The production of relatively large amounts of pure monoclonal antibodies (MAb) has facilitated the development of MAb-based immunometric assays for a variety of clinically important analytes. 'Two-site' heterogeneous assays are now available which incorporate a labelled MAb and a second, different MAb coupled to a solid support. These assays possess certain advantages over the corresponding immunoassays including speed, precision, working range and specificity. They are largely dependent on two specific molecular interactions (labelled MAb-antigen; antigen--solid support MAb) and thus one might expect them to be particularly sensitive to assay conditions. With reference to two MAb--based two-site immunoradiometric assays (for human growth hormone and prolactin) which are being developed in this laboratory we wish to report the effect of various conditions including pH, ionic strength, buffer species etc. on assay response in order to emphasize the need for careful optimisation of monoclonal antibody based assays.
Abstract. A hybridoma (ES‐15) was obtained by fusing the NS‐1 cell line with spleen cells from a mouse immunised with soluble blood group A 2 substance. The cloned hybridoma culture supernatant was shown to contain an IgM class antibody which strongly agglutinates group A cells and weakly agglutinates group B cells. The serological specificity of this antibody is described as anti‐A,(B) in this report. The abilities of unconcentrated monoclonal anti‐A,(B), a commercial human polyclonal anti‐A,B (group O serum) and a commercial monoclonal anti‐A reagent to detect 15 examples of A x cells were compared by both slide and tube techniques. Using a slide technique monoclonal anti‐A,(B) agglutinated 14 examples of A x cells, human anti‐A,B 2 examples, while monoclonal anti‐A failed to detect any of the A x cells tested. Similar differences in the reactivity of the three antibodies were observed using a tube technique. Data are also presented which show that a 1:1 (v/v) mixture of monoclonal anti‐A,(B) with a monoclonal anti‐B reagent is an effective replacement for human anti‐A,B in ABO grouping procedures.
Balb/c mice were immunized with human blood-group A2 active cyst fluid glycoprotein. Fusion of spleen cells with NS-1 myeloma cell line produced a total of 11 blood group antibody secreting hybridomas of which seven were apparently specific for blood-group A and were subjected to further evaluation. Of these 2 were IgM class and 5 IgG class. Two anti-A supernatants showed a significant decrease in avidity time against A2B cells when ionic strength was increased to 0.25. Tube titres of all anti-A supernatants were unaffected by ionic strength. pH had no effect on the avidity time or tube titre of any of the antibodies tested. The supernatant from one hybrid (ES-9) was selected for further evaluation as a blood grouping reagent. This supernatant had a tube titre of 1:128 and was used without concentration for comparison of its serological reactivity with two examples of commercial monoclonal anti-A and one example of human anti-A. Monoclonal anti-A (ES-9) was found to be a potentially useful red cell grouping reagent.
A tissue culture system using chick embryo cells gave bacteria-free vaccinia virus suspensions of sufficient potency to use as a vaccine. Clinical trials with vaccines prepared by this method gave similar results to those with sheep lymph vaccine.
Hybridoma cells secreting anti-PAP were produced by fusion of NS-1 myeloma cells with spleen cells from immunized Balb/c mice. Three of 32 hybrids secreted antibodies. Dilution cloning of the two hybrids producing the highest antibody titres showed that each antibody was monoclonal. One clone from each hybrid (clones ES2 and ES8) was selected for further study. The specificity of the antibodies appeared satisfactory, no inhibition of 125I-PAP binding to antibody being seen with extracts of bone, intestine, kidney, leucocytes, liver or lung. The association constants of the antibodies from ES2 and ES8 were 1.7 X 10(8) and 1.7 X 10(9) l/mol respectively, both were of the IgG1 class. For the immunoradiometric (IRMA) assay of serum PAP 125I-labelled monoclonal antibody was incubated with serum and the PAP-labelled antibody complex was separated by addition of solid-coupled polyclonal anti-PAP. The wide working range of the response curve (0.3-400 micrograms/l) and the rapid analysis time (4 h) offer practical advantages over RIA procedures. Clinical evaluation of the assay is in progress. ES8 antibody also appears to have good specificity for immunocytochemical applications. Localisation of micrometastases in bone in prostatic carcinoma was readily achieved.
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