Chemical nature of mouse antibodies homologous to the 3-pyridylazo group: the fine specificities of hybridoma and serum antibodies.

Rabbit antibodies which bind aromatic annular nitrogen-containing haptens exhibit a specificity wherein such nitrogens are distinguished from the closely related aromatic CH group. The mouse hybridoma system was used to extend this work producing hybridoma antibodies homologous to the 3-pyridylazo group. Fine specificity mapping by double antibody radioimmunoassay revealed differences among the individual hybridomas, as well as a greater resemblance of mouse serum antibodies to rabbit serum antibodies than to hybridoma antibodies. Quantitative structure-activity relationships applying the parameters of hapten molar refractivity had hydrophobicity were used to help elucidate the types of intermolecular forces involved in the interaction of pyridine derivatives with the antibodies. The results are consistent with the interpretation that pyridine binding to antibody does not involve desolvation.

Further characterization of two "DR supertypic" specificities. Additional evidence that they reside on molecules different from those carrying HLA-DR locus specificities.

Peripheral lymphocytes from a panel of individuals who had been assayed for DR specificities by the conventional cytotoxicity assay were typed for DR "supertypic" specificities, DC1 and BR4 x 7, by the radioimmunoassay. A positive and a negative population were clearly distinguished for both specificities and the strong association of the DC1 specificity with DR1, 2, and w6 was confirmed as well as the BR4 x 7 specificity with DR4 and 7. Family study also supported this strong association. Appropriate papain digestion separated molecules carrying DC1 determinant from those carrying DR2 as well as from those carrying DRw6, and separated molecules carrying BR4 x 7 from those carrying DR4. Specificity analysis of the 8th Workshop antisera by use of these separated antigen preparations showed that some anti-DC1 antisera do not possess appreciable anti-DR1, 2, or w6 activity and vice versa. The same was found for DR4 x 7 in its relationship with DR4 and 7. The existing evidence could be explained most economically by assuming a genetic model of two loci in linkage disequilibrium each coding for analogous but distinct forms of the small (beta) subunits of Ia molecules.

Experimental anti-alveolar basement membrane antibody-mediated pneumonitis. I. The role of increased permeability of the alveolar capillary wall induced by oxygen.

Attempts to produce in animals lung lesions mediated by anti-alveolar basement membrane (ABM) antibodies have thus far been inconclusive. The aim of the present study was to test the hypothesis that increased permeability of the endothelial cell lining is needed before circulating antibodies can gain access and bind to ABM. A goat antiserum was prepared against purified rabbit ABM. After i.v. injection of the gamma-globulin fraction into rabbits, goat IgG was detected in glomerular basement membrane but not in ABM. However, 17 of 19 rabbits injected with anti-ABM antibody after exposure for 62 to 66 hr to 100% oxygen had a diffuse linear binding of goat IgG in ABM and died with pulmonary edema and hemorrhagic pneumonitis. By the paired label isotope technique, uptake of anti-ABM antibodies in lung far exceeded that in kidney. Semiquantitative histologic studies indicated that the lesions in the lung of these rabbits were more severe then those found in rabbits exposed to 100% oxygen and injected with normal goat serum gamma-globulin. None of the latter animals died with pulmonary edema; none was found to have binding of goat IgG in the lung. The results indicate that under normal physiologic conditions the endothelium is a barrier that prevents binding of IgG antibodies to ABM. The increased permeability induced by oxygen in the alveolar capillary wall is a nonimmunologic factor allowing the development of a reproducible model of severe anti-ABM antibody-mediated pneumonitis.

Succinylation of hapten-protein conjugates facilitates coupling to erythrocytes by water soluble carbodiimide: preparation of stable and sensitive target cells for use in hemolytic assays.

A facile method is described for the preparation of haptenated sheep red blood cells (SRBC) for use as targets in hemolytic spot and plaque assays for the detection of anti-hapten antibody. The method involves the use of the water soluble 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDCI) as a reagent to couple hapten-succinyl-rabbit serum albumin conjugates to SRBC. The presence of the succinyl groups on such conjugates is shown to increase the efficacy of the resulting target cells, presumably by acting as a substrate for the EDCI and thus increasing the extent of coupling to SRBC.

Cells isolated from the embryonic neural retina differ in behavior in vitro and membrane structure.

Several subpopulations of cells were isolated from trypsin-dissociated embryonic (14 days) chick retinas. The cells of each subpopulation differed in associative behavior measured by cell aggregation and stationary culture assays and in glycoproteins that contain glucosamine. Freeze-fracture analysis showed that these populations also differed in intramembrane particle content.

The development and use of radiolabeled antitumor antibodies.

The use of radioactive tumor-localizing antibodies is developed historically, starting with the demonstration that antibodies can be radioiodinated without destroying antibody activity. Then, antibodies against normal organs were shown to contain antibodies which localize in the normal organ. Subsequently, antibodies capable of localizing in tumors were demonstrated, and these localized well enough to permit their use diagnostically by scanning for radioactivity and their use therapeutically by localizing sufficient radioiodine. Various relevant problems are discussed.

HLA-DR typing by radioimmunoassay.

A radioimmunoassay procedure is described by which peripheral blood lymphocytes can be typed for HLA-DR specificities. The major advantages of this method are the following: simple and reproducible procedure, no need for B lymphocyte separation, no need for optimal viability, and no need for preabsorption of antisera with platelets. This method will find an application in the genetic and biochemical analysis of the HLA complex, and in the clinical tests of Ia antigens for diagnostic or prognostic purposes and in retrospective transplant studies.

Purification and separation of subsets of human Ia molecules by papain digestion.

Papain digestion of human Ia(-like) molecules was performed under various conditions using 125I-labelled preparation of non-ionic-detergent-solubilized Ia antigens of Daudi cells. The products were examined for their allospecificities by a direct binding reaction with human Ia alloantisera. The Daudi Ia preparation is known to contain Ia molecules of DRw6 specificity, an HLA-DR specificity and also Ia molecules of DC1 specificity, a putative non-HLA-DR specificity. Limited papain digestion cleaved off the hydrophobic portion of human Ia molecules and gave smaller sized Ia products. The cleavage did not affect the Ia alloantigenic determinants and occurred much more readily with molecules of DC1 specificity than with molecules of DRw6 specificity. As a consequence, limited papain digestion of the Daudi Ia pool yielded an Ia preparation with DRw6 specificity but lacking DC1 specificity and another Ia preparation which was enriched in DC1 specificity. The limited papain digestion of the Daudi Ia pool followed by gel filtration and LcH affinity chromatography also produced Ia REPARATIONS OF HIGH PURITY. Extensive papain digestion damaged the Ia alloantigenic determinants but the DC1 determinant was much more resistant than the DRw6 determinant. Thus extensive papain digestion yielded an Ia preparation which was relatively rich in DC1 specificity and essentially devoid of DRw6 specificity.

Molecular identification of human Ia antigens coded for by a gene locus closely linked to HLA-DR locus.

Human Ia(-like) specificities controlled by gene loci other than HLA-DR were searched for at the molecular level in cells of human B-cell-type cell lines which carry two established DR specificities. Chevalier cells of DRw3 and 7 and U698M cells of DRw2 and 4 were used. Their Ia molecules were partially purified, radioiodinated and analyzed for Ia specificities by the direct binding and sequential binding assays with a selected panel of human Ia alloantisera. It was possible in both the cell lines to define a third subset of Ia molecules carrying a new specificity in addition to two Ia subsets carrying the established DR specificities. The new specificity was detected by putative anti-DRw4 and anti-DRw7 antisera and was closely associated with DRw4 and DRw7 at population level. It was thus designated provisionally as BR4X7. These results suggest that the BR4X7 specificity is coded for by a separate Ia locus closely linked to HLA-DR locus. The determinant(s) responsible for BR4X7 was located on the small subunit of Ia molecules.

Human cell membrane components dominant in T cell lineage: identification and characterization of human TL-like antigens.

Cell membrane components that contain beta 2-microglobulin were purified from cells of a human T cell-type leukemia cell line, HPB-ALL. They contained membrane components that have the same molecular size and the same subunit structure as HLA(A,B,C) antigens but are separable from the typical beta 2-microglobulin-containing cell membrane components, i.e., the HLA (A,B,C) antigens, by xenoantibody reagents. A sensitive radioimmunoassay was constructed for detection of the T cell membrane components. The assay revealed that the cell membrane components are expressed exclusively on cells of T cell-type leukemia cell lines among the human lymphoid cell lines tested, predominantly in thymus, among the human organs and tissues tested. They were not present on cells of human B cell-type cell lines or on cells of nonlymphoid organs and tissues. No alloantibodies directed to the T cell membrane components, the putative human homologues of mouse TL antigens, were found in any of the human tissue typing sera tested.

Human Ia-like antigens in non-lymphoid organs.

Human Ia-like antigens in liver and kidney were shown by the immunofluorescence assay to be present mostly in the endothelial-mesenchymal cells of these organs. The parenchymal cells apparently contained no human Ia-like antigens. The antigens in liver and kidney were purified and shown to have the same subunit structure as human Ia-like antigens of cultured B-lymphoid cells. The human Ia-like antigens in non-lymphoid organs, not only in liver and kidney but also in testis, heart, muscle and brain, carried all the xenoantigenic characteristics of human Ia-like antigens expressed on lymphoid cells of B-cell lineage.

Carcinoembryonic antigen-binding immunoglobulin isolated from normal human serum by affinity chromatography.

A human immunoglobulin that binds carcinoembryonic antigen (CEA) was isolated from four individual normal human sera by affinity chromatography with the use of a CEA-Sepharose solid adsorbent. The yield of isolated protein, termed human CEA-binding protein (HCBP), ranged from 1.8 to 10 microgram/ml serum. HCBP is a gamma-globulin of restricted electrophoretic heterogeneity as shown by immunoelectrophoresis. HCBP was shown to bind radioiodine-labeled CEA both by a radioimmune precipitation assay and by a radioimmunoelectrophoresis assay. This protein was of practical interest because of its potential usefulness as a carrier of radioactivity or therapeutic agents to a CEA-producing tumor for therapeutic or diagnostic purposes.