摘要:

The function of MHC class‐I molecules is to sample peptides from the intracellular environment and present them to CD8+cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide‐ß2m‐class‐I complexes a biochemical peptidc‐class‐I binding assay has been generated recently and this paper reports on a similar assay for the interaction between ß2m and class I. As a model system human ß2m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, KD, is close to 0.4 nw. The rate of association at 37 C is very fasi (the kais around 5 × 104/M/s) whereas the dissociation is slow (the kdis around 8 × 10−6/s); the ratio of dissociation to association yields a calculated KDclose to the observed value. At 37° C almost all of the purified class I participates in binding of the exogenously offered ß2m showing that a considerable exchange of the endogenous ß2m occurs. Finally, it was demonstrated that exogenous ß2m enhances binding to MHC class‐I of short perfectly‐matching peplides

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03341.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Recently, a panel of monoclonal antibodies (MoAbs) was developed that identified a novel tumour‐cell antigen conserved across species (mouse, rat and man). Fluorescence‐activated cell sorter (FACS) analysis demonstrated that this antigen was expressed at highest levels on human tumour cell lines sensitive to natural killer (NK)‐cell lysis. These MoAbs inhibited NK cell lysis of K562 target cells (by up to 90%), as well as a variety of other NK‐sensitive target cells. Biochemical analyses revealed that the MoAbs reacted with a polypeptide of 42 kDaitons, distinct from other known cell surface antigens [1]. Now the expression of this antigen has been analysed further with a panel of 24 tumour cell lines to determine its role in NK cell function. The expression of target cell major histocompatibility complex (MHC) molecules in conjunction with sensitivity to NK cell lysis was examined also. For each of the 24 cell lines it was found that the level of expression of the novel target cell antigen determined the sensitivity of the celt line to NK cell lysis. However, the level of MHC antigen expression could modulate target cell sensitivity to NK cell lysis, in that high levels of MHC class‐I molecule expression resulted in a target cell that was insensitive to NK cell lysis regardless of the level of expression of the novel antigen. Thus, for most transformed cell lines, sensitivity to NK cell lysis appeared to be determined by the expression and levels of the novel antigen in association with MHC class‐

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03342.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

This paper describes a procedure for analysing multiple antibody reactivities that explores a commercially available immunoblot system, and is based on a double staining of nitrocellulose membranes, revealing both antibody reactivities and the migration position of the blotted proteins in the membrane. Quantification of both stainings by densitometry allowed the accurate superposition of the immunoreactivity and total protein profiles of each Line. Moreover, the protein stainings ofthe different lanes could be adjusted with a simple‐scale transformation algorithm, correcting for possible distortions during electrophoretic migration, and allowing for the precise comparison ofthe immunoreactivity profiles in different lanes. The procedure is discriminatory enough to identify unique reactivity patterns in random pools of 104activated B cells, and to define strain‐specific natural antibody repertoires. The utilily of this immunoblot method as an assay for simultaneously scoring multiple reactivities to hundreds of antigens in complex mixtures of antibodies, and thus defining antibody repertoires in a global manner, is discus

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03343.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Serum amyloid A (SAA) protein was isolated from acute phase sheep sera by ultracentrifugation, gel filtration and ion‐exchange chromatography. The purified protein was characterized by sodium dodecylsulfate polyacrylamidc gel electrophoresis (SDS‐PAGE), isoelectric focusing, amino acid composition and Edman degradation. Protein SAA sheep consists of 112 amino acid residues and has a blocked N‐terminus. The amino acid sequence showed a high degree of homology with SAA proteins from other species, especially at positions 32 to 54, indicating that this particular part of the protein is important for its function. When compared to human protein SAA, nine inserted amino acids could be demonstrated, located in regions 69 to 77. Similar observations have been seen in cow, horse, dog, cat, and mink protein SAA. Heterogeneities were found in positions 28, 55, 63, 64, 66, 75, 77, 78, 80 and 89. Positions 63, 64, 66, 75, 77, 78 and 80 revealed the existence of a minor gene product of protein SAA sheep. The minor variant of protein SAA sheep is identical in these positions with the corresponding positions in protein SAA cow. By comparing the amino acid sequences of the different SAA proteins, two separate branches in the evolutionary pattern of protein SAA appear. One of the branches includes the species with the insertion which represents also one of the more heterogeneous part of the pr

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03344.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Polymerase chain reaction (PCR) has increased dramatically the speed of cloning and characterizing numerous genes. However, its application to identifying and analysing new germline Ig‐variable (V) gene families has been hampered by the lack of sequence information in the downstream flanking regions of the concerned V genes, which are deleted during V(D)J rearrangements. To circumvent this problem, the possibility was explored that a degenerate downstream primer may be used in conjunction with a specific upstream primer, to clone members of new Vβ gene families, as much less is known about Vλ genes than Vh and Vk genes in humans. Firstly the feasibility and the specificity of a degenerate primer was examined by comparing it with an established downstream primer in amplifying known Vλ1 genes. The results were positive. Thus, the degenerate primer was used to clone and characterize germline Vλ genes of the recently defined Vλ8 and Vλ9 gene families. This current strategy may help speed up the identification and characterization of all human Vλ genes. Moreover, a similar strategy can be applied to identify and characterize rapidly new V genes of either known or unknown Ig and T‐cell receptor V gen

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03345.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

The major histocompatibility complex (MHC) class‐II alleles at the DRBI, DQBl and DPBl loci were investigated in 40 patients with primary biliary cirrhosis (PBC) and 43 local healthy controls. Restriction fragment length polymorphism (RFLP) was used for DRBI typing. DQBl and DPBl regions were amplified using the polymerase chain reaction (PCR) and then probed with32P‐labelled allele‐specific oligonucleotide probes. There was an increased frequency of DR8 (10% compared to 4% in controls), and a decreased frequency of DR2 (18% compared with 28% in controls) in patients with PBC, but the differences were not significant. There were no significant differences for the other DR alleles, the seven DQ alleles, or the eight DP alleles. In conclusion, no significant MHC class‐II associations with primary biliary cirrhosis have been demon

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03346.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

N12.12 is a monoclonal immunoglobulin (Ig) kappa light chain (KLC) secreted by a B‐celI hybridoma derived from spleen cells of a normal SJA mouse. No heavy chain was detected in the culture supernatant of this hybridoma using an enzyme immunoassay (EIA) and after polyacrylamide gel eleclrophoresis (SDS‐PAGE) of the35S‐methionin biosynthetically labelled proteins secreted by the cells. It was shown that NI2. I2 KLC reacted with mouse actin, trinitrophenylated bovine serum album in (TNP25‐BSA) and weakly with bovine myoglobin. The binding of the NI2.12 ‘monoclonal antibody’ to mouse actin or to TNP25‐BSA was inhibited specifically by both antigens with a dissociation constant (KD) for binding to mouse actin of 10−7M. The results indicate that a free KLC can bind both to mouse and to non‐mouse molecules, thus exhibiting binding characteristics usually attributed to natural multire

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03347.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

A novel activation‐dependent lymphocyte cell‐surface antigen which is recognized by a MoAb, BL Ac(F2), is described. Although not found on resting lymphocytes the antigen is induced rapidly wiihin 2–4 h following stimulation of the cells using mitogens or antibodies against the T‐eell CD3 antigen and sIgM on B cells, respectively. Immunopreeipitation and Western Blotting indicated that the MoAb recognizes a molecule in a range of 78 85 kDa. Beyond its activalion‐dependent expression on lymphocytes the antigen was detected also on myelo‐monocytic cells. Expression kinetics and cellular distribution of this molecule suggest that it is distinct from previously described activation‐dependent cell‐surface antigens sueh as CD6

ISSN:0300-9475    

DOI:10.1111/j.1365-3083.1994.tb03348.x

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY