A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Monoclonal antibodies directed against mammalian RNA polymerase I. Identification of the catalytic center

Mouse myeloma cells were fused with splenocytes from a mouse that had been immunized with RNA polymerase I purified from a rat hepatoma. Hybridoma cells were selected and colonies secreting antibodies directed against the enzyme were detected by analysis of cell culture supernatants in a solid phase radioimmunoassay. Two of these cell lines were grown on a larger scale and the interaction between the immunoglobulins obtained from them and RNA polymerase I was studied in detail. Antibodies from both of the hybridoma cell lines were able to inhibit DNA-dependent RNA synthesis catalyzed by RNA polymerases I and III, but not that catalyzed by polymerase II. The antibodies were also capable of reducing the RNA chain elongation reaction catalyzed either by RNA polymerase I associated with isolated nucleoli or by enzyme preinitiated in vitro on calf thymus DNA. Inhibition of RNA polymerase I activity by the monoclonal antibodies was inversely related to the nucleotide concentration. In contrast, the DNA concentration had relatively little effect on inhibition by the antibodies. Analysis of immune complex formation between the antibodies and isolated individual enzyme subunits demonstrated that the monoclonal antibodies were directed against the largest (Mr = 190,000) polypeptide of the polymerase I. These data indicate that the largest subunit of RNA polymerase I contains an immunological determinant in common with RNA polymerase III and suggest that the polymerase I polypeptide of Mr = 190,000 contains a catalytic center involved in RNA chain elongation.     

Conservation of a DNA-binding site in the largest subunit of eukaryotic RNA polymerase II

Using a monoclonal antibody to a DNA-binding site of calf RNA polymerase II, we found that this site occurs on the largest subunit and is structurally similar in RNA polymerase II of widely divergent eukaryotes. In immuno-blotting of electrophoretically separated subunits, the monoclonal antibody recognized a determinant on the largest polypeptide of all RNA eukaryotic polymerase II forms tested, with a preference for the IIA enzyme subunit of 215 X 10(3) Mr over the partially proteolyzed 180 X 10(3) Mr form. This site is conserved on human, chicken, Drosophila, wheat germ and yeast RNA polymerase II, all of which reacted strongly with the monoclonal antibody. These results contrasted with those obtained with polyclonal antibodies to non-functional determinants of the calf enzyme. The reactivity of the polyclonal antibody with eukaryotic RNA polymerase II steadily decreased with increasing evolutionary distance from the original antigen; the yeast enzyme showed no cross-reactivity. These results suggest that a basic functional feature of eukaryotic RNA polymerase II has been strongly conserved and support the view that divergence of RNA polymerase II has taken place mainly in other, perhaps regulatory, sites of the enzyme.     

Production and characterization of a monoclonal antibody against the seed lectin of the Dolichos biflorus plant

Spleen cells from mice immunized with the Dolichos biflorus seed lectin were fused with cells from the mouse myeloma Sp2/O-Ag14 cell line to form hybridomas. Those hybridomas producing antibodies against the seed lectin were cloned at least four times and the monoclonal antibodies from clone C11/64-56.28 were characterized and found to be specific for Subunit I of the lectin; they do not react with the structurally similar Subunit II. In previous studies, we have shown that although these two subunits appear to differ only at their COOH-terminal ends, only Subunit I has carbohydrate binding activity. Using a solid phase enzyme immunoassay, the antigenic determinant fr the monoclonal antibody was found to be located on the COOH-terminal cyanogen bromide fragment of this subunit. The monoclonal antibody inhibits the ability of the lectin to agglutinate erythrocytes and N-acetyl-D-galactosamine, the specific hapten for the lectin, inhibits the ability of the antibody to combine with the lectin. These results suggest that the monoclonal antibody recognizes a determinant that is located either at or near the active site of the lectin or that is conformationally interdependent with the active site.     

Anti-RNA polymerase I antibodies: potential role in the induction and progression of murine lupus nephritis

Antibodies against RNA polymerase I were detected in plasma and kidney eluates of NZB/W mice. Plasma concentrations of the antibodies were the highest in mice with incipient nephritis and the lowest in mice with progressive nephritis. Mice with attenuated nephritis due to immunosuppressive therapy had intermediate plasma concentrations of the antibodies. The specific concentrations (ng/microgram IgG) of anti-RNA polymerase I antibodies in kidney eluates were significantly (10- to 70-fold) greater than the corresponding plasma concentrations. These results indicated that the decreased plasma concentration of the antibodies in mice with more advanced disease was at least partially due to selective concentration of anti-RNA polymerase I antibodies in the kidneys. The degree of this selective concentration was directly proportional (R2 = 0.9962) to the severity of renal disease, as reflected by the concentration (microgram/g tissue) of IgG eluted from the kidneys. The concentration (microgram/g tissue) of anti-RNA polymerase I eluted from the kidneys also was increased in mice with more severe renal disease. Further, the extent of this increase was greater than that of total IgG, again suggesting that anti-RNA polymerase I antibodies had been selectively concentrated in the kidneys. These findings are strongly suggestive of an important role for the RNA polymerase I/anti-RNA polymerase I antibody system in the pathogenesis of murine lupus nephritis.     

Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies

GTP cyclohydrolase I, the first enzyme in the de novo biosynthesis of tetrahydrobiopterin, was enriched more than 13,000-fold from human liver by preparative isoelectric focusing using Sephadex G-200 SF gels. The pI of the active enzyme was determined as 5.6 by analytical isoelectric focusing in the same matrix. The native enzyme has an apparent molecular mass of 440 kDa and appears to be composed of eight 50-kDa subunits as estimated from SDS/PAGE. The enriched enzyme preparation was used to produce specific monoclonal antibodies. From 11 monoclonal antibodies obtained, one was extensively characterized for further applications. This monoclonal antibody belongs to the IgM class and shows immunoreactivity with GTP cyclohydrolase I both from man and from Escherichia coli. It is capable of highly sensitive detection of GTP cyclohydrolase I by ELISA and by Western blot analysis. The monoclonal antibody was used for the immunoenzymatic localisation of GTP cyclohydrolase I in human peripheral blood mononuclear cells. Furthermore, it was possible to demonstrate the absence of immunoreactivity in cells with GTP cyclohydrolase I deficiency. The antibody's use as a tool either for differential diagnosis of atypical phenylketonuria due to GTP cyclohydrolase I deficiency or prenatal diagnosis of this severe inherited metabolic disease is now under investigation.     

High level expression of a functional human/mouse chimeric anti-CD20 monoclonal antibody in milk of transgenic mice

Rituximab, a chimeric anti-CD20 monoclonal antibody, is one of the most successful biomedicines and has been used to treat at least 370,000 patients with indolent, aggressive non-Hodgkin's lymphoma and other malignant diseases. However, the global demand for rituximab and other therapeutic monoclonal antibodies is exponentially increasing and barely able to be met by current manufacturing capacities of mammalian cell culture. The mammary gland bioreactor has been regarded as an ideal substitute for mammalian cell culture to mass-produce recombinant monoclonal antibodies at the lowest possible cost. Here, we show a feasible model to produce recombinant anti-CD20 antibodies in the mammary glands of transgenic animals. Six lines of transgenic mice were generated by co-microinjection of the two expression cassettes that can specially express the chimeric light and heavy chain of anti-CD20 mAbs in the milk of transgenic animals. The recombinant antibodies were detected in the milk of transgenic mice with the highest expression level up to 17 microg/mul and could specifically bind the CD20 surface antigens on human B-lymphoma cells.     

消减免疫法制备克伦特罗单克隆抗体

目的：制备克伦特罗（CL）单克隆抗体。方法：重氮化法制备克伦特罗完全抗原,消减免疫法免疫BALB／c小鼠,常规杂交瘤技术制备抗克伦特罗单克隆抗体。结果：消减免疫法使小鼠获得了对BSA的耐受,单抗筛选过程中获得了高达8．2％的阳性率。最后得到了针对CL的特异性抗体。结论：用消减免疫法制备针对小分子污染物的单克隆抗体,可减少在制备单克隆抗体时筛选的工作量,可增加获得预期抗体的机会。     

Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase.

Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit. In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase. It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M., nd Goldfarb, A. (1990) Gene 89, 7-12). The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876. This region may be important in enzyme assembly or subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093. This region may be involved in the catalytic function of RNA polymerase.     

Monoclonal antibodies against a specific nonhistone chromosomal protein of Drosophila associated with active genes

Hybridomas secreting monoclonal antibodies have been produced by fusion of NS-1 mouse myeloma cells with the spleen cells of mice inoculated with a 60-65,000-mol wt fraction of proteins released from Drosophila embryo nuclei treated with DNase I. The antibodies secreted by the hybridomas were examined with polytene chromosomes of formaldehyde- fixed salivary gland squashes by an immunofluorescence assay. Most of the clonal antibodies obtained resulted in specific staining of the chromosomes relative to the cytoplasmic debris. In the case of clone 28, the antibodies showed a preferential association with sites of gene activity, both puffs and loci identified as puffing at some time during the third instar and prepupal period. In larvae that were heat shocked (exposed to 35 degrees C for 15 min before removal and fixation of the glands), the antibodies of clone 28 stained preferentially the induced heat-shock loci while continuing to stain most of the normal set of loci. The antigen for clone 28 was identified as a single protein of approximately 62,000 mol wt by using the antibodies followed by 125I- rabbit anti-mouse Ig to stain nitrocellulose replicas of SDS polyacrylamide gels of total chromosomal proteins. This study demonstrates that monoclonal antibodies can be used successfully in immunofluorescence staining of formaldehyde-fixed polytene chromosomes. The results verify the hypothesis that a specific nonhistone chromosomal protein is preferentially associated with the set of loci that includes both active sites and those scheduled to be active at some time in this developmental program. Such proteins may play a general role in the mechanisms of cell determination and gene activation.     

Localization of vimentin in myoepithelial cells of the rat mammary gland

Summary Myoepithelial cells in the virgin rat mammary gland have been shown to contain vimentin, using a polyclonal antiserum to vimentin purified from hamster fibroblasts. This antiserum has been shown to be specific for vimentin by immunoblotting and ELISA techniques. Similar results were obtained with a monoclonal antibody to vimentin. In the mammary glands of pregnant rats, the staining with vimentin antibodies is much weaker in the myoepithelial cells of the developing alveolar buds than in the main ducts. Similarly, in lactating glands, the staining of myoepithelial cells is much weaker in the secretory alveoli than in lactiferous sinuses. In each case, staining with antivimentin co-localizes with staining with polyclonal antisera to callous keratin (which specifically stain myoepithelial cells in the rat mammary gland).     

The recombinant chimeric antibody chHAb18 against hepatocellular carcinoma can be produced in milk of transgenic mice

Biologically active recombinant monoclonal antibodies (mAbs) and their derivatives are in demand as therapeutic agents against a variety of cancers. The antibodies are generally produced by mammalian cell culture, but their production in the milk of transgenic animals would help meet the increasing demand. The mouse-human chimeric antibody chHAb18 has been proven to inhibit the invasion and metastasis of human hepatocellular carcinoma (HCC) cells by recognizing the HAb18G/CD147 molecule that is highly expressed on the surface of HCC tissue. Here, we report that transgenic mice generated by co-microinjection of two cassettes encoding the heavy and light chain genes of chHAb18 could highly express functional chHAb18 in their mammary glands. The expression level range of 1.1–7.4 mg ml⁻¹ was independent of transgenic copy number. Immunoassays demonstrated the ability and specificity of chHAb18 to bind purified antigen (i.e., HAb18G) or HCC cells. Recombinant chHAb18 from transgenic milk exhibited affinity almost equal to chHAb18 derived from CHO cells, and was 68% of that of the parental murine antibody, HAb18. In light of successful clinical application of HAb18, the chHAb18 expressed in mammary glands of transgenic mice constitutes an important step towards high-yield and scaled-up production of this antibody.     

Murine monoclonal antibody against aldosterone: production, characterization and use for enzymoimmunoassay

Hybridomas secreting monoclonal antibodies to aldosterone were obtained by fusion of myeloma cells and spleen cells from Balb/c mice immunized with aldosterone-3-carboxylmethyloxime-bovine serum albumin. A monoclonal antibody was purified from ascites fluid and characterized. An affinity constant of 1.61 x 10(9) M-1 has been measured and no cross-reactivity with tetrahydroaldosterone (THA), cortisol, cortisone, corticosterone, deoxycorticosterone (DOC), dehydroepiandrosterone (DHA), progesterone and estrone, could be detected. A peroxidase conjugated-antibody (1.5 mole of enzyme per mole of antibody) was obtained and used for microwell enzyme immunoassay and Immun-Blot assay. The high affinity and specificity of this antibody should make the direct determination of aldosterone in biological fluids possible at concentrations as low as 5 x 10(-10) M.     

Characterization of nine monoclonal antibodies against the Escherichia coli cyclic AMP receptor protein

Nine hybridoma clones producing antibodies against the Escherichia coli cAMP receptor protein (CRP) have been isolated. Five of the monoclonal antibodies (Class I) had a much higher affinity for native CRP while the remaining four (Class II) bound equally well to native or denatured CRP. Using native N-terminal CRP cores, it was shown that none of the Class I monoclonal antibodies cross-reacted with the 15,000-Da CRP core, and only two bound to the 18,800-Da CRP core. The positions of the antigenic determinants for the Class II monoclonal antibodies were found by Western blotting analysis to reside in the N-proximal region of CRP. Only one monoclonal antibody strongly inhibited cAMP binding by CRP, and this was accompanied by a consequent strong inhibition of both lac DNA binding and abortive initiation by RNA polymerase. Each of the Class I monoclonal antibodies inhibited abortive initiation, and four of these antibodies also blocked the binding of cAMP X CRP to the lac DNA fragment. One Class I and one Class II monoclonal antibody bound to the cAMP X CRP X DNA complex. Two of the Class II monoclonal antibodies were without apparent effect on any of the assays used.