Production and characterisation of mouse monoclonal antibodies reactive with the lipopolysaccharide core of Pseudomonas aeruginosa.

Monoclonal antibodies (MAbs) to the core antigen region of lipopolysaccharide (LPS) of Pseudomonas aeruginosa were produced from mice immunised with whole cells of heat-killed rough mutants of Pseudomonas aeruginosa expressing partial or complete core LPS. MAbs were screened in an enzyme-linked immunosorbent assay (ELISA) against three different antigen cocktails: S-form LPS from three P. aeruginosa strains, R-form LPS from six P. aeruginosa strains and, as a negative control, R-form LPS from Salmonella typhimurium and Escherichia coli. Selected MAbs were subsequently screened against a range of extracted LPS and whole cells from both reference strains and clinical isolates of P. aeruginosa. The antibodies were also screened in ELISA against whole-cell antigens from other Pseudomonas spp. as well as strains of Haemophilus influenzae, Neisseria subflava and Staphylococcus aureus. Five MAbs reacting with the core component of P. aeruginosa LPS were finally selected. Two of these, MAbs 360.7 and 304.1.4, were particularly reactive in immunoblots against unsubstituted core LPS, including that from O-antigenic serotypes of P. aeruginosa. The MAbs also reacted with some of the other Pseudomonas spp., but not with P. cepacia or Xanthomonas (Pseudomonas) maltophilia. Cross-reactivity with whole cells from other bacterial species was minimal or not observed. Reactivity of MAbs with some Staph. aureus strains was observed, and binding to the protein A component was implicated. The reactivity of the MAbs was investigated further by flow cytometry and immunogold electronmicroscopy. The suitability of the MAbs for an immunological assay for detection of P. aeruginosa in respiratory secretions from CF patients is discussed.

Human atrial natriuretic factor (ANF). Characterisation of a monoclonal antibody panel and its use in radioimmunoassay.

The production of nine monoclonal antibodies to human atrial natriuretic factor (ANF 1-28) is described. All possible combinations of two antibodies failed to reveal any which could simultaneously bind ANF. Studies with ANF analogues and the antibodies having the three highest affinity values (KD = 5, 25 and 21 pM) indicated that the antibodies are directed to the central portion of the antigen molecule. The highest affinity antibody was able to replace polyclonal antisera in the radioimmunoassay of ANF in extracts of plasma.

Stability of murine monoclonal anti-A, anti-B and anti-A,B ABO grouping reagents and a multi-centre evaluation of their performance in routine use.

We have previously reported the production of 3 murine monoclonal reagents for ABO typing (designated ES-9, ES-4 and ES-15). This study presents results of tests of stability of these 3 reagents, together with a fourth murine monoclonal antibody (LM103/107). In addition, data are also presented from a multi-centre evaluation of the performance of the murine monoclonal reagents in routine ABO typing of both donors and patients using a wide variety of techniques, both manual and automated. The potency and stability of the 4 monoclonal antibody based reagents is compared with a broad selection of monoclonal and polyclonal ABO typing reagents. The reagents used for comparison were produced by European and United States manufacturers in both the public and private sector and are widely used in routine ABO typing. The Scottish monoclonal reagents have been used successfully to ABO type over 500,000 blood samples in 7 centres within the UK, with no discrepant results.

The use of rat mixed-thymocyte culture-conditioned medium for hybridoma production, cloning and revival.

Allogeneic rat mixed-thymocyte 48 h culture-conditioned medium (MTM) was used successfully in place of feeder cells for hybridoma production with the NS-1 and NS-0 plasmacytoma lines. It permitted lower concentrations of fused cells to be seeded, and supported the transition from 96 to 24 well plates. MTM improved the performance of poor sera during cloning. It also assisted the survival of cells that were sensitive to thawing from liquid nitrogen storage, and cells that had inadvertently been allowed to overgrow. Two rats could produce the equivalent of 1500-5000 ml feeder cell suspension according to the dilution used; 150-500 mice would be required to produce such a quantity of cells. Thus use of MTM entailed a considerable saving in mice and provided a secure supply of 'reagent', since a batch could be prepared, checked for sterility, frozen and stored indefinitely.

Immunoaffinity purification of factor VIII complex.

Murine monoclonal antibodies to human von Willebrand factor (vWf) were immobilised on Sephacryl S-1000. Various solutes were screened for their ability to elute 125I-vWf from the immobilised antibodies. The most effective solutions were then tested to determine which allowed retention of factor VIII procoagulant activity (VIII:C) and activity of vWf measured by platelet aggregation in the presence of ristocetin (Ristocetin cofactor activity R. cof.). Finally, F VIII complex was purified from both plasma and cryoprecipitate by immunoaffinity chromatography under the selected conditions. The product had a specific activity of 45 units of VIII:C per mg of protein and 60 units of R. cof. per mg representing a 4000-fold purification from plasma. The fibrinogen and fibronectin content were each less than 4% of the total protein with vWf accounting for 60% of the total protein in the final product. Multimer analysis of the product showed a similar pattern to normal plasma and contamination by murine monoclonal antibody was less than 300 ng per mg of protein. A novel product is thus obtained containing both clinically relevant VIII:C and R. cof. in a single vial whilst using only one specific monoclonal antibody.

Comparison of radioimmunoassay and immunoradiometric assay for serum prostatic acid phosphatase.

We have compared the laboratory performance of immunoradiometric (IRMA) and radioimmunoassay (RIA) methods developed in this laboratory for measurement of serum prostatic acid phosphatase (PAP). The IRMA utilizes a radiolabelled mouse monoclonal anti-PAP and a solid phased rabbit polyclonal anti-PAP. The same rabbit antibody is used in the RIA. The IRMA shows excellent precision over a much wider working range (0.25-1000 micrograms/l) than the RIA (0.73-14.0 micrograms/l), and can be completed in 5 h, while the RIA requires 3 days. Levels in healthy males and in patients with benign prostatic hypertrophy are similar in both assays, upper limits of normal being 1.8 micrograms/l (IRMA) and 4.7 micrograms/l (RIA). The two assay methods correlate very well (r = 0.97) when PAP is measured in serum from prostatic cancer patients, although IRMA results are generally lower than those obtained by RIA. About 20% of patients with non-metastatic prostatic carcinoma had elevated serum PAP, whereas about 80% of those with metastatic disease had raised levels. The diagnostic efficiencies of the RIA and IRMA appeared similar. The value of the IRMA in follow-up and staging remains to be determined.

The production and characterisation of a panel of ten murine monoclonal antibodies to human procoagulant factor VIII.

A panel of 10 murine monoclonal antibodies to procoagulant FVIII has been developed from the fusion of a single spleen. Balb/c mice were injected with a purified preparation of FVIII: Ag, and antibody production in sera and hybrid culture supernatants was monitored using a specific radiometric screening assay. The antibodies all inhibit FVIII clotting activity in normal plasma, and when immobilised on agarose retain their ability to recognise and bind the FVIII procoagulant protein. Studies on protein A-purified immunoglobulins demonstrate a range of properties within the panel of antibodies with regard to species cross-reactivity, clotting inhibition and immunoadsorption. The panel of antibodies has been used to screen heat-treated FVIII concentrates for the occurrence of heat-induced neoantigens.

Characterisation of mouse monoclonal antibodies produced by immunisation with a single serotype component of a polyvalent Pseudomonas aeruginosa vaccine.

Mouse monoclonal antibodies raised by immunisation with a protective antigen extract from Pseudomonas aeruginosa serotype 1 varied in immunoglobulin isotype, in passive protective properties against infection by homologous P. aeruginosa serotype 1, and in cross-reactions in ELISA against antigen preparations from 15 other P. aeruginosa serotypes. All monoclonal antibodies with specificity in ELISA for the immunising antigen gave some degree of protection to mice against lethal infection by the homologous P. aeruginosa serotype. The IgG antibodies were more protective than the IgM antibodies.

Enhancement of factor VIII-von Willebrand factor ristocetin cofactor activity by monoclonal antibodies.

Five monoclonal antibodies to human von Willebrand factor were selected for characterization from 18 produced in murine hybridomas. All showed a high and specific affinity for human von Willebrand factor (vWf) but exhibited little if any cross-reaction with sera from other species. The antibodies defined four epitopes on vWf, none of which were involved in platelet binding. Binding of two distinct antibodies at one of these epitopes was associated with enhancement of the rate of vWf-dependent platelet agglutination in the presence of ristocetin. This effect was more noticeable when cryosupernatant plasma was used in place of normal plasma as the source of vWf, and was not explicable simply in terms of antibody-induced cross-linking of vWf.

Characterisation of epitopes on human tissue plasminogen activator recognised by a group of monoclonal antibodies.

Seven mouse monoclonal antibodies have been produced against human melanoma tissue plasminogen activator (t-PA). They were specifically bound to 125I t-PA but not 125I urokinase (u-PA) and inhibited t-PA, but not u-PA, activity in plasminogen-rich 125I fibrin wells. Three of the antibodies directly inhibited the amidolytic activity of t-PA and the two most effective also bound near the active site histidine residue as determined by competition experiments using active site blocking agents. Several antibodies interfered with the fibrin binding properties of t-PA. One antibody neither interacted with the active site nor inhibited fibrin binding but still effectively quenched t-PA activity in fibrin wells suggesting that it masks another region of the molecule necessary for effective biological activity.

Monoclonal antibodies directed against human alpha-thrombin and the thrombin-antithrombin III complex.

Human alpha-thrombin was poorly immunogenic in Balb/c mice. Nevertheless, following fusion of spleen cells from a responding mouse with NS-1 cells, 8 mouse monoclonal antibodies against alpha-thrombin were isolated, and 6 were characterised. Five of these were isotype IgG2a, and one was IgG1. One, EST 1, bound thrombin only minimally, and was directed against a neoantigen on the thrombin-ATIII (T-AT) complex. This antibody also recognised a site on prothrombin, though with much lower affinity. Its binding was markedly temperature-dependent, indicating a requirement for molecular mobility. A second antibody, EST 4, would not bind the T-AT complex. It inhibited both the clotting and amidase activities of thrombin, and modification of the active site histidine, but not the active site serine, reduced the affinity constant of binding to EST 4. This antibody appears to be directed against an epitope in the vicinity of the enzyme active site. The epitopes for EST 1 and EST 4 were both remote from those of the other monoclonal antibodies, EST 2, 6, 7 and 8. These four competed with each other for binding to thrombin, and all inhibited clotting but not amidase activity. Thrombin binding was not affected by modification of the active site, though formation of the T-AT complex reduced the affinity of binding to EST 6 and EST 8. These monoclonals recognise epitopes in the region of the fibrinogen binding site.

A monoclonal antibody-based immunoradiometric assay for h-LH.

An immunoradiometric assay (IRMA) for h-LH based upon an 125I-labelled mouse monoclonal antibody (MAb) to h-LH with an effective equilibrium constant of 5.8 X 10(9) l/mol is described. A total incubation time of 3 h at room temperature was required, separation by means of the sucrose layering procedure took a further 1 h and counting times were 1 min/tube. Using the first IRP for h-LH (prep. 68/40) as standard, the detection limit was 0.1 U/l serum and the within-assay CV for duplicate determinations was less than 10% over the range 1-280 U/l and less than 3% at 10-100 U/l. The epitope, with which the MAb reacted, shared structures, on the alpha- and beta-subunits of LH since the assay responded to the intact hormone, but not to either of the subunits. Specificity was greater than 100 000:1 for h-LH vs. h-FSH and greater than 10 000:1 for h-LH vs. h-TSH. h-CG and h-LH were approximately equipotent. The results on 604 unselected samples were generally very similar to those found by RIA except at levels below 2 U/l for which the IRMA regularly gave lower results suggesting relative freedom from non-specific serum effects. The new assay, based upon potentially limitless supplies of a very stable reagent offers advantages of speed, sensitivity, range, and precision over conventional RIA. The specificity appears to be excellent. Although there are marginally more steps the total staff involvement is less than with conventional methods employing centrifugation.

The preparation, characterization and application of monoclonal antibodies to human prostatic acid phosphatase (PAP).

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.

Optimization of assay conditions in monoclonal-based immunoradiometric assay.

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.

Evaluation of monoclonal antibodies to blood group A.

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 monoclonal antibody to human blood group B. Performance, evaluation and optimisation.

A monoclonal anti-B antibody (ES-4) was obtained by fusing spleen cells from a mouse immunized with soluble human blood group B substance with mouse myeloma cell line NS-1. The antibody was shown to agglutinate optimally group B cells at pH 7.2 and 0.15 ionic strength. Increasing the ionic strength to 0.24 gave optimal reactivity over a wider pH range. Culture supernatant containing anti-B (ES-4), after pH and ionic strength adjustment could be used in unconcentrated form as a red cell typing reagent. Anti-B (ES-4) agglutinated five examples of cells of the Bweak (Bw) phenotype and one example of acquired B phenotype. In contrast three of the Bw cells and the acquired B phenotype were not agglutinated by a commercial monoclonal anti-B by a tube technique. The data suggested that the equilibrium constant of the monoclonal anti-B (ES-4) was higher than that of the commercial reagent.

A mouse monoclonal antibody with anti-A,(B) specificity which agglutinates Ax cells.

A hybridoma (ES-15) was obtained by fusing the NS-1 cell line with spleen cells from a mouse immunised with soluble blood group A2 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 Ax cells were compared by both slide and tube techniques. Using a slide technique monoclonal anti-A,(B) agglutinated 14 examples of Ax cells, human anti-A,B 2 examples, while monoclonal anti-A failed to detect any of the Ax 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.