Monoclonal antibodies to bovine serum albumin: affinity and specificity determinations

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.     

Biological and Chemical Characterization of Endotoxin from Capnocytophaga sputigena

An endotoxin was isolated from Capnocytophaga sputigena strain 4 by a modification of the hot phenol-water method. The extraction procedure yielded a lipopolysaccharide which accounted for approximately 1.5% of the dry weight of the cells. The material was composed of 18.6% lipid (as C₁₅ fatty acid), 46.5% neutral sugar including 9.6% hexose, 18.3% 6-deoxy sugar, 1.0% 2-keto-3-deoxy sugar, and 4.8% heptose. Hexosamine, protein, and phosphorus were found in quantities amounting to 9.0, 2.9, and 2.0% of the dry weight, respectively. No pentose or nucleic acid was detected. Acid hydrolysis resulted in the release of the constituent sugars and the formation of an insoluble precipitate. The lipopolysaccharide was tested for numerous biological activities characteristic of endotoxins. The pyrogenicity was relatively low; the fever index 40 was 17 μg, and 10 μg was required to give the characteristic biphasic fever response. The toxicity of the extract was very low, with a 50% chicken embryo lethal dose of 15.6 μg and a 50% mouse embryo lethal dose of greater than 8 mg. Similarly, the C. sputigena endotoxin had modest effects on leukocytes when compared with endotoxin standards from other organisms. The extract exhibited little or no mitogenicity when tested on mouse spleen lymphocytes. It was not toxic to human peripheral polymorphonuclear leukocytes and caused the release of only a small (13%) portion of lysosomal enzymes. Although the C. sputigena lipopolysaccharide caused significant activation of mouse peritoneal macrophages, the dose required was twice that of an Escherichia coli endotoxic standard. However, the Limulus amoebocyte lysate clotting activity of the lipopolysaccharide was comparable to that of an Serratia marcescens lipopolysaccharide standard, and passive hemagglutination tests revealed that 1 μg of the lipopolysaccharide was capable of sensitizing 1 ml of a 2% sheep erythrocyte suspension for agglutination with an antiserum prepared against C. sputigena whole cells.     

Evaluation of the single radial haemolysis (SRH) technique for rubella antibody measurement.

Sera from 1258 individuals have been tested by four laboratories for rubella antibody by both the haemagglutination-inhibition and single radial haemolysis techniques. There was good agreement between the results obtained by the two methods. Although sheep red blood cells were used in the single radial haemolysis plates, no problems were encountered with sera from patients with infectious mononucleosis. The single haemolysis technique was found to be simple, convenient, and reliable, and suited to the rapid screening of large numbers of sera to assess susceptibility to rubella in the context of a vaccination campaign. However, since the technique does not detect anti-rubella IgM, it should not be used as the only test to investigate suspected recent infection.     

Diagnosis of cytomegalovirus pneumonitis on bronchoalveolar lavage fluid: comparison of cytology, immunofluorescence, and in situ hybridization with viral isolation.

Forty-three bronchoalveolar lavage (BAL) specimens from 40 immunocompromised patients were studied for the presence of cytomegalovirus (CMV) by rapid diagnostic methods. DNA in situ hybridization, cytology, and immunofluorescence were compared to conventional cell culture. Eleven (25%) of the 43 BAL samples grew CMV in culture. In situ hybridization detected 6 of these 11 for sensitivity, specificity, and predictive values of positive and negative of 55%, 94%, 75%, and 86%, respectively. Cytology had a sensitivity of 73% and specificity of 100%. Six Papanicolaou-stained cytospins were screened cytologically versus one hybridization cytospin, and the higher sensitivity of cytology may reflect this extensive sampling. The immunofluorescent method had a sensitivity equal to that of cytology (73%): however, the specificity (72%) was significantly less than that of either the probe or cytology. These data suggest that although in situ hybridization can be a rapid, useful method for detecting CMV in BAL specimens, cytology appears to be a more sensitive method.     

CD4 monoclonal antibody pairs for immunosuppression and tolerance induction

A pair of rat anti-mouse CD4 monoclonal antibodies (mAb) have been selected which bind to different epitopes of the molecule. Both the mAb are rat IgG2b and show clear synergistic activity in complement lysis in vitro. When injected together in vivo, they exhibit an improved immunosuppressive effect, compared to each antibody alone, on allogeneic graft rejection, humoral responses and on tolerance induction. Limiting dilution analysis indicates that the in vivo depletion of interleukin 2-producing cells is improved using both mAb by 2-3-fold over that obtained with the individual antibodies. As little as 60 ng per mouse of the CD4 antibody pair was sufficient to allow the induction of tolerance to human gamma-globulin, even without elimination of the CD4+ cells. The results suggest that appropriate antibody pairs may be good candidates for effective immunosuppressive serotherapy in man.     

HLA-B*35-Restricted CD8+-T-Cell Epitope in Mycobacterium tuberculosis Rv2903c

Few human CD8(+) T-cell epitopes in mycobacterial antigens have been described to date. Here we have identified a novel HLA-B*35-restricted CD8(+) T-cell epitope in Mycobacterium tuberculosis Rv2903c based on a reverse immunogenetics approach. Peptide-specific CD8 T cells were able to kill M. tuberculosis-infected macrophages and produce gamma interferon and tumor necrosis factor alpha.     

LIN-39/Hox triggers cell division and represses EFF-1/fusogen-dependent vulval cell fusion

General mechanisms by which Hox genes establish cell fates are known. However, a few Hox effectors mediating cell behaviors have been identified. Here we found the first effector of LIN-39/HoxD4/Dfd in Caenorhabditis elegans. In specific vulval precursor cells (VPCs), LIN-39 represses early and late expression of EFF-1, a membrane protein essential for cell fusion. Repression of eff-1 is also achieved by the activity of CEH-20/Exd/Pbx, a known cofactor of Hox proteins. Unfused VPCs in lin-39(-);eff-1(-) double mutants fail to divide but migrate, executing vulval fates. Thus, lin-39 is essential for inhibition of EFF-1-dependent cell fusion and stimulation of cell proliferation during vulva formation. Supplemental material is available at http://www.genesdev.org.