A simple method of HLA-DRB typing using enzymatically amplified DNA and immobilized probes on microtiter plate.

We have developed a simple and economical method for HLA-DNA typing, called microtiter plate hybridization (PCR-MPH), which could replace standard PCR-SSO. This method is similar to that of an ELISA. Briefly, the PCR products labeled at the 5' termini with biotin were hybridized with probes immobilized on a microtiter well, and the bound PCR products were detected by streptavidin-conjugated enzymes followed by color development. A system for HLA-DRB1 "generic" typing (e.g., DR1, DR2), using microtiter wells coated with 12 different SSOs has been established. The HLA-DRB types classified using this method agreed well with those obtained by conventional serologic typing. The advantages of this microtiter plate-hybridization method for routine HLA-DNA typing are a short assay time, easy processing of large numbers of samples, and the potential for automation.

A simple method of detecting amplified DNA with immobilized probes on microtiter wells.

We have developed a simple hybridization method for the detection of specific DNA sequences amplified by polymerase chain reaction (PCR). This method is similar to an enzyme-linked immunosorbent assay (ELISA) format in that labeled PCR products at the 5' termini are hybridized with probes immobilized on a microtiter well and the bound PCR products are detected in a manner similar to that of an enzyme immunoassay (EIA). Two improvements have been made in immobilizing the probe to the microtiter wells, in terms of increasing both immobility and hybridization efficiency. One is that single-stranded (ss) DNA, without the complementary strand, is used. The other is that instead of a single copy, a tandem array of the probe is used for immobilization and hybridization. Using of ssDNA containing about a 60-repeat array of a relevant sequence as an immobilized probe, the sensitivity increased 10-fold over that of a single oligonucleotide unit. We also found that the hybridization conditions such as time, temperature, and solution composition could be simplified. Therefore this method is especially suited for handling of a large number of samples, for example detection of viruses, bacteria, and other pathogens, as well as mot human genetic disorders.

Routine low and high resolution typing of the HLA-DRB gene using the PCR-MPH (microtitre plate hybridization) method.

We describe HLA-DRB1 typing using polymerase chain reaction-based microtitre plate hybridization (PCR-MPH), which can process large numbers of samples. MPH typing is similar to an enzyme-linked immunosorbent assay (ELISA), in which a tandemly ligated sequence-specific oligonucleotide is immobilized on microtitre wells. The typing procedure consisted of two steps. In the first, PCR-MPH with 16 probes was performed to determine the specificities of the serological levels (DR1, DR2, DR3, DR4, DR11, DR12, DR13, DR14, DR7, DR8, DR9 and DR10) after generic amplification ("low resolution typing'). In the second step, DR1, DR2, DR4, DR12/8 and DR3/11/13/14 were group-specifically amplified based on the results of the first PCR-MPH, and microtitre plate hybridization proceeded in a similar manner to the first step ("high resolution typing'). Low resolution typing was completed within 2 h after generic amplification, and the results of high resolution typing were obtained in another 3.5 h after amplification. The allelic types classified using PCR-MPH were completely concordant with those obtained by PCR-single-strand conformation polymorphism or PCR-restriction fragment length polymorphism.

Large-scale DNA typing for human platelet alloantigens by PCR-PHFA (preferential homoduplex formation assay).

Alloimmunization against human platelet alloantigens (HPA) is known to be involved in disorders such as neonatal alloimmune thrombocytopenic purpura, posttransfusion purpura, and refractoriness to platelet transfusion therapy. HPA typing is essential in diagnosis and management of patients. Therefore a reliable and speedy method is necessary for HPA typing. We have successfully applied a new DNA typing method, PCR-preferential homoduplex formation assay (PHFA) method, to typing for the HPA-1, -2, -3, -4, -5 and -6 systems. This method is based on DNA strand competition during hybridization under a precisely controlled temperature gradient between a double-labelled amplicon (standard DNA), prepared from biotin- and DNP-labelled primers, and an unlabelled amplicon (sample DNA). The results obtained by PCR-PHFA typing were in good agreement with the allotypes determined by serological typing and by other DNA typing methods. The PCR-PHFA method can be easily automated, is suitable for typing both small and large numbers of samples, and thus is applicable to routine HPA typing.