摘要:

Abstract:Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single‐chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2–3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly‐G tailing the first strand cDNA followed by anchor PCR with a forward poly‐C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL‐link‐VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge‐CH2‐CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti‐human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02509.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:DNA typing of HLA class II alleles of the DRB1/3/4/5 and DQB1 loci using sequence‐specific oligonucleotide probes and polymerase chain reaction amplified DNA has been used for the large‐scale typing of donors for the National Marrow Donor Program unrelated donor registry. The results of quality control analysis for the second year of the project (10/1/939/30/94) show the typing continues to be highly accurate, specific, and reliable. The average percent of correctly classified HLA oligotypes (groups of alleles defined by a hybridization pattern with a panel of sequence‐specific oligonucleotide probes) based on 9,244 DRB1 and 7,244 DQB1 assignments was 99.8% (range 99.4%100.0%) for DRB1/DRB3/DRB4/DRB5 and 99.8% (range 99.6%100.0%) for DQB1. This level of accuracy is particularly remarkable because the 4,636 DRB quality control samples were tested blindly and could not be distinguished from 57,580 donor samples tested at the same time by the laborat

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02510.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The utility of the MLC assay as a test of HLA‐D region matching and predictor of graft‐versus‐host disease (GvHD) was evaluated in 435 patients receiving marrow grafts from unrelated donors. Donors and recipients were phenotyped for HLA‐A, B and DR antigens by serology, tested in MLC, and retrospectively genotyped for DRB1, B3, B4, B5, DQB1 and DPB1 alleles by PCR/SSOP. Of the 244 HLA‐A, B, DR‐identical donor‐recipient pairs with evaluable MLC and DRB1 typing results available, 208 were matched for HLA‐A, B and DRB1, while 36 were matched for HLA‐A and B and mismatched for a DRB1 allele. Donor anti‐recipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from 7.2 to 100%, with a median of 4.0%. A comparison of reactivity in MLC between pairs matched versus mismatched for DRB1 alleles showed a significant overlap in the distribution of RRs. Using optimally‐defined RR cutoffs of 4 and 16%, no correlation between MLC results and risk of developing clinically significant grades III‐IV GvHD (p=0.6 and 0.5, respectively) was found when the contribution of DRB1 mismatch was accounted for. Matching for DRB1 alleles, in contrast, was a better predictor of clinically significant GvHD, with DRB1‐matched transplant recipients less likely to develop grades III‐IV GvHD than DRB1‐mismatched recipients (p=0.14). Among the 208 patients and donors matched for DRB1 alleles, the MLC, although reactive (RR>4.0%) in 45% of cases, did not predict GvHD. Overall, these results underscore the limitations in using the MLC to predict DRB1 matching or risk of clinically significant GvHD among patients receiving unrelated marrow grafts. The availability of DRB1 allele matching by sequence‐specific oligonucleotide probes (SSOP) or by direct sequencing provides a method for donor matching that is rapid, precise and superior to the MLC for predict

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02511.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Previous studies have indicated that certain alleles of HLA‐DR and ‐DQ genes were strongly associated with susceptibility and resistance to insulin‐dependent diabetes mellitus (IDDM), and the role of DQ molecule in IDDM has been suggested. To further clarify the association of DQ alleles with IDDM, we determined the nucleotide sequences of full‐length cDNA from 13 DQA1 alleles and 14 DQB1 alleles. The sequencing analysis revealed sequence polymorphisms outside the hypervariable region of DQ genes. We then analyzed the DQA1 and DQB1 polymorphisms along with that of DRB genes in 86 B‐lymphoblastoid cell lines (B‐LCLs) from various ethnic groups and in healthy unrelated Japanese and Norwegian individuals. The allelic and haplotypic distributions in each population revealed the characteristic haplotypic formation in the HLA class II region. HLA genes in 139 Japanese and 100 Norwegian IDDM patients were analyzed. DQB1*0301 was negatively associated with IDDM in both ethnic groups, irrespective of associated DRB1 and DQA1 alleles. In DQB1*0302 positive populations, which represented a positive association with IDDM in both ethnic groups, DRB1*0401, *0404, *0802 haplotypes increased in the patients, whereas DRB1*0406 haplotype decreased. Considering about the hierarchy in DRB1 alleles with IDDM susceptibility (DRB1*0401>*0404>*0403 in Norwegian and DRB1*0802>*0403>*0406 in Japanese), the genetic predisposition to IDDM is suggested to be defined by the combination of DR‐associated susceptibility and DQ‐associated susceptibility and by the DQ‐associated resistance which is a domin

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02512.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:We have characterized two novel HLA‐B alleles, B*7802 and B*51022. The Caucasian‐derived variant B*7802 most resembles the African‐derived B*7801, from which B*7802 differs by two nucleotides. Only one of these modifications, however, is translated: a tyrosine for aspartate substitution occurs at residue 74 in B*7802, while the second nucleotide difference reflects a proximal synonymous substitution in codon 23. A second variant, B*51022, differs synonymously only at codon 23 from B*51021. Comparative analysis of the B5 CREG demonstrates that other pairs of B5 alleles differ synonymously only at codon 23 or synonymously at codon 23 and non‐synonymously at a second more distal location. Contrary to the genesis of like pairs of B5 alleles via introduction of coordinate yet distant mutagenic events onto a single B5 progenitor, we postulate that synonymously different B5 progenitor molecules, B5ATT and B5ATC, are evolving in convergence to generate homologous B5 allele pairs differing silently at codon 23. Our finding that B*7802 is a single amino acid away from complete convergence with B* 7801 and that B*51022 and B*51021 are in complete convergence is exemplary of such ev

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02513.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:The HLA‐B70 antigen is among the most common antigens present in African Americans; however, monospecific serologic reagents defining B70 and its subtypes, B71 and B72, are rare. We have recently reported the molecular characterization of a B71 allele (B*1510) from an African American individual carrying the haplotype HLA‐A30, Cw3, B71(w6). In order to better define the degree of polymorphism of molecules carrying the B71 serological specificity in the human population, we have used serology, cDNA sequencing, and PCR/SSOP typing to characterize B71 alleles from additional individuals from different ethnic populations and carrying different class I haplotypes. All carried either B*1510 or B*1518 alleles. Other HLA‐B alleles isolated from these individuals (B*5001, B*4901, B*3501, B*3701) were identical to previously reported sequences except for a novel B41 allele (B*4102) identified in one Hispanic individual. This allele has concurrently been identified by Rufer and colleagues in Caucasian individuals. The B*4102 allele differs from B*4101 at codons 95 (Leu/Trp) and 97 (Ser/Arg). In addition, the B*4102 allele differs from B*4101 by two silent substitutions at codons 94 (ACC/ACT) and 99 (TAC/TAT). Since the polymorphic sequence present in B*4102 is also present in other HLA‐B alleles (e.g., B*2707, B*4002, B*0702), it may represent a gene conversion cassette. The allelic diversity at the class I loci and the scarcity of monospecific alloantisera support the importance of the application of molecular based methods to identify HLA class I alleles in matching unrelated donor/recipient pairs for bone marrow transpla

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02514.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:HLA‐DR and DQ alleles have been detected by DNA typing in Ashkenazi and non‐Ashkenazi Jews from Israel. Allele frequencies, characteristic DR/DQ linkage disequilibria, population distances and their corresponding dendrogram by using the Neighbor‐Joining method were used to study relatedness between Jewish and other Mediterranean and non Mediterranean populations. Closest relatedness is observed between Ashkenazi and non‐Ashkenazi Jews, and, in decreasing order, also with Algerians, Spaniards (including Spanish‐Basques), French and Italians. Also, particular characteristic Central European alleles are observed in Ashkenazi Jews and Mediterranean/African alleles in non‐Ashkenazi Jews. This is consistent with historical data, Jews being an ancient Mediterranean population, who have had a certain degree of admixture with their 2000–3000 years old neighbors in spite of cultural and religious traditions which have preserved identity o

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02515.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

Abstract:Sequencing‐based HLA typing (SBT) is a PCR based high resolution HLA typing method in which polymorphic regions of the gene are sequenced and directly used for typing. Currently, for class II SBT, alleles are identified by comparison of the exon 2 sequence with their corresponding allele sequence library. Routine SBT requires reliable identification of heterozygosity, and automated assignment of the alleles. In sequencing strategies different enzymes can be used for primer extension. The most characteristic difference between sequences obtained by two protocols using Sequenase®, or Taq‐cycle sequencing, respectively, is a difference in incorporation of nucleotides in the primer extension leading to different sequence profiles. In Taq‐cycling sequencing variable nucleotide incorporation results in irregular, but reproducible peak patterns, whereas Sequenase® incorporates nucleotides in nearly equal amounts, resulting in more even peak patterns. In a previously published multi‐center study we evaluated HLA‐DPB1 SBT using Taq‐cycle sequencing, and showed that typing can reliably be performed, considering the specific sequence profiles. In this study the applicability of Sequenase® for HLA‐DPB1 SBT was tested. A panel of samples were typed by SBT at five test sites which participate in the Sequencing Based Typing component of the 12thInternational Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. The assignment of homozygosity and heterozygosity was validated by Multi‐Sequence Analysis, performing cluster analysis of chromatographic data of all sequences at each position. Sequence characteristics were examined and considered for appropriate assignment. Data reveals that Sequenase® sequencing can also reliably be used

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02516.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02517.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

ISSN:0001-2815    

DOI:10.1111/j.1399-0039.1996.tb02518.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY