Definition of glomerular antigens by monoclonal antibodies produced against a human glomerular membrane fraction.

Experimental animal models of glomerulonephritis (GN) produced by direct antibody binding to non-basement membrane glomerular capillary wall antigens do not to date have human parallels. To examine the potential for this form of humoral glomerular injury in man, we sought to define discrete human non-GBM glomerular antigenic targets using hybridoma technology. Mice were immunised intraperitoneally with 20-100 micrograms of a human glomerular membrane fraction (HGMF). Six fusions have yielded 12 stable reagents defined by positive glomerular indirect immunofluorescence (IF) and microELISA using HGMF as the screening antigen. Subclass analysis of ascitic McAbs indicated several IgG1, one IgG2b, and three IgM reagents. Distinctive IF patterns of reactivity with epithelial, endothelial or mesangial structures have been observed, with or without peritubular capillary, tubular basement membrane and vessel wall reactivity. Seven normal non-renal human organs and the kidneys of rat, rabbit and sheep have shown patterns characteristic of each individual McAb, restricted to human or with species cross reactivity. To partially characterise McAb-reactive antigens, detergent-solubilised renal cortex and collagenase-solubilised GBM (CS-GBM) extracts have been probed by immunoblot. A unique McAb 7-5Q, reactive with glomerular and tubular epithelial structures, binds major bands of approximately 107 KD and 93 KD in detergent solubilised cortex and a single band of similar size by immunoprecipitation (110 KD). 5-3A (a human-restricted linear-reacting McAb) binds bands of 20-200 KD (major band 58 KD) in CS-GBM. In conclusion, distinct species-restricted and more broadly disposed glomerular epitopes are definable in man by McAbs and are potential targets for humoral injury. Purification of these antigens will allow assay for circulating putative nephritogenic auto-antibody and potentially, McAbs may be useful in screening urine for evidence of occult structural renal disease.

Monoclonal antibodies to crosslinked fibrin degradation products (XL-FDP). I. Characterization and preliminary evaluation in plasma.

Monoclonal antibodies (mabs) were raised against X-oligomers, the earliest soluble fragments released from crosslinked fibrin (XL-FN), by the action of plasmin. Two of the mabs (NIBn 52 and NIBn 123) were monospecific for X-oligomers in that they showed no binding to fibrinogen, the plasmic fragments of fibrinogen (D and E) and non-crosslinked fibrin (X, Y, D and E), or the terminal digestion product of XL-FN, fragment DD-E. One other mab (NIBn 178) was panspecific for X-oligomers in that it exhibited a weak affinity for fibrinogen. The mabs were used to develop a two-site immunoradiometric assay (IRMA) and an enzyme-linked immunospecific assay (ELISA) which permitted the specific measurement of X-oligomers directly in plasma, rather than in serum. This immunoassay is a true assay of fibrinolysis as distinct from fibrinogenolysis and may be a potential aid in the diagnosis and evaluation of thrombosis. In preliminary studies, the assay detected low levels of X-oligomers in normal plasma and elevated levels in patients with disseminated intravascular coagulation.

Monoclonal antibodies to crosslinked fibrin degradation products (XL-FDP). II. Evaluation in a variety of clinical conditions.

Plasmas from patients with a wide variety of thrombotic and presumed prethrombotic conditions were examined for high molecular weight crosslinked fibrin degradation products (known as X-oligomers) using a two-site enzyme-linked immunospecific assay (ELISA). This assay employed a catcher-tag principle using two monoclonal antibodies (mabs) directed towards different epitopes on the complex X-oligomer fraction. In general, thrombotic events (pulmonary embolism, PE, myocardial infarction, MI, peripheral vascular disease, PVD, and disseminated intravascular coagulation, DIC) were accompanied by elevated levels of X-oligomers in the plasma. During pregnancy the value of X-oligomer assays was demonstrated to be a clear-cut marker for pre-eclampsia. Patients following a variety of forms of surgery present with heterogeneous plasma levels of X-oligomers and this may merely reflect the formation and lysis of the fibrin formed during and after surgery. The possible value of this ELISA procedure in monitoring thrombolytic therapy is discussed with a critical analysis of the data presented herein. While the assay of X-oligomer was demonstrated to be a valuable marker of fibrinolysis in plasma, more extensive data are required in order to assess whether such an assay is of diagnostic value in thrombosis-related conditions.

The use of monoclonal antibodies raised against a human IgE myeloma paraprotein for the study of allergen extracts and sera from allergic patients.

Murine monoclonal antibodies have been produced against a single human IgE myeloma preparation. A single fusion yielded six different monoclonal antibodies which bound strongly to the immunogen. Two of these failed to react with 'normal' human IgE or with a second IgE paraprotein. The other four antibodies reacted with normal and myeloma-derived IgE and were found to be specific for antibodies of this class. Two of these antibodies were potent reagents for the detection of IgE by two site immunoradiometric assay, immunoblotting, and radioallergosorbent test. The other two antibodies were considerably less potent reagents when used in these assay systems. Our findings suggest that care should be taken in selection of suitable monoclonal antibodies for immunochemical study of allergens and sera from allergic patients.

Structural differences around hormonogenic sites on thyroglobulins from different species detected by monoclonal antibodies.

Thyroxine remains attached to its synthetic site in thyroglobulin until it is released by proteolysis. Strong homology in the primary sequence surrounding thyroxine-forming residues in thyroglobulins from various species suggests a unique three-dimensional structure at hormonogenic sites. To examine this, two thyroxine-binding mouse anti-(chicken thyroglobulin) monoclonal antibodies, 1A10 and 5F6, were used as probes for this region in an enzyme-linked immunosorbent inhibition assay. The thyroxine content of thyroglobulins had a marked positive influence on the monoclonal antibody binding: when the thyroxine content of human thyroglobulin rose by 6.6-fold, cross-reactivities rose 25-fold for the 1A10 monoclonal antibody and 17.6-fold for the 5F6 monoclonal antibody. However, interspecies comparison of thyroglobulin preparations with similar thyroxine content showed lower than expected cross-reactivities for human, pig and sheep thyroglobulins when compared with chicken thyroglobulin. Only when the thyroxine content of heterologous thyroglobulin preparations was two or three times higher did the cross-reactivities equal or surpass that of chicken thyroglobulin. It is concluded that in thyroglobulin there are structural differences in the different animal species near the thyroxine-forming sites bound by these monoclonal antibodies. The known primary sequence similarity does not seem to result, therefore, in identical three-dimensional structures about this site. These differences may reflect species-specific variations in distant regions brought close as a result of chain folding to form the hormonogenic site, such as those around the donor diiodotyrosine residue or in polysaccharide structures. These monoclonal antibodies provide information about the structure of thyroglobulin, which cannot be obtained from knowledge of the amino acid sequence alone.

Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex.

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin. Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.

The effect of primary immunization and concanavalin A on the production of monoclonal natural antibodies.

Hybridomas derived from mice sham-immunized with saline were found to secrete 'natural' antibodies with a wide range of specificities. A high response to bovine serum albumin (BSA) and in particular BSA conjugated with oxazolone was observed routinely. The oxazolone-BSA response was probably directed toward antigenic sites exposed as a result of the coupling procedure; only 2% of the oxazolone-BSA-binding supernatants also bound to oxazolone-ovalbumin. An unexpected cross-reactivity was seen between the oxazolone-BSA-binding supernatants and an 18-amino-acid peptide that forms part of the VP1 capsid protein of poliovirus serotype 3. Some supernatants were also found to react with all proteins tested, including the synthetic poliovirus peptide; this reactivity was maintained following cloning. Primary immunization resulting in the generation of antibodies to the injected antigen nevertheless had no effect on the repertoire of 'natural' antibodies produced. Injection of the T-cell mitogen concanavalin A increased the frequency of 'natural' antibodies. This effect was enhanced when ConA was injected together with antigen.

Interleukin-2 in vivo: production of and response to interleukin-2 in lymphoid organs undergoing a primary immune response to heterologous erythrocytes.

In this report, we describe experiments which demonstrate that antigenic stimulation in vivo causes the appearance of cells in both spleen and lymph node which secrete interleukin-2 (IL-2). Cells also appear in these organs which proliferate in response to IL-2. For these experiments, sheep red cells (SRBC) were injected into the spleens or footpads of mice, and cell suspensions from spleens or popliteal lymph nodes prepared at various times after antigenic stimulation. These cells were assayed for their ability to respond to IL-2, and their cell culture supernatants for secreted IL-2. The proliferative response to IL-2 steadily increased following SRBC injection to reach a peak at Day 2 for spleen cells and at Day 3 for lymph node cells. Maximal production of IL-2 was displaced from the maximal response to the lymphokine by peaking one day later for both organs. Our results strongly implicate the participation of IL-2 in the in vivo immune response and suggest the existence of in vivo regulatory mechanisms, which can control the time of IL-2 production and also the appearance of cells with receptors for IL-2.

Single shot intrasplenic immunization: an advantageous procedure for production of monoclonal antibodies specific for human fibrin fragments.

We describe the use of the single shot intrasplenic immunization technique as a particularly effective procedure for the production of specific monoclonal antibodies against a high molecular weight antigen. We found that with this technique several different, completely specific monoclonal antibodies could be produced against high molecular weight crosslinked fibrin degradation products. These results contrasted with those obtained using conventional multidose immunization, which only produced monoclonal antibodies that were cross-reactive with fibrinogen and/or noncrosslinked fibrin degradation products.