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HLA oligotyping by Elisa
146
Abstracts
B-7.4 #175
B-7.4 #176
HLA OLIGOTYPINGBY ELISA. D Kostyu, M Ghanayem, N Chasen, M Baldwin, H Hanson, K O'Connell, F Ward. Department of Immunology, Duke Medical Center, Durham NC. Last year we described an enzyme-linked immunosorbent assay where PCR amplified DNA was immobilized in 96-well microtiter trays, exposed to FL-labelled probes and then detected using an alkaline-phosphatase labelled anti-FL FAB, substrate and amplifier (Hum Imm 38:148 1993). Probes and antisense controls were identified for generic DRB1 typing. Since then, we have applied this technique to high resolution DRB, DQA1, DQB1 typing and to identification of some class I alleles, including HLA-A*8001. Testing a variety of probes and loci has allowed us to identify several variables that may apply to oligotyping in general. (a) Probes that are18 bases tong with 9 or 10 GCs have given highly specific reactions. Probes with lower GC content have been weakly reactive, warranting an increase in probe length. Probes with a high GC content (especially for DQB1) may be shortened to 15 bases to minimize cross-reactivity. (b) The amount of DNA amplified can be quantitated by ELISA. Each PCR amplified product is tested with a probe that recognizes a conserved internal sequence of the amplified exon. Serial dilutions (. 15pmol to .01pmol) of a biotin-labelled antisense oligo are also tested at the same time, establishing a reference curve and allowing extrapolation of concentration. Standardizing the amount of DNA added/well improves resolution. (c) Biotinylated antisense oligos that are exactly complementary to each probe have proven to be interesting positive controls. These have for instance allowed us to identify probes that are able to bind to their complementary antisense oligo, but not to PCR amplified DNA. This seems to occur in particular regions of some amplified exons and we believe is due to internal folding of the PCR strand and thus unavailability of the probe binding site. Brief annealing at high temperature (10 min,56-65C; l Omin, RT) seems to eliminate this problem. Stringency washes are still performed at lower (38C,47C,56C) temperatures. Overall, attention to probe length, annealing temperature, amplified DNA concentration and other variables optimizes a simple, quick (<90 minutes) and inexpensive assay.
A NOVEL DRB SSO-TYPING SYSTEM FOR ROUTINE HLA DNA TESTING. M Ballas, M Dzwonkowski, A Gr~ibert & V Lenhard, Biotest AG, Dreieich, Germany A novel DRB-SSO typing system designed for application to routine HLA DNA testing is presented. The special features of this system are a nylonmembrane-coated microtiter plate plus 28 sequence specific oligonucleotide probes with identical hybridization and washing temperatures. The test is based on the conventional dot blot format, i.e. PCR amplified DNA is spotted onto the membrane-coated microtiter plate and hybridized to the 28 oligonucleotide probes. Unspecifically bound probes are removed by a stringent wash step. The oligonucleotides are Digoxigenin-labelled, and specifically bound probes are detected by means of an alkaline phosphatase conjugated anti-Digoxigenin antibody. The enzyme converts a colourless substrate into a dye precipitate in the last step of the test. The entire test including DNA extraction and PCR amplification is accomplished in less than five hours. At least 12 DNA samples can be typed in parallel, and one person can comfortably type 24 DNA samples per day. Only standard technical equipment, i.e. thermal cycler and waterbath, is required. The 28 oligonucleotide probes discriminate between 49 DRB alleles or allelic groups, respectively. This is the maximum resolution capacity achievable from DRB generic amplified DNA. In heterozygotes, certain allelic combinations remain unresolved. These unresolved combinations, however, are, in most csase, restricted to the subtypes of the same serological specificity, i.e. DRBI*1305 and "1306. The use of the microtiter plate format renders this typing system very flexible for both single individual typing and large donor panel testing. Accuracy and reliability of this system have been demonstrated in Japanese and German DNA workshops, and in clinical trials conducted in the U.S., France and Germany with more than 800 specimen analyzed so far.
Abstracts
B-7.4 #175
B-7.4 #176
HLA OLIGOTYPINGBY ELISA. D Kostyu, M Ghanayem, N Chasen, M Baldwin, H Hanson, K O'Connell, F Ward. Department of Immunology, Duke Medical Center, Durham NC. Last year we described an enzyme-linked immunosorbent assay where PCR amplified DNA was immobilized in 96-well microtiter trays, exposed to FL-labelled probes and then detected using an alkaline-phosphatase labelled anti-FL FAB, substrate and amplifier (Hum Imm 38:148 1993). Probes and antisense controls were identified for generic DRB1 typing. Since then, we have applied this technique to high resolution DRB, DQA1, DQB1 typing and to identification of some class I alleles, including HLA-A*8001. Testing a variety of probes and loci has allowed us to identify several variables that may apply to oligotyping in general. (a) Probes that are18 bases tong with 9 or 10 GCs have given highly specific reactions. Probes with lower GC content have been weakly reactive, warranting an increase in probe length. Probes with a high GC content (especially for DQB1) may be shortened to 15 bases to minimize cross-reactivity. (b) The amount of DNA amplified can be quantitated by ELISA. Each PCR amplified product is tested with a probe that recognizes a conserved internal sequence of the amplified exon. Serial dilutions (. 15pmol to .01pmol) of a biotin-labelled antisense oligo are also tested at the same time, establishing a reference curve and allowing extrapolation of concentration. Standardizing the amount of DNA added/well improves resolution. (c) Biotinylated antisense oligos that are exactly complementary to each probe have proven to be interesting positive controls. These have for instance allowed us to identify probes that are able to bind to their complementary antisense oligo, but not to PCR amplified DNA. This seems to occur in particular regions of some amplified exons and we believe is due to internal folding of the PCR strand and thus unavailability of the probe binding site. Brief annealing at high temperature (10 min,56-65C; l Omin, RT) seems to eliminate this problem. Stringency washes are still performed at lower (38C,47C,56C) temperatures. Overall, attention to probe length, annealing temperature, amplified DNA concentration and other variables optimizes a simple, quick (<90 minutes) and inexpensive assay.
A NOVEL DRB SSO-TYPING SYSTEM FOR ROUTINE HLA DNA TESTING. M Ballas, M Dzwonkowski, A Gr~ibert & V Lenhard, Biotest AG, Dreieich, Germany A novel DRB-SSO typing system designed for application to routine HLA DNA testing is presented. The special features of this system are a nylonmembrane-coated microtiter plate plus 28 sequence specific oligonucleotide probes with identical hybridization and washing temperatures. The test is based on the conventional dot blot format, i.e. PCR amplified DNA is spotted onto the membrane-coated microtiter plate and hybridized to the 28 oligonucleotide probes. Unspecifically bound probes are removed by a stringent wash step. The oligonucleotides are Digoxigenin-labelled, and specifically bound probes are detected by means of an alkaline phosphatase conjugated anti-Digoxigenin antibody. The enzyme converts a colourless substrate into a dye precipitate in the last step of the test. The entire test including DNA extraction and PCR amplification is accomplished in less than five hours. At least 12 DNA samples can be typed in parallel, and one person can comfortably type 24 DNA samples per day. Only standard technical equipment, i.e. thermal cycler and waterbath, is required. The 28 oligonucleotide probes discriminate between 49 DRB alleles or allelic groups, respectively. This is the maximum resolution capacity achievable from DRB generic amplified DNA. In heterozygotes, certain allelic combinations remain unresolved. These unresolved combinations, however, are, in most csase, restricted to the subtypes of the same serological specificity, i.e. DRBI*1305 and "1306. The use of the microtiter plate format renders this typing system very flexible for both single individual typing and large donor panel testing. Accuracy and reliability of this system have been demonstrated in Japanese and German DNA workshops, and in clinical trials conducted in the U.S., France and Germany with more than 800 specimen analyzed so far.