Antibody-mediated activation of T cell clones as a method for screening hybridomas producing antibodies to the T cell receptor

In this study, supernatants (SN) of hybridomas established by fusing P3X63.Ag8.653 to spleen cells from C57L mice (Vß8 family of T cell receptor (TcR) gene negative) immunized with the H-Y specific Vß8 allotype positive helper T cell (HTL) clone OI6 were screened for the capacity to activate cloned T cells in the absence of interacting stimulator cells. In the first assay, SNs were mixed with Vß8⁺ H-Y specific CTL OH2 and ⁵¹Cr-labelled non-specific B lymphoma (L10.A). In this system, antibodies (Ab) which can bind to L10.A by Fc-Fc receptor interaction and recognize TcR can facilitate lysis of L10.A target cells by OH2 CTL. In the second assay, OI6 clone cells were cultured in microtiter well, previously coated with hybridoma supernatants (SN). In this assay, Ab recognizing OI6 TcR complex and bound to plastic plates can stimulate OI6 cells to proliferate in the absence of stimulator cells. Using these two screening methods, nine hybridomas were established. Analysis of these hybridoma SN using surface staining, inhibition of T cell function and immunoprecipitation of radiolabelled surface molecules followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) showed that five Ab were directed to the allotypic determinant (Vß8) of TcR and four Ab were specific to the clonotypic determinant of OI6 TcR. These results suggest that this Ab-mediated activation of T cell clone can be used for the screening of hybridomas secreting anti-TcR Ab and the immunogenicity of OI6 clonotypic determinants is similar to that of the Vß8 allotypic determinant even in strains which do not express the Vß8 TcR allotype.     

An ELISA method for the quantification of anti-streptolysin-O antibodies

This paper describes a new assay, based on the ELISA technique, for the quantification of antibodies to streptolysin-O (ASLO). We have compared its performances with that of a standard method (inhibition of hemolysis). Using a panel of 137 sera covering the whole range of ASLO titers, the results showed a good correlation between both methods but the ELISA method was more reproducible than the standard technique, thus represents a convenient alternative for the quantification of ASLO.     

An ELISA for the detection of anti-acetylcholine receptor antibodies using biotinylated alpha-bungarotoxin

An antibody-capture enzyme immunoassay has been developed for the detection of anti-acetylcholine receptor (AChR) antibodies in tissue culture supernatants using biotinylated alpha-bungarotoxin (B alpha BGT). Immunoglobulins in culture supernatants were bound indirectly to microtitre plates via an anti-globulin antibody already coupled to polyvinyl plates. Anti-AChR antibodies were then detected by incubation with AChR crude extract. Bound AChR was revealed by incubation with B alpha BGT followed by horseradish peroxidase-conjugated avidin. This assay is specific, more sensitive than the commonly used double antibody radioimmunoassay, avoids the use of radioactive material, is practical for large numbers of samples and is particularly suitable for detecting anti-AChR antibodies in tissue culture supernatants.     

An evolutionary and structural perspective on T cell antigen receptor function

After a brief overview of the themes and variations that occur in the family of receptors containing immunoreceptor tyrosine‐based activation motifs (ITAMs), and of recent structural data on the ligand‐binding subunits of these receptors, we use these data to revisit how information on the state and quality of occupancy of the binding site of the T cell antigen receptor (TCR) is conveyed to the proximal components of the TCR transduction cassette.     

Insulin-specific T cell help to TNP-specific B cells requires activation via the antigen T cell receptor

In the present study, the production of large numbers of insulin (Ins)-specific, H-2-restricted T helper (Th) cell clones is described. Among 148 clones analyzed, 121 clones had Th cell function, and these were divided into 75 Ins-specific Th cell clones and 52 autoreactive clones. The 148 clones were isolated from Ins-specific T cell lines produced by in vitro stimulation of T lymphocytes from (b X d)F1 mice immunized with Ins 7, 14, 32 or 56 days before. The following characteristics were tested with regard to the Th cell clones: restriction specificity and antigen requirement for optimal help or interleukin 2 (IL2) production. No differences in these characteristics were found among clones originating from day 7, 14, 32 or 56 T cell lines. A preference for H-2b as restriction element and an antigen concentration of about 0.01 microgram trinitrophenylated (TNP)-Ins/ml for optimal help were general traits. Optimal IL2 release is not yet obtained with 100 micrograms TNP-Ins/ml. Thus, the antigen requirement for optimal help and IL2 release differs by a factor 10(4) at least. Certain twice-cloned Th cell lines were tested for IL2 production when stimulated with anti-Thy-1 monoclonal antibodies (mAb). All clones analyzed were stimulated by mAb H155 but not with mAb H140 nor with mAb against Lyt-1, L3T4, LFA-1 or H-2K/D molecules. Therefore, we determined whether TNP-conjugated H155 mAb would mediate Th cell-B cell collaboration as well as TNP-Ins. The results with nine different Th cell clones and six different TNP-conjugated mAb used in a 10(7)-fold concentration range showed that Th cell clones have to be triggered via the T cell receptor for expression of helper function to B cells. Thus, though IL2 gene activation, synthesis or release apparently can be activated via at least two pathways: T cell receptor or Thy-1, it seems that activation of the genes responsible for synthesis and release of the helper factors, which ensure antigen-specific B cell proliferation and differentiation, needs Th cell-B contact mediated via the antigen-specific T cell receptor.     

An ELISA method for quantification of Tamm-Horsfall protein using monoclonal antibodies

Eight hybridoma cell lines secreting monoclonal antibodies (MoAbs) to Tamm-Horsfall protein (THP) were established. The isotype and reaction pattern of the MoAbs with THP from rat, rabbit, guinea pig and man were employed for the selection of clones. At least four epitopes were recognised on human THP. One of these epitopes differed from the others in its dependence on the state of aggregation of the THP. An ELISA procedure was developed for quantification of THP in urine requiring no other sample treatment than dilution in the assay buffer. In this ELISA, THP showed an increased immunoreactivity after freezing.     

ELISA is the superior method for detecting antineutrophil cytoplasmic antibodies in the diagnosis of systemic necrotising vasculitis

BACKGROUND: Antineutrophil cytoplasmic antibodies (ANCA) have been used as a diagnostic marker for systemic necrotising vasculitis, a disease classification which includes Wegener granulomatosis, microscopic and classic polyarteritis nodosa, and Churg Strauss disease. OBJECTIVE: To compare the diagnostic value of the two methods for detecting these antibodies--immunofluorescence and enzyme linked immunosorbent assay (ELISA)--with respect to biopsy proven active systemic necrotising vasculitis in a clinically relevant population. METHODS: A prospective study to ascertain the patient's diagnosis at the time of each of the 466 requests for ANCA received at one laboratory over a nine month period, and allocate each to one of five diagnostic groups: active and inactive biopsy proven systemic necrotising vasculitis, suspected systemic necrotising vasculitis, low probability systemic necrotising vasculitis, and not systemic necrotising vasculitis. RESULTS: ELISA was superior to immunofluorescence in the diagnosis of systemic necrotising vasculitis because it was less likely to detect other diseases. This was reflected in its specificity of 97% and positive predictive value of 73%, compared with 90% and only 50% for immunofluorescence (p = 0.0006 and p = 0.013, respectively). ELISA had a negative predictive value of 98% which was not significantly different to immunofluorescence. ELISA was technically superior. CONCLUSIONS: ELISA is the superior method of ANCA detection in the diagnosis of systemic necrotising vasculitis and should be used in conjunction with a compatible clinical picture and histological evidence.     

A stable haemagglutinating antigen for detecting toxoplasma antibodies

The production of a toxoplasma-sensitized cell preparation stable for at least one year at 5 degrees C and its performance in the diagnosis of toxoplasmosis is described.     

A human T cell tumor which expresses the putative T cell antigen receptor

A monoclonal antibody (mAb) called H1-2D4 which reacts only with the T cell line HPB-ALL and not with other T cell lines, normal or activated peripheral blood cells, B lymphoblasts (B-LCL), or thymocytes has been developed. This mAb cocaps the T3 antigen on HPB-ALL and anti-T3 mAb cocaps the antigen which reacts with H1-2D4. Purified H1-2D4 precipitates a heterodimer from HPB-ALL cells which has components with molecular weights of 51000 and 39000. The properties of the molecule recognized by H1-2D4 suggest that it is the HPB-ALL equivalent of the putative human T cell antigen receptor.     

A simple method for detecting antibodies to rubella.

A simple microplate method of enzyme linked immunosorbent assay for Rubella antibody is described. This Micro-ELISA was compared with haemagglutination inhibition in a study of 188 human sera. The total discrepancy rate between the two tests was only 3-7%.     

Signal transduction by the T cell antigen receptor.

The T cell antigen receptor (TCR) must recognize antigen, and translate this recognition event into intracellular signal transduction events. Two signal transduction events are regulated by the TCR: the activation of a protein tyrosine kinase (PTK) and phospholipase C (PLC). Recent studies suggest that the TCR-activated PTK regulates PLC activation by the phosphorylation of tyrosine residues of PLC gamma 1. The complex structure of the TCR is now being related to its signal transduction function. Studies with chimeric receptors reveal that the antigen binding Ti heterodimer communicates with the subunits involved with signal transduction, the CD3 chains and zeta dimers, through the carboxy-terminal regions of the Ti chains that surround and include the transmembrane domains. Other chimeras have helped demonstrate that the zeta chain family of dimers function to couple the TCR to intracellular signal transduction mechanisms. The signal transduction function of the TCR can be regulated in a number of ways and by other T cell surface molecules. The plasma membrane tyrosine phosphatase CD45, plays a critical role to specifically regulate TCR-mediated activation of PTK's and PLC. Thus, an understanding of the complex structure of the TCR and the intricacies of its signal transduction function is rapidly emerging.     

An ELISA for detecting pestivirus antigen in the blood of sheep persistently infected with border disease virus

A monoclonal antibody capture enzyme-linked immunosorbent assay (ELISA) has been developed to detect a pestivirus-specific antigen in leucocytes of sheep persistently infected with border disease virus. A blind trial was conducted to compare the specificity of the ELISA with conventional tissue culture virus isolation on blood samples from 58 sheep, aged 3 to 48 months. There was total agreement between the two tests; 27 sheep were shown to be BDV-infected. The ELISA OD values of the positive samples ranged from 0.12 to 0.86 and were not related to age, strain of virus with which they were infected or presence of serum neutralising antibody. Negative samples had OD values between 0 and 0.02.     

An 'antigen capture' ELISA for secretory immunoglobulin A antibodies to hepatitis B surface antigen in human saliva

An enzyme-linked immunosorbent assay (ELISA) was developed which permits the assay of specific secretory immunoglobulin A (sIgA) antibodies against hepatitis B surface antigen (HBsAg) in saliva. The assay is based on the binding of sIgA antibodies present in saliva to microtitre plates coated with excess of F(ab')2 anti-secretory component antibodies, followed by the addition of specific antigen, HBsAg and finally peroxidase-labelled anti-HBsAg. The assay is fast, simple, reproducible and antigen specific as shown by total absence of inhibition of specific antigen by unrelated antigens but significant inhibition of labelled anti-HBsAg by unlabelled anti-HBsAg. The values obtained for hospital personnel exposed to hepatitis infections (0.068 +/- 0.083 U/ml) and for post-icteric hepatitis B patients (0.062 +/- 0.033 U/ml) were significantly higher than values in control subjects (0.013 +/- 0.006 U/ml).     

Requirement of the T cell antigen receptor occupancy for the target cell lysis by cytolytic T lymphocytes

We have constructed a bivalent bifunctional F(ab)2 fragment with binding specificity for a V beta 8 T cell antigen receptor and human tumor-associated antigen. Using the bifunctional antibody to focus cytolytic activity of mouse CTL to a human carcinoma cell line and anti-V beta 8 TCR Fab' as a competitor, we demonstrate that only a small percentage (-0.5%) of the TCR engaging on the target molecule is sufficient to deliver a lytic signal to the target cells.     

Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton

T cell receptor (TCR) engagement leads to actin polymerization at the site of T cell contact with antigen-presenting cells. Here we have studied the dynamic activity of proteins involved in regulating actin polymerization in live T cells after activation. Two such adaptor proteins, Nck and the Wiskott-Aldrich syndrome protein (WASp), were recruited to the TCR during initial T cell activation, where they colocalized with the tyrosine kinase Zap70. The recruitment of Nck and WASp depended on TCR-induced tyrosine phosphorylation and the LAT and SLP-76 adaptors. Nck and WASp migrated peripherally and accumulated at an actin-rich circumferential ring. Thus, actin polymerization regulated by the TCR begins at the TCR. Molecules recruited to the TCR regulate actin polymerization and this process drives plasma membrane movement and cellular spreading.