Analysis of HLA specificity of human monoclonal antibodies by cytofluorimetry and cell ELISA.

In this study we report on the characterization of cytotoxic human monoclonal antibodies (HmAb) detecting polymorphic HLA class II specificities using cytofluorimetric analysis in combination with micro cell ELISA. In both techniques, five anti-HLA HmAb were tested against HLA-transfected murine L cells as target cells and the bound antibody was detected, either by cytofluorimetry or by cell ELISA reader, after addition of fluoresceinated or peroxidase-conjugated anti-human IgG+IgM antibodies, respectively. The results demonstrate that HmAb directed against HLA-DR, -DQ and -DP molecules can be efficiently discriminated by cytofluorimetry and cell ELISA, which appear to be highly sensitive and perfectly comparable to the standard cytotoxicity assay.

Production of two human hybridomas secreting antibodies to HLA-DRw11 and--DRw8+w12 specificities.

In this study we describe the production of two human monoclonal antibodies (mAbs) HMP12 and HMP14, that recognize polymorphic HLA-DR specificities. These mAbs have been produced by hybridization of antibody-secreting Epstein-Barr virus-transformed cells with SHM-D33 human-mouse heteromyeloma. By microcytotoxicity assay HMP12 mAb was found to react with all DRw11-positive cells and HMP14 mAb with all cells bearing the DRw8 or the DRw12 specificity. Cytotoxic activity of HMP14 was completely removed after absorption with DRw8- or DRw12-positive cells and unaffected by absorption with cells carrying different DR specificities. The HLA specificity was further analyzed by cytofluorometry on mouse transfectant cells. The reactivity of the two mAbs was correlated with the presence of a particular polymorphic amino acid residue in the DR beta chain and by this approach the epitopes possibly involved in the antibody binding sites were predicted.

Human anti-HLA-DQw2 monoclonal antibody secreted by an Epstein-Barr-virus--transformed lymphoblastoid cell line: assessment of the monoclonality, allospecificity, and target.

IgM molecules were purified by the use of anti-IgM antibody-coupled Sepharose from the culture supernatant of an Epstein-Barr-virus-transformed lymphoblastoid cell line, MP1, that secretes alloantibodies possessing HLA-DQw2 specificity as defined by the cytotoxicity assay. The obtained IgM preparation was labeled with radioactive iodine-125I and fractionated by gel filtration. It contained pentameric IgM and smaller oligomeric IgMs. When tested by the direct cellular binding assay against a panel of HLA-typed cell lines, they all showed the DR3 and DR7 association pattern characteristic of DQw2. A weak but significant binding was detected for DR1, DR6, and DR9. On isoelectrofocusing, MP1 pentameric IgM gave a restricted banding pattern comparable to monoclonal IgM obtained from a patient with Waldenström's syndrome. Moreover, the pattern was identical to that of IgM purified from the culture supernatant of a defined hybrid clone, 162, that was generated by fusing MP1 cells with heteromyeloma D33 cells. The target class II molecules showed the dimeric structure that conforms to DQw2 molecules.

Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity.

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of "cluster picking," and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class II determinants.