Elsevier

Transplant Immunology

Volume 1, Issue 3, September 1993, Pages 192-197
Transplant Immunology

A screening assay to simultaneously determine the presence and specificity of HLA anti-idiotypic antibodies

https://doi.org/10.1016/0966-3274(93)90046-BGet rights and content

Abstract

HLA sensitization is generally associated with an increased risk of graft failure. However, in many cases, highly sensitized patients with a negative current serum crossmatch may be successfully transplanted despite the high levels of alloantibodies (Ab1) in their serum. Sensitized patients may be divided into two groups. The group with a high-risk of early graft failure produces a negative current serum crossmatch as a result of antibody attrition, but upon transplantation the reactivation of Ab1 by the donor organ results in graft failure. The low-risk group gives a negative current serum crossmatch due to the abrogation of Ab1 by anti-idiotypic antibodies (Ab2). This specific inhibition results in the protection of the graft and improved graft survival.

In this paper we describe a screening method which enables large numbers of patients to be assessed for the presence of Ab2 in pretransplant sera, while simultaneously determining the specificities of these antibodies. The pretransplant assessment of sensitized patients for the presence of Ab2 would enable low-risk patients to be distinguished from high-risk patients, while information regarding Ab2 specificity would enable permissible mismatches to be considered. With this information at hand, the pretransplant waiting time for these patients may be greatly reduced.

In our modification of the inhibition assay, selected dilutions of peak sera (P/n) were tested in the presence of either platelet absorbed current serum (P/n + Cabs) or an equal volume of fetal calf serum (P/n + FCS) as a dilution control. Using the microlymphocytotoxicity assay, these serum combinations were screened against a large panel of T lymphocytes, and Ab1 specificities for each serum combination were assigned based on the reactivity patterns with the cell panel. Inhibition by Ab2 in Cabs was said to be present when Ab1 reactivity observed in (P/n + FCS) was not observed in (P/n + Cabs). The specificity of the lost activity was assigned to Ab2. When reactivity was lost in (P/n + FCS), the presence of Ab2 could not be determined because inhibition could not be distinguished from a dilution effect. To increase the chances of detecting inhibition, dilutions close to the peak serum end-point titre were selected for testing.

Fifty-two renal transplant recipients with peak serum panel reactive activities (PRA) of >45% were tested. Of these, 36 had current PRAs of <45% while 16 had current PRAs of >45%. Inhibition was observed in 1752 patients and Ab2 specificities were assigned to 15. Six sera contained Ab2 directed towards more than one HLA specificity. Twenty-seven sera did not display inhibition and were assumed not to contain Ab2, while eight sera could not be analysed because Ab1 reactivity was lost in (P/n + FCS) due to the dilution effect. There was no significant difference in the number of sera displaying inhibition between the patients with current PRAs of <45% and those with current PRAs of >45% (30% versus 37%).

To conclude, our modification of the inhibition assay has enabled us to simultaneously analyse 52 renal transplant recipients for the presence and specificity of Ab2 in pretransplant sera. It is hoped that the application of the method may enable a greater proportion of highly sensitized patients to receive kidney transplants and reduce their time on the renal transplant waiting lists.

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