DR antigens on melanoma cells: analysis with monoclonal antibodies

Two monoclonal antibodies (691-13-17 and 37-7) can precipitate DR antigens from radioiodinated, detergent solubilized melanoma cells. However, after complete depletion of lysates with one antibody (691-13-17) alpha-like chains can be still be precipitated from some melanoma cells by the other antibody (37-7). Antibody 37-7 also precipitates an additional distinct antigen from SK-MEL 37 but not from any five other melanoma cell lines.     

Invariant proteins associated with guinea-pig Ia antigens

Analysis of guinea-pig Ia immunoprecipitates by two-dimensional gel electrophoresis demonstrated the specific association of Ia molecules with several types of invariant proteins. These include a 33,000 mol. wt basic protein homologous to murine invariant chain (I_i), and a set of 34,000–36,000 mol. wt proteins more acidic than I_i (acidic invariant chain). Two 23,000–25,000 mol. wt nonpolymorphic proteins with pls of 6.0 and 6.5 were also observed in association with Ia, as was a basic protein of mol. wt 42,000. Pulse/chase studies using [³⁵S]methionine demonstrated that I_i, but not acidic invariant chain, was associated with newly synthesized Ia molecules. The amount of ³⁵S-I_i decreased greatly throughout the chase period. ³⁵S-acidic invariant chain was clearly present in Ia precipitates by 30 min after Ia synthesis, but was not detected 4hr after synthesis. Only acidic invariant chain was associated with mature Ia antigens bound by the lectin Ricinus communis I. Our results indicate that guinea-pig invariant proteins are differentially bound by Ia molecules during maturation of Ia - and β-chains, and suggest that acidic invariant chain could be a processed form of I_i.     

Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes

We compared bovine serum albumin, commercial non-fat dry milk, and Tween 20 as blocking agents for immunologic probing of bacterial proteins transferred to nitrocellulose sheets. There were quantitative and qualitative differences in antigens detected that depended on which blocking agents were used. We suggest that several methods for blocking and washing nitrocellulose should be compared when Western blotting is used to detect immunologically reactive proteins.     

Serological precipitation method for studying biosynthesis and secretion of immunoglobulins by human peripheral blood lymphocytes

A reproducible, serological method to quantify radiolabeled immunoglobulin (Ig) synthesized and secreted by human peripheral blood lymphocytes is described. In contrast to indirect precipitation (rabbit antihuman Ig and goat antirabbit Ig), the direct coprecipitation method using human IgG and rabbit antihuman IgG F(ab)₂ showed much smaller, non-specifically precipitable radioactivity (i.e., 2～14%) without affecting Ig radioactivity.     

A monoclonal antibody that recognizes an antigenic determinant shared by HLA A2 and B17

A hybridoma monoclonal anti-HLA antibody has been produced by the technique of Kobler and Milstein [1]. This antibody recognizes a new specifity common to HLA A2 and B17. It was shown to be a single antibody by isoelectric focusing and absorption experiments.     

Partial purification and characterization of chicken immune-associated antigen in serum coded for by the major histocompatibility complex

Chicken serum contains 3 molecular species of immune-associated (Ia) antigen coded for by the major histocompatibility complex (MHC). These molecules are named B-L alloantigens [1]. They vary in electrophoretic migration velocity and molecular size [2]. The aim of this study was to characterize one of the antigen species - the low molecular size form. Therefore, we performed a partial purification by: (i) affinity chromatography; and (ii) ammonium sulfate precipitation of serum B-L antigen from the chicken plasma. Since the MHC-antigens were known to be glycoproteins, the purification was based on lectin affinity chromatography, as previously used for the membrane-bound MHC-antigens [3]. Electro-immunochemical analysis using rabbit antibodies against the chicken lymphocyte plasma membrane and the partially purified antigen were employed to monitor the purification and to characterize the different molecular forms of the Ia molecules. The partially purified preparation was then analyzed to elucidate the biochemical structure of the serum B-L antigen. Finally, rabbit antiserum was raised against this preparation to evaluate its level of purity and to follow further purification of this molecule.     

Biochemical purification of detergent-solubilized H-2 alloantigens

A multistep Chromatographic fractionation scheme is described for purifying the H-2K^b and H-2D^b major histocompatibility antigens as isolated from the murine lymphoblastoid cell line EL4 (H-2^b haplotype). The membrane-integrated antigen molecules were solubilized with the non-ionic detergent NP-40 and were purified by gel filtration chromatography, ion exchange chromatography and affinity chromatography with lentil lectin conjugated to Sepharose. During the latter procedure, use of a linear gradient monosaccharide elution effected partial separation of the H-2K^b and H-2D^b antigens. At this stage the H-2 glycoproteins were highly purified based on several criteria. Upon polyacrylamide gel electrophoresis in SDS the major band migrates with a mol. wt of approximately 45,000 daltons corresponding to the mol. wt of antigens obtained by immunoprecipitation. Moreover, near identity of the profiles of the arginine-containing tryptic peptides from chromatographically-purified and immunoprecipitated H-2K^b preparations suggests that a high degree of homogeneity has been achieved in the Chromatographic purification. As is demonstrated, mg quantities of the H-2K^b and the H-2D^b antigens can be isolated and partially separated from each other by this purification scheme thereby opening the way for structural studies of the H-2K and H-2D molecules by a variety of biochemical methods.     

Cell surface glycoproteins of rabbit lymphocytes: characterization with monoclonal antibodies

The structural characteristics of antigens recognized by a panel of monoclonal antibodies prepared against a rabbit T-lymphocyte cell line have been investigated. Those antigens which could be isolated using immunoadsorbents prepared from the monoclonal antibodies had mol. wts of 42,000, 90,000 and 120,000. The 42,000 mol. wt molecule is similar or identical to a rabbit class I major histocompatibility complex antigen and its characterization has been reported elsewhere. Three different 90,000 mol. wt proteins can be distinguished by their reactivity with lectins and by sequential immunoprecipitation. The 120,000 mol. wt protein is a very abundant surface glycoprotein that appears to be a specific marker for T-cells in the rabbit. It is the immunodominant antigen in a lentil lectin bound glycoprotein pool. Over half of the antibodies were directed against this antigen. All antigens detected by the panel of monoclonal antibodies have been detected on normal lymphoid cells.     

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one M_r 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent M_r 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32–34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.     

Characterization of major histocompatibility antigens on trinitrophenyl-modified cells.

Cell membrane proteins encoded for by the major histocompatibility complex (MHC)¹ are associated with the antigenic determinant(s) recognized on trinitrophenyl (TNP)-modified cells by syngeneic murine cytotoxic T lymphocytes and by hapten-reactive guinea pig T cells. To investigate the relationship of the TNP moiety on TNP-modified cells to these major histocompatibility antigens, peritoneal exudate cells or splenocytes from two inbred guinea pig strains and one inbred murine strain were TNP-modified, radioiodinated and lysed in detergent. TNP-derivatized proteins were then isolated using an anti-TNP immunoabsorbent, and the presence on TNP-derivatized histocompatibility antigens in the eluted proteins was determined by immunoprecipitation experiments and SDS-polyacrylamide gel electrophoretic analysis. Whereas most of the various histocompatibility antigens examined were found to be TNP-derivatized in amounts proportional to the degree of membrane protein derivation as a whole, only small amounts of TNP-modified strain 2 guinea pig Ia antigens were found, and no hapten-modified strain 13 guinea pig Ia antigens were detected. It is concluded that, in contrast to most MHC gene products, strain 13 Ia antigens are not derivatized on TNP-modified cells and, thus, represent an important exception demonstrating that histocompatibility antigens need not be directly TNP-derivatized for T cell recognition and activation.     

HLA-DR antigens in Chinese-American families

This paper contains results of a study on HLA-DR antigens in Chinese-American families. DR2, DR4, DRw9, and DRw6Y were the most common DR specificities encountered, and DR1 occurred with the lowest frequency. All recognized DR antigens were observed. The frequency of a blank allele was 6.4–12.8%. Weak serologic reactions with sera primarily of Caucasian origin were not infrequently observed. These findings suggested that ethnic-related antigens were present in this population. Two families showed segregation of a new serologic pattern based on polyspecific sera. The gene frequencies of the Bf^F allele and the GLO¹ allele were low as compared to Caucasians. A method is described for improving the yield of viable B cells from frozen B-lymphocyte preparations.     

Identification of murine H-2Db histocompatibility antigens in cells transfected with cloned H-2 genes

Clones of mouse L-cells transformed with 21 cosmids containing 15 major histocompatibility complex class I genes of C57BL10 (H-2b) sperm cell DNA were analyzed for the expression of their transfected H-2 and Qa/Tla genes. Three cosmids contained a single gene, mapping to the H-2D region. This gene encodes the H-2Db alloantigen: mouse L-cells transfected with cosmids containing this gene reacted with monoclonal antibodies and alloantisera specific for the H-2Db antigen and expressed a 46-kd H-2 heavy chain associated with beta 2-microglobulin in their cell membranes. Furthermore, these transfected cells were stimulators of, and targets for, anti-H-2Db cytotoxic T-lymphocytes. Eighteen cosmids contained 14 different genes mapping to the Qa and Tla regions. L-cells transfected with these genes did not express class I genes reacting with alloantisera or monoclonal antibodies against Qa2, Qa4 or TL differentiation antigens. In particular, the Qa2,3 gene of C57BL10 was not identified.     

Guinea-pig peritoneal macrophage receptor for IgG--II. Purification of the receptor and its partial characterization

The Fc gamma receptor of guinea-pig peritoneal macrophages was purified by affinity chromatography by using rabbit IgG or guinea-pig IgG2 coupled to Sepharose. Lysates prepared by treatment of 125I-labeled macrophages with NP-40 were first applied to BSA-Sepharose and then to IgG-Sepharose and eluted with 0.5 M acetic acid containing 1% NP-40. The specific binding was determined by interaction of the 125I-labeled receptor with IgG-Sepharose in the presence and absence of soluble IgG. The specific binding of the purified receptor was 42-82%. Interactions of the purified receptor with IgG-Sepharose were equally well inhibited by soluble rabbit IgG or guinea-pig IgG2, but not by F(ab')2 fragments. Inclusion of NP-40 in the buffer used in the assay reduced nonspecific binding of the receptor to the affinity gels. The purified receptor can be stored for 20 days at 4 degrees C without a significant loss of the specific binding activity. Analysis of the receptor by SDS-polyacrylamide gel electrophoresis, under nonreducing and reducing conditions, revealed two major peaks of radioactivity corresponding to mol. wts of about 50,000 and 25,000, and one very minor peak corresponding to a mol. wt of about 30,000. The results obtained suggest that the protein of the second major peak is a product of the dissociation of the protein of the first major peak rather than a product of its reduction by 2-mercaptoethanol.     

In vitro development of human monoclonal antibody-secreting plasmacytomas

A polyclonal human lymphoblastoid cell line transformed in vitro with Epstein-Barr virus produced specific anti-Rhesus D antibody. It was repeatedly enriched by rosetting procedures and subsequently cloned. The cloning conditions employed a combination of mouse macrophage feeder layers, antimycoplasma agents, and low density passage. Formal evidence of monoclonality was obtained in one case which was of human IgG1 isotype and was secreted at the level of 15-20 micrograms/ml. All clones showed long-term stability in culture after 10 months of continuous passage. Both polyclonal and monoclonal cell lines possessed antigens characteristic of highly differentiated B cells, yet they also expressed Epstein-Barr virus nuclear antigen (EBNA). This study exemplifies a simple method for obtaining monoclonal antibody secreting plasmacytomas of human origin.     

Protein micelles of human histocompatibility antigens (HLA-A and HLA-B)

Human histocompatibility antigens (HLA-A and -B) are membrane proteins which have large hydrophilic domains outside the cell membrane and a small hydrophobic portion in the lipid bilayer. In this paper we describe optimal conditions for preparing micelles of detergent-solubilized HLA-A2 and -B7 antigens. These homogeneous protein aggregates are water soluble and free of detergent and lipid. Hydrophobic interactions between the intramembraneous portions of the HLA antigens are the driving forces in the formation of these protein micelles. The papain-solubilized fragment of the HLA antigens is not included in the micelle. The average molecular weight of the HLA micelles is around 9 × 10⁵ daltons, which suggests sixteen HLA-A2 and/or HLA-B7 antigenic molecules per protein aggregate. Electron microscopic studies revealed that the most frequent size of the micelles is 12 mm and that HLA-micelles are similar but not identical to micelles from Sindbis Virus glycoproteins (E1 and E2) The HLA-A2 and -B7 micelles retained full antigenic activity as judged by precipitations with allo- and heteroantisera. Such micelles will no doubt be important tools in further studies of the role of histocompatibility antigens.     

Recognition of HLA-B27 and related antigen by a monoclonal antibody

A monoclonal antibody that binds specifically to HLA-B27, B7, and B22 is described. Binding to B27 appeared to be slightly stronger than to B7 and stronger than to B22 in an indirect binding assay, but no difference in B7 and B27 binding could be detected by Scatchard analysis. No distinction could be made between B27 on cells from normal and from ankylosing spondylitis patients in any assay system. The antibody, which was not cytotoxic, blocked complement-dependent cytolysis mediated by human HLA typing sera specific for B7 and B27. Competitive binding studies with other monoclonal antibodies showed that ME1 could block the binding of antibodies that recognized different antigenic sites on HLA. ME1 did not bind to Klebsiella pneumoniae. This reagent will be useful in further analysis of the relationship between B27 and ankylosing spondylitis.     

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (± weak DC) (22 MoAbs); Dr + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.     

Identification by monoclonal antibody of a major (28 kDa) surface membrane antigen of Schistosoma mansoni

Monoclonal antibody M.1 was generated from mice immunized with membrane enriched extracts of mechanically transformed schistosomula. M.1 bound to the surface membranes of cercariae and young (0-24 h post-transformation) schistosomula but did not bind to older schistosomula or cultured worms. M.1 immunoprecipitated an antigen of approximate molecular weight 27-28 kDa from schistosomula. Experiments using metabolic labeling showed that the antigen was actively synthesized by developing schistosomula. Further M.1 immunoprecipitated a similar 27-28 kDa antigen from membrane-enriched extracts of miracidia, lung and adult worms as well as from schistosomula.     

The molecules recognized by anti-MT2 alloantisera and anti-MT2-like (DRw52) monoclonal antibodies are different

The human class II alloantigens include the HLA-DR, DQ, and MT determinants. Previous reports in the literature suggest that while the DQ determinants appear to be on a molecule separate from DR, the MT determinants are variably present on DR or DQ molecules. We have previously reported, using the homozygous DR5 cell line Swei, that the MT4 determinant defined by the allosera, MGH88B, was only on the DQw3 molecule, while MT2, defined by the functionally monospecific anti-MT2 alloantiserum MGH87B, was present on both the DR5 and DQw3 molecules. We now report using the monoclonal antibody ILR2 directed against an MT2-like determinant DRw52, that DRw52 is present on the DR molecules only. The MT2 determinant(s) recognized by the functionally monospecific alloantisera MGH87B appear to include the DRw52 determinant(s) recognized by the monoclonal antibody ILR2.     

Monoclonal antibodies recognizing determinants specific for the promastigote stage of Leishmania mexicana

Two monoclonal antibodies (IX-IF9-D8 and IX-5H9-CI) produced to a membrane enriched fraction of Leishmania mexicana amazonensis promastigotes have been demonstrated to be specific for the promastigote (insect) form and not the amastigote (mammalian host) form of the parasite. The antigens recognized by these monoclonal antibodies are not found on amastigotes isolated from infected animals or on amastigotes isolated from a macrophage cell line J774 infected initially with promastigotes. The antigens are not re-expressed by amastigotes cultured at 34°C; however, amastigotes cultured at 24°C that have begun transformation into promastigotes do express these antigens. The level of expression of these antigens in cultures of amastigotes undergoing transformation into promastigotes, increases with time from 16 to 36 h and appears to correlate with the percentage of promastigotes. Two protein molecules with apparent molecular weights of 40 000 and 92 000 have been identified by radioimmune precipitation as associated with L. mexicana promastigote stage specific determinants.