RESUMEN
A common problem facing HLA-typing laboratories is to substitute genomic typing for serology without having to handle a large number of oligoprobes, primers, or restriction enzymes. A protocol is described for HLA-DRB1 genomic typing using a combination of PCR amplification, DNA heteroduplex analysis, and restriction enzymes. Because the core of the procedure is the analysis of the DNA heteroduplexs, it shall be termed HET typing. There are two stages: the first stage comprises two rounds of PCR amplification of the polymorphic second exon of the HLA-DRB genes directly on lysed blood cells. The first amplification is with DRB generic primers, and the second amplification with seven HLA-DRB1 group-specific primers at the 5' end and a common 3' primer. The latter is designed with two nucleotide mismatches, thus creating an artificial restriction site to differentiate between both HLA-DRB1 variants at position 86, which is of critical importance in antigen presentation. The second stage involves subjecting the final amplified product to both DNA heteroduplex formation and digestion by two single-cutter restriction endonucleases. The digested or heteroduplexed samples are run on the same polyacrylamide gel. A total of 25 HLA-DRB1 alleles can thus be differentiated with a total of 10 primers and two restriction enzymes and without the use of probes. This protocol is ideally suited to preliminary HLA class II typing of bone marrow donors.
Asunto(s)
Genes MHC Clase II , Antígenos HLA-DR/genética , Ácidos Nucleicos Heterodúplex/análisis , Polimorfismo de Longitud del Fragmento de Restricción , Alelos , Secuencia de Bases , Células Sanguíneas , ADN/análisis , Prueba de Histocompatibilidad/métodos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
22 patients awaiting bone marrow transplantation (BMT) and 65 unrelated healthy HLA-A, B and DR serologically identical donors were studied. 21 patients were non reactive on mixed lymphocyte culture (MLC) towards at least one BMT donor, resulting in 32 pairs MLC-negative. 33 other HLA-matched donors gave proliferative responses on MLC. The phenotype DR3/DR7 was significantly higher in patients (P) and donors (D) studied (p < 0.00001). P and D pairs were DNA typed by RFLP analysis for DRB, DQA and DQB genes, and DNA matched by PCR Fingerprinting (PCRF) for DRB, DQA, DQB and DPB. Six patients and 18 donors were also oligotyped for the subtypes of DR1, DR3, DR4 and DR5. Patients and donors were divided according to identity on RFLP, PCRF and responsiveness on MLC, represented by RRI. In the group of MLC non responder pairs, 11% had differences on DRB PCRF compared to 57% in the group of MLC responders (p = 0.0002). The mean RRI value of PCRF DRB incompatible pairs was significantly higher compared to RRI of compatible pairs (p = 0.012). With respect to PCRF for DPB, 75% of MLC-ve pairs were different. Also the mean RRI value was significantly lower in DPB identical pairs compared to non identical ones (p = 0.048). The compatibility between P/D pairs assessed by oligotyping was in accordance with DRB PCRF. PCRF for DQB, but not for DQA, corresponded to MLC responses. Our findings confirm that PCRF offers a precise and fast alternative in DNA matching for DRB. We also suggest that PCRF for DQB and DPB, together with DRB, could eventually substitute MLC.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Histocompatibilidad/inmunología , Donantes de Tejidos , División Celular , Células Cultivadas , ADN/genética , Antígenos HLA/análisis , Antígenos HLA/genética , Antígenos HLA-DP/análisis , Antígenos HLA-DP/genética , Antígenos HLA-DP/inmunología , Antígenos HLA-DQ/análisis , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Cadenas alfa de HLA-DQ , Cadenas beta de HLA-DQ , Antígenos HLA-DR/análisis , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Antígenos de Histocompatibilidad Clase II/análisis , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Prueba de Cultivo Mixto de Linfocitos , Linfocitos/citología , Linfocitos/inmunología , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
In 36 women with unexplained primary recurrent abortion, 13 with secondary unexpained recurrent abortion, 25 with primary unexplained infertility, 7 with secondary unexplained infertility and two groups of control women, autoantibodies to soluble cellular antigens were measured by Western blotting to a disaggregated HeLa cell antigen preparation, by counter immunoelectrophoresis and by indirect immunofluorescence. Using Western blotting the women with primary infertility and those with secondary recurrent abortion had a significantly higher prevalence of autoantibodies (P less than 0.01 in each case). This was not shown using the other methods. It is possible that these antibodies could be causally related to the pathology of the conditions studied.