The Detection of Serum Hla Antibodies by Luminex Bead Assays Can Be Improved by the Addition of Edta or Dithiotreitol

Abstract

The detection and specification of human leukocyte antibodies (HLA antibodies) has improved in the recent years through the introduction of solid phase techniques. The Luminex x-MAP technology with single HLA antigen coated beads allows a more precise and reproducible specification of HLA antibodies than cell based methods or ELISA using antigen extracts from cells. However, there are some drawbacks. The sensitivity is very high and the correlation of the results with clinical data and standard test systems like the complement dependent cytotoxicity (CDC) assay is not always satisfactory. Recently it has been shown, that Luminex-based HLA antibody assays might be disturbed by prozone phenomena resulting from binding of C1q and/or IgM, when sera with a high content of HLA antibodies are analyzed. Dilution, treatment of the sera with DTT or addition of EDTA seem to decrease these effects but may have marked influences on the results of these assays. We tested sera from five highly immunized patients for in CDC (for HLA-Class I antibodies), LABScreen Mixed and LABScreen single antigen tests (for HLA-Class I and II antibodies) using untreated, DTT-treated serum or serum supplemented with 8 mM EDTA. 4 CDC (-/+DTT) positive sera showed a high increase (DTT>EDTA) in reaction strengths for some HLA specificities, which was more pronounced for the antibody specificities reactive in CDC. DTT or EDTA treatment changed the number of HLA antibody specificities detected to a variable extent in all five sera. The CDC negative serum showed minor changes in reaction strength. The signal strenghs of the different specificities mainly remained in the order of the untreated serum. In summary Luminex bead assay systems are sensitive to prozone phenomena which might be diminished by DTT treatment or addition of EDTA. By using these methods a further increase of sensitivity and a change of detected specificities with results differing from the results with untreated sera have to be expected. However, the use of DTT or EDTA might improve the results. In our cases we found, that specificities confirmed by data from the immunizer and/or found in the CDC-assay are better represented in the results. Further efforts should be taken to verify, if one of these methods could replace the regular Luminex bead assay with untreated serum.