Structural characterization of developmentally expressed antigenic markers on chicken erythrocytes using monoclonal antibodies

Monoclonal antibodies selected for embryonic and adult erythrocyte specificity have been used to characterize developmentally expressed markers on the surfaces of mature circulating erythrocytes of young and adult chickens. The data presented demonstrate that the antigenic changes which occur on the avian erythrocyte membrane with organismic maturation can be accounted for, at least in part, by changes in the expression of structurally different, but possibly related polypeptides. Monoclonal antibodies selected for specific reactivity with the erythrocytes of newly hatched chicks recognize a glycoprotein of 48,000 daltons apparent molecular weight. On two-dimensional isoelectric focusing gels, this antigen, which appears identical in all strains studied, displays microheterogeneity; consisting of eight to nine closely spaced spots with an isoelectric midpoint of approximately 5.5. This antigen is not expressed on the circulating erythrocytes of mature birds; however, an antigen with similar, but perhaps not completely identical structure, can be detected within the adult bone marrow. The monoclonal antibodies which show preferential binding to the circulating erythrocytes of adult birds also immune precipitate an antigen of 48,000 daltons apparent molecular weight, but this antigen has a more basic isoelectric point. The adult antigen is polymorphic. Slightly different patterns were obtained on two-dimensional gels with erythrocytes from inbred birds having different major histocompatibility genotypes. It has a major component near pH 7.0 and additional focusing spots usually occurring at a slightly lower molecular weight near pH 6.8 or 6.6 depending upon strain. Competitive radiobinding assays with B-system-specific alloantisers suggest that these antigens may in fact be antigens of the polymorphic BG locus of the chicken major histocompatibility complex. One-dimensional peptide mapping of the immune precipitated embryonic and adult erythrocyte polypeptides demonstrate that the antigens are borne on distinct but possibly related polypeptides. Both common and unique peptide fragments are found in the digestion products. Selective solubilization of the chicken erythrocyte membrane suggests that the antigens are integral membrane proteins extractable with nonionic detergent but not with reagents which remove peripheral proteins.     

Bi-specific monoclonal antibodies: selective binding and complement fixation to cells that express two different surface antigens

A new dimension of immunotherapeutic selectivity might be achieved if antibodies could distinguish cells that co-express two different surface antigens. Bi-specific monoclonal antibodies (BSMAB) with two different antigen combining sites that share a common Fc region theoretically might have such a potential. Two such BSMAB were produced by hybrid-hybridoma clones prepared by fusion of pre-existing hybridomas and were purified by isoelectric focusing. CD3,4 (IgG2a, IgG2b) recognizes the T cell surface antigens CD3 and CD4, and CD3,8 (IgG2a, IgG2a) recognizes CD3 and CD8. These BSMAB promote complement-mediated lysis of target cells that bear both surface antigens 25 to 3125 times more efficiently than those that express only one of the antigens. This selectivity results from the increased avidity of these antibodies for cells with both antigens, as reflected by the increased surface immunoglobulin concentration detected by flow cytometry. It was also demonstrated that there exists a threshold surface immunoglobulin density necessary for antibody-dependent complement-mediated cytotoxicity microtiter assays for the various IgG antibodies tested in both bivalent and monovalent binding. These results support the associative model of IgG-mediated complement fixation.     

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.     

Concurrent expression of basal and luminal epithelial markers in cultures of normal human breast analyzed using monoclonal antibodies

The aim of this study was to investigate the retention in culture of the antigens characteristic of the two mammary epithelial subclasses, basal and luminal epithelium. Primary and secondary cultures of normal human mammary-gland cells were used for immunolocalization experiments with monoclonal antibodies to luminal and basal epithelium. In contrast to the in vivo situation, in which reactivity was only seen in basal cells that were negative for the luminal antigen, we found the homogeneous expression of the basal marker by all of the cultured cells at second passage, and the simultaneous expression of the luminal marker by some of these cells. Characterization of the basal antigen expressed in culture using sodium-dodecyl-sulfate/polyacrylamide gel electrophoresis and immunoblotting techniques showed it to be a 51-kilodalton keratin peptide with an isoelectric pH of 5.4, and confirmed its similarity to the antigen expressed in vivo. Our findings thus demonstrated the coordinate expression of the basal and luminal antigens in cells cultured on solid substrates. The availability of monoclonal antibodies to epithelial-subclass-specific markers of the human mammary gland now makes it feasible to search for culture conditions that would allow the maintenance and manipulation of cell differentiation in vitro.     

Antigen-dependent heterogeneity of human migration inhibitory factor

Human migration inhibitory factor (MIF) produced by peripheral blood mononuclear cells stimulated with purified protein derivative, tetanus toxoid, streptokinase-streptodornase, or Candida albicans antigen was analyzed by gel filtration and isoelectrofocusing. In all cases, supernatants harvested after a 24-hr exposure of the mononuclear cells to the antigen yielded only one MIF species with an isoelectric point of 5. In contrast, isoelectrofocusing of supernatants obtained from cells exposed to the antigen for an additional 24 hr demonstrated that different antigens induce the elaboration of different MIF species. Streptokinase-streptodornase and tetanus toxoid induced the production of one MIF species with an isoelectric point of 5 (pH 5-MIF). Stimulation of cells with Candida antigen elaborated a MIF species with an isoelectric point of 3 (pH 3-MIF). In contrast, stimulation of cells with purified protein derivative induced the production of both pH 3-MIF and pH 5-MIF.     

Molecular characterization of the murine T cell antigen receptor and associated structures

Previous studies have demonstrated that the T cell antigen-specific receptor is a disulfide-linked heterodimer with subunits of 40-48 kilodaltons. We have produced a series of antiserums and monoclonal antibodies to epitopes carried by the molecule, including clonotypic epitopes specific to individual T lymphomas as well as epitopes shared by different T cell lines. Using these reagents we have isolated the heterodimers from a variety of T cells for comparison of primary structure via two-dimensional peptide mapping. The results indicate that the peptide maps of the alpha and beta subunits are extremely different, indicating that the subunits are encoded by different genes, and both subunits contain constant as well as variable peptides. To determine whether the murine T cell receptor is associated with other cell surface structures, C6VL lymphoma cells were radioiodinated, cross-linked with the cleavable reagent dimethyl-3,3'-dithiobispropionimidate, solubilized, and subjected to immunoprecipitation with the clonotypic antibody 124-40, and the precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linked samples, but not sham-treated precipitates, contained structures similar to the human Leu-4/T3 structure in addition to the receptor subunits. These results indicate that similar structures may be associated with the receptor in the human and the mouse.     

Antigenic differences between tumor-associated fetal antigens and rat histocompatibility antigens

Certain molecular properties of purified tumor-associated fetal antigens (TAFA) were analyzed by sequential immune precipitation (SIP), isoelectric focusing, and high-pressure liquid chromatography (HPLC). The antigenic relatedness of rat histocompatibility antigens and the various TAFA were determined by SIP. SIP of chloramine-T-labeled purified TAFA or lactoperoxidase-iodinated tumor cell membranes, in the presence of rat alloantisera and monospecific rabbit anti-TAFA sera demonstrated no antigenic cross-reactivities or similarities between H-antigens and TAFA. TAFA were also compared with histocompatibility antigens for isoelectric point optima and molecular weight. Rat H-antigens had isoelectric points in the 7.0–8.5 pH range, whereas all TAFA focused at pH 5.0–6.5 or above pH 8.0. Molecular weights were determined by HPLC. TAFA-I and TAFA-III had molecular weights of 16,000–17,500 daltons, whereas TAFA-II had a molecular weight of 12,000. The antigens were not coprecipitated by the rat alloantisera. Each TAFA was also isolated (via immune precipitation) from NP-40-solubilized tumor cell membranes. These TAFA were identical to the chloramine-T-labeled TAFA which had been previously extracted and purified from rat fibrosarcomas and osteosarcomas. These studies demonstrated that although TAFA and H-antigens cocap on embryonic and transformed cell membranes, these determinants are different molecules; they are not covalently linked on cell membranes; and TAFA are not cleavage products of normal NBR H-antigens.     

Mechanisms of heat-induced antigen retrieval: does pH or ionic strength of the solution play a role for refolding antigens?

We investigated the effects of pH and ionic strength of solutions used for antigen retrieval to elucidate the mechanism of heat-induced antigen retrieval (HIAR) in immunohistochemistry. The immunostaining intensity of nuclear, cytoplasmic, cell membrane, and extracellular matrix antigens with 17 different antibodies was evaluated in formaldehyde-fixed and paraffin-embedded mouse and human tissues. Deparaffinized sections were autoclaved for 10 min in buffers with different pH values ranging from 3.0 to 10.5. To test the influence of ionic strength on immunoreactions, the sections were autoclaved for 10 min in 20 mM Tris-HCl buffers (TB) at pH 9.0 and 10.5 with or without 25, 50, and 100 mM NaCl. There were two immunostaining patterns for pH dependency of HIAR. First, the majority of antibodies recovered their antigenicity when heated in the buffers with both acidic pH (pH 3.0) and basic pH (pH 9.0 and 10.5). Second, some antibodies showed strong immunostaining only at basic pH values (pH 9.0 and 10.5). When the sections were autoclaved in TB at pH 9.0, immunostaining of all eight antibodies examined decreased as the NaCl concentration increased. On the other hand, when the sections were treated with TB at pH 10.5, all antibodies yielded stronger reactions in the buffer containing NaCl than in the buffer without NaCl; five antibodies exhibited the strongest immunoreaction at concentrations from 25 to 50 mM. These results suggest that the extended polypeptides by heating are charged negatively or positively at basic or acidic pH, and that an electrostatic repulsion force acts to prevent random entangling of polypeptides caused by hydrophobic attractive force and to expose antigenic determinants, during cooling process of HIAR solution.     

Human renal renin. Complete purification and characterization

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.     

The in vitro antibody response to cell surface antigens. II. Monoclonal antibodies to human leukemia cells.

A method has been developed for the production of monoclonal mouse antibody responses in vitro against human cell surface antigens. Limiting numbers of immune spleen cells were transferred to syngeneic, irradiated recipients whose spleen fragments were then cultured in vitro and stimulated to produce antibody. The majority of the antibody from any one fragment culture was likely to be the product of a single donor B cell and thus monoclonal. Evidence for this included a linear relationship between donor cell transferred and spleen fragments producing antibody, extremely restricted isoelectric focusing patterns of the individual antibody products, and unique reactivity patterns of these antibodies against a panel of human lymphoid cells. Different human B leukemia cells were seen as immunogenically distinct by the mouse. By using the monoclonal mouse antibodies as probes, a fine analysis of cell surface antigens is jow possible.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Fractionation of polyclonal antibody by isoelectric focusing--differences in cross-reactivity and affinity of rabbit clonotype anti-human thyrotropin antibody

Immunoglobulin G (IgG) fractions prepared from three different batches of rabbit antihuman thyrotropin (hTSH) antisera were fractionated by agarose isoelectric focusing (IEF) in the pH ranges 3 to 10 and 5 to 8. Staining of protein in agarose gel after IEF showed that polyclonal IgG separated into more than 20 protein bands with isoelectric points (pIs) ranging from 6 to 9. The clonotype antibodies to hTSH were recovered from the fractions and subjected to radioimmunoassay for determination of the binding-affinity for hTSH and the cross-reactivity with human chorionic gonadotropin (hCG). The affinity constants of the antibodies recovered ranged from 6.4 X 10(9) M-1 to 3.1 X 10(10) M-1, and the cross-reactivities of the clonotype antibodies differed greatly. A good correlation was observed between the pIs of antibody molecules and their cross-reactivities: antibodies with higher pIs bound hCG more strongly than those with lower pIs. The correlation coefficients between the pIs and cross-reactivities were 0.83, 0.84, and 0.87 in three batches of antibody.     

Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.

The objective of this study was to identify those immunodominant sperm antigens recognized by antisperm antibodies (ASA) in the serum samples of infertile men and women. High-resolution two-dimensional gel electrophoresis was employed to separate human sperm proteins using isoelectric focusing or nonequilibrium pH gradient electrophoresis, followed by PAGE. Serum samples from 15 infertile male subjects and 6 infertile female subjects that contained ASA as assayed by the immunobead binding test (IBT) were analyzed by Western blotting followed by enhanced chemiluminescence (ECL). Serum samples from 10 fertile subjects (5 males and 5 females) that were ASA negative by IBT were used as controls. The ECL blots were analyzed by computer scanning to compare the immunoreactivity between serum samples from fertile and infertile subjects and to identify the antigens unique to the sera of the infertile subjects; 98 sperm auto- and iso-antigenic protein spots were recognized by sera from infertile males and females but not from fertile subjects. Based on vectorial labeling with 125I at the sperm surface, a subset of 6 auto- and iso-antigens was identified as possibly relevant to antibody-mediated infertility.     

Activation of "silent" antibodies and their interaction with antigens

Animal and human blood serum contains great amount of blocked (or "silent") immunoglobulins which, being activated by heating to 60 degrees C, pH decrease to 2.0-2.5 or treatment with 5M KSCN acquire a capacity to interact with different antigens. This interaction may be equally prevented or weakened by both identical and serologically non-related antigen, i.e. activated immunoglobulins are polyspecific. Polyspecific immunoglobulins show less affinity in comparison with monospecific antibodies, their interaction with antigens depends considerably on temperature.     

Intermediate filament proteins immunologically related to desmin in astrocytes: A study of chicken spinal cord by two-dimensional gel electrophoresis and immunoblotting

Co-migration experiments by two-dimensional SDS-PAGE using chicken spinal cord extracts and desmin purified from chicken gizzard showed that desmin is not present in spinal cord. However, by the immunoblotting procedure, desmin antibodies recognized 3 spinal cord antigens with different molecular weights and isoelectric points than desmin and the glial fibrillary acidic (GFA) protein. These antigens which also reacted with GFA protein antibodies were not identified in chicken gizzard extracts. The reactivity of the antigens with a monoclonal antibody recognizing an epitope common to most intermediate filament proteins (1) suggests that immunostaining of astrocytes with desmin antibodies (2, 3) is due to the presence of new intermediate filament proteins immunologically related to desmin.Special issue dedicated to Dr. Paola S. Timiras     

Analysis of the B-G antigens of the chicken MHC by two-dimensional gel electrophoresis

The B-G antigens of the chicken major histocompatibility complex (MHC) have been analyzed by high resolution two-dimensional (2-D) gel electrophoresis. Monoclonal antibodies recognizing a widely shared B-G determinant were used for immunoprecipitating the B-G antigens from radioiodinated, detergent-solubilized erythrocyte membrane preparations. The B-G antigens produce a variety of patterns on 2-D gels. The number of polypeptides within a B-G pattern varies among haplotypes from single polypeptide arrays showing slight microheterogeneity to complex patterns which contain as many as four or five polypeptide arrays differing in relative mobility and isoelectric point. Many of the patterns, but not all, include a polypeptide of Mr =48 kd focusing near pH 6.9. At present it is not understood whether the multiple polypeptides within some B-G patterns represent the expression of multiple B-G genes or whether they are the result of modifications of single gene products during biosynthetic processing. 2-D gel analyses were also used to confirm the assignment of the same B-G haplotype in several different inbred flocks and the fate of the B-G antigens in two B system recombinant haplotypes. The 2-D gel patterns of these highly polymorphic antigens provide evidence for a complexity of the B-G locus not previously demonstrated. This technique may serve to define more objectively the diverse chicken MHC haplotypes which are now recognized and characterized only by serological techniques using alloantisera and monoclonal antibodies with varying cross-reactivities.     

Purification and characterization of class II histocompatibility antigens from a homozygous human B cell line

Human class II histocompatibility antigens were purified from the Epstein-Barr virus-transformed human B lymphoblastoid cell line LG-2 by immunoaffinity chromatography. This is the first time all three subsets have been prepared as nonradioactive materials on a milligram scale. The yields of DR, DQ, and DP from 10 g of cells were approximately 12, 2, and 0.2 mg, respectively. Cross-contamination of the subsets was found to be less than 2% when assayed by measuring the binding of antigen-specific monoclonal antibodies to antigen immobilized on fixed erythrocytes. The three purified subsets were extensively characterized. They contained no detectable invariant chain. The three proteins were distinguished by their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The denatured antigens were susceptible to partial removal of carbohydrate by endoglycosidase H and apparently complete removal of carbohydrate by endoglycosidase F. The isolated, denatured chains differed in their affinities for radiolabeled lectins, suggesting differences in carbohydrate structures. A water-soluble form of each antigen was prepared by a controlled papain digestion of the native antigen. Both native and denatured antigens were analyzed for their reactivities with a panel of class II antigen-specific monoclonal antibodies, allowing a precise definition of the specificities of the antibodies.     

Charge variants of tubulin,tubulin S,membrane-bound and palmitoylated tubulin from brain and pheochromocytoma cells

Isoelectric focusing (IEF) of only approximately 1 microg of rat brain tubulin yields 27-30 distinct charge variants in the pH range of 4.5-5.4 with band separations of 0.01-0.02 pH units as detected by silver staining. Variants can be efficiently transferred from the immobilized gradient strip to polyvinylidene difluoride (PVDF) membranes for reaction with monoclonal antibodies. C-terminal-directed antibodies to alpha- and beta-tubulin yield patterns similar to N-terminal-directed antibodies. Removal of the acidic C-termini with subtilisin to form tubulin S increases the pI values by approximately 1 pH unit, leads to a loss in the isoelectric distinction between the alpha- and beta-tubulin variants seen by N-terminal-directed antibodies, and abolishes reactions with all beta-variants and all but three alpha variants by C-terminal-directed antibodies (TU-04 and TU-14). Many, but not all, of the variants are substrates for autopalmitoylation of rat brain tubulin. The distribution of isoelectric variants differs between cytoplasm and membrane fractions from PC12 pheochromocytoma cells. A potential role for different variants is suggested.     

Glutamate dehydrogenase in the first leaf of wheat

Glutamate dehydrogenase extracted from wheat leaves ( Triticum aestivum L. cv. Capitole) taken at two different physiological stages was analysed by electrophoretic and immune-chemical techniques. Two NAD-dependent antigens were identified which bear the balk of the glutamate dehydrogenase activity in the two extracts. The first enzyme was found in much larger amounts in young than in senescent leaves and the reverse situation was observed for the second antigen. The possible relationships between this antigenic polymorphism and the heterogeneity detected by isoelectric focusing from the two extracts were investigated. A charge heterogeneity (isoelectric points about 5.7 and 4.8) was found for the first antigen in both extracts. The second antigen appeared homogeneous (isoelectric point about 5.7) at least in senescent leaves. The last result indicates that two quite different antigens appear in the same isoelectric focusing zone.