Simple elisa method for the evaluation of soluble hla class i antigens in human serum

Abstract

A simple sandwich ELISA method has been developed for the quantification of soluble HLA class I antigens (s-HLA) in human serum. The assay utilizes the monoclonal antibody Q6/64, directed to a monomorphic determinant of the HLA alpha-chain, to capture the antigen and the biotinylated NAMB1 monoclonal antibody, directed to beta 2-microglobulin, as the detection antibody. The extract of the LG-2 lymphoid cell line and pooled sera from 100 healthy subjects are utilized as standards. The arbitrary value of 100 s-HLA Relative Units/mL (RU/mL) is given to the 1:20 dilution of pooled human sera whose optical density value corresponds to the one of the extract of 1 x 10(6) LG-2 cells (6.25 micrograms/mL protein concentration). The assay is easy to perform, specific, reproducible (intra- and inter-assay variations ranging from 3.2% to 8.87%), sensitive (detection limit of 6 RU/mL), and needs a small amount of serum (0.1 mL). The mean serum levels of s-HLA found in 100 healthy normal subjects are 41.9 +/- 13.4 RU/mL. The potential uses of the method are discussed.

In vitro immunosuppressive activity of soluble HLA class I and Fas ligand molecules:do they play a role in autologous blood transfusion?

“…The concentrations of sHLA‐I, sHLA‐I‐HC, sHLA‐II, and sFasL molecules were determined by double‐determinant immunoassay (DDIA), as described, 20,38,47,48 with minor modifications. W6/32, TP 25.99, LGII‐612.14, and NOK‐2 MoAbs and biotinylated NAMB‐1, HC‐10, Q5/13, and NOK1 MoAbs were utilized to capture and detect sHLA‐I, sHLA‐I‐HC, sHLA‐II, and sFasL molecules, respectively.…”

Derivation and Application of Monoclonal Antibodies Recognizing Several Epitopes on Bovine Serum Albumin.

“…The sensitivity was higher than that in other ELISA systems (Isoai et al 1989;Spertini et al 1992) and it would be increased using more sensitive substrates such as chemoluminescent materials. Sandwich ELISA is found to be more advantageous than regular ELISA: it could specifically detect native BSA in biological samples in the presence of other proteins (Takata et al 1989;Comuzio et al 1991;Ho 1992).…”

Increased serum concentration of soluble HLA‐DR antigens in HIV infection and following transplantation

“…HLA class I1 molecules were purified from HUT 78 cells lysate by immunoaffinity chromatography using an EAE-Sepharose beads column conjugated with CR 10/343 MoAb. SDS-PAGE electrophoresis and Western blot analysis (using the anti-HLA class I1 p chain LGII-612.14 MoAb and an anti-mouse Ig peroxidase-linked antibody) were performed on the lysate and on the eluate to identify the band corresponding to HLA class I1 p chain (Figure 1 The serum level of soluble HLA class I antigens (sHLA-I) was determined utilizing a previously described DDIA, with minor modifications (25). The serum level of soluble intercellular adhesion molecule-1 (SICAM-1) was determined utilizing SICAM-1 -EASIA kit from Medgenix Diagnostics SA.…”