title: Determination of total, free and complexed protein S in plasma by ELISA, and comparison with a standard electroimmunoassay.
abstract: An enzyme-linked immunosorbent assay (ELISA) for measuring total, free and complexed protein S in plasma was developed. To assay free protein S, C4b-binding protein-bound protein S (C4b-BP-PS) was extracted by addition of polyethyleneglycol (PEG) 6000 (5%, final concentration) to plasma samples. Microtiter plates were coated with rabbit anti-human protein S, and bound protein S was detected with labelled anti-protein S antibody. Diluted plasma samples were incubated in the plates overnight at 22 degrees C to permit C4b-BP-PS complexes to dissociate. Mean variation coefficients of 2.1 and 3.2% (intra-assay) and 4.3 and 7.9% (inter-assay) were found for total and free protein S assays, respectively. The ELISA measures free and complexed protein S with equal efficiency as is demonstrated by the fact that the sum of free protein S and C4b-BP-PS complex levels in normal individuals, women in their third trimester of gestation and patients with acute deep vein thrombosis (DVT), equaled the level of total protein S present in the corresponding plasma. Total protein S values obtained with the ELISA, in all groups studied, correlated well with those obtained with a standard electroimmunoassay (EIA) (r = 0.93; n = 40). However, total protein S levels measured by EIA were lower than those assayed by ELISA in pregnant women and in DVT patients. Furthermore, addition of several amounts of purified C4b-BP to NHP, which reduced the recovery of free protein S, did not influence the total protein S values measured by ELISA but slightly decreased the recovery of total protein S measured by EIA. These results indicate the necessity of using assays which accurately and reliably measure the total amount of protein S antigen. After addition of C4b-BP to NHP, the residual functional protein S level was lower than the residual level of free protein S antigen; this lends support to the idea that C4b-BP-PS complex inhibits the activated protein C cofactor activity of protein S.
authors.affiliation: Research Center, Hospital La Fe, Valencia, Spain.
authors.forename: F
authors.initials: F
authors.lastname: España
authors.forename: S
authors.initials: S
authors.lastname: Hendl
authors.forename: J
authors.initials: J
authors.lastname: Aznar
authors.forename: J
authors.initials: J
authors.lastname: Gilabert
authors.forename: A
authors.initials: A
authors.lastname: Estellés
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D003169:Complement Inactivator Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D007118:Immunoassay; D008297:Male; D011247:Pregnancy / Q000097:blood; D011263:Pregnancy Trimester, Third; D017293:Protein S; D013924:Thrombophlebitis / Q000097:blood
chemical_list: D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017293:Protein S