Sensitive antigenic determinations of high molecular weight kininogen performed by covalent coupling of capture antibody.
Knowledge of the role of high-molecular-weight kininogen (HK) in disease states has been limited by the lack of easy, reliable, and rapid assays for HK antigen. We have developed two immunochemical determinations, which use covalent coupling of the capture antibody, that will greatly facilitate the measurement of HK in plasma and cells. The first is an enzyme-linked immunosorbent assay (ELISA) that is performed in 96-well microplates and can be prepared up to 5 months in advance; it can, therefore, be available when needed. The second is a very rapid method that uses the technique known as particle concentration fluorescence immunoassay (PCFIA) and can be performed on 96 samples in less than 30 minutes. The HK-ELISA correlated well with HK coagulant activity in 22 normal donors (r = 0.88). The interassay coefficient of variation (CV) of 22 samples assayed on 5 separate days was 12%, whereas the interassay CV was 4%. The HK-PCFIA demonstrated an excellent correlation (r = 0.97) with HK coagulant activity in 22 normal donors, with an interassay CV of 7% and an intraassay CV of 1.5%. The HK ELISA was linear between 6 and 80 ng/ml, whereas the HK-PCFIA was linear between 5 and 800 ng/ml. Using the HK-PCFIA, we found a difference in HK antigen levels between two groups of patients with sepsis. The mean of those who survived (n = 10) was 70 micrograms/ml, whereas the mean of those who died (n = 6) was 48 micrograms/ml. Both HK-ELISA and HK-PCFIA were able to detect HK antigen in baboon plasma, enabling the monitoring of HK during experimental protocols. The HK-PCFIA was sensitive enough to detect HK antigen in the supernatant of hepatoma G2 at levels of 1 to 4 ng/ml. These assays should enable convenient and frequent measurement of HK antigen in various physiologic and pathophysiologic states, as well as in cell fractions.
Scott
C F
CF
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Colman
R W
RW
D000818:Animals; D000906:Antibodies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007239:Infections / Q000097:blood; D007704:Kininogens / Q000097:blood* / Q000737:chemistry; D008113:Liver Neoplasms / Q000097:blood / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology; D008970:Molecular Weight; D010215:Papio / Q000097:blood; D014407:Tumor Cells, Cultured
D000906:Antibodies; D007704:Kininogens