIA system and found that the values measured by the previous system were 1.2-5.8-fold higher than the new system values. These results suggest that the previous system detected "hEGF" in excess owing to the nonspecificity of the antibody used. We investigated the molecular nature of immunoreactive hEGF detected in serum using our new system and confirmed that considerable amounts of immunoreactive hEGF exist as a high molecular weight form through S-S linkage with some macromolecule(s) in human blood as reported previously (Biochem Int 12:677-683, 1986).
Takei
Y
Y
Department of Molecular Biology, Gifu Pharmaceutical University, Japan.
Kurobe
M
M
Ezawa
H
H
Hayashi
K
K
D000328:Adult; D000818:Animals; D000906:Antibodies / Q000032:analysis; D001550:Benzamidines; D002851:Chromatography, High Pressure Liquid; D004815:Epidermal Growth Factor / Q000097:blood / Q000276:immunology / Q000652:urine; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques / Q000592:standards*; D008297:Male; D008875:Middle Aged; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012685:Sepharose; D015842:Serine Proteinase Inhibitors
D000906:Antibodies; D001550:Benzamidines; D015842:Serine Proteinase Inhibitors; D004815:Epidermal Growth Factor; D012685:Sepharose; C032157:benzamidine