Correlation of specific keratins with different types of epithelial differentiation: monoclonal antibody studies

We have prepared three monoclonal antibodies against human epidermal keratins. These antibodies were highly specific for keratins and, in combination, recognized all major epidermal keratins of several mammalian species. We have used these antibodies to study the tissue distribution of epidermis-related keratins. In various mammalian epithelia, the antibodies recognized seven classes of keratins defined by their immunological reactivity and size. The 40, 46 and 52 kilodalton (kd) keratin classes were present in almost all epithelia; the 50 kd and 58 kd keratin classes were detected in all stratified squamous epithelia, but not in any simple epithelia; and the 56 kd and 65-67 kd keratin classes were unique to keratinized epidermis. Thus the expression of specific keratin classes appeared to correlate with different types of epithelial differentiation (simple versus stratified; keratinized versus nonkeratinized).     

Fine specificity and idiotype diversity of a murine anti-HLA-DQw3 monoclonal antibody elicited with a syngeneic antiidiotypic monoclonal antibody

A total of 469 hybridomas was generated from a BALB/c mouse immunized with the syngeneic antiidiotypic mAb KO3-34 that recognizes an idiotope within the Ag-combining site of the immunizing syngeneic anti HLA-DQw3 mAb KS13. One of the hybridomas, named S2B154, was shown with a number of serologic and immunochemical assays to mimic the specificity of anti HLA-DQw3 mAb KS13. This result was unexpected, because the antiidiotypic mAb had been classified as gamma because of the lack of detection of anti-HLA-DQw3 antibodies in sera from BALB/c mice immunized with the antiidiotypic mAb KO3-34. The antiidiotypic mAb S2B154, an IgG1, displays a lower affinity constant to HLA-DQw3 Ag-bearing lymphoid cells than mAb KS13, an IgG2b, but a higher one to antiidiotypic mAb KO3-34. Testing with a panel of antiidiotypic mAb elicited with the mAb KS13 showed that both antiidiotypic mAb S2B154 and mAb KS13 express in their Ag-combining site the idiotopes recognized by the antiidiotypic mAb KO3-256, KO3-335, and R1-38. However, the antiidiotypic mAb S2B154 does not react with the mAb R18-9 that recognizes an idiotope outside the Ag-combining site of mAb KS13. The results we have presented question the validity of the classification of antiidiotypic antibodies based on their ability to inhibit the binding of the corresponding antibodies to Ag and on the specificity of antisera elicited with antiidiotypic antibodies. Furthermore, the hybridomas we have developed in this Id cascade provide us the opportunity to analyze the structural basis of idiotypic Ag mimicry and to evaluate the regulatory properties of antiidiotypic antibodies in the immune response to HLA class II Ag.     

Generation and characterization of a monoclonal antibody with specificity for Mycoplasma arginini

Previously, we generated monoclonal antibodies (MAbs) that bound to the surface of human embryonic stem cells (hESCs) in an attempt to discover new hESC-specific surface markers. In this study, MAb 47-235 (IgG1, kappa) was selected for further characterization. The MAb bound to the surface of undifferentiated hESCs but did not bind to mouse ESCs or mouse embryonic fibroblast cells in flow cytometric analysis. The antibody immunoprecipitated a 47 kDa protein from the lysates of cell surface-biotinylated hESCs. Identification of the protein by quadrupole time of flight tandem mass spectrometry revealed that 47-235 binds to Ag 243-5 protein of Mycoplasma arginini. BM-Cyclin treatment of the hESCs that reacted with 47-235 resulted in loss of mycoplasma DNA and the reactivity to 47-235. Nevertheless, the hESCs that were reactive to 47-235 maintained self-renewal and pluripotency and thus could be differentiated into three embryonic germ layers.     

A monoclonal anti-idiotypic antibody against a human monoclonal IgM with specificity for myelin-associated glycoprotein

A human monoclonal IgM lambda antibody, directed against MAG, obtained from a patient with polyneuropathy associated with a gammopathy, was used as an immunogen to generate mouse monoclonal anti-idiotype antibodies. One hybridoma antibody, designated A8F2, reacts uniquely with the M-IgM of the patient, shows high affinity binding to the patient's M-IgM, and dose-dependently inhibits binding of the patient's M-IgM to its specific antigen MAG. Thus, A8F2 is a monoclonal anti-idiotype antibody that recognizes a region of the MAG binding site of the patient's IgM. Use of this anti-idiotype antibody in a competition RIA revealed the presence of naturally occurring anti-idiotype in the patient's serum. Because anti-idiotype antibodies may be part of a mechanism for down-regulation of antibody production, the use of A8F2 to induce a specific immunosuppression should be considered.     

Determination of monoclonal antibody concentration in cell culture by capture ELISA

Methods for analyzing capture ELISA results to estimate antibody concentrations are reviewed and experimentally compared. Good correlation of data to the theoretical isotherm was obtained with a nonlinear regression method, but some linearization methods resulted in very poor correlation of the data. Reasons for the poor correlations are discussed, and strategies are recommended for calculation of antibody concentrations.     

Determination of monoclonal antibody specificity by mixed precipitation ingel

The enzyme-immunodiffusion technique is advanced which permits testing monoclonal antibodies included in the precipitate line formed in gel by polyclonal antibodies with the corresponding antigen. Rat or mouse monoclonal antibodies were mixed with polyclonal rabbit antiserum to the antigen at issue. The precipitate formed by immunogen and rabbit polyclonal antibodies included monoclonals.     

SFR3-DR5, a monoclonal antibody with HLA-DR5 specificity

We have developed a monoclonal antibody with HLA-DR5 serologic specificity. The antibody, SFR3-DR5, binds specifically to DR5-positive lymphoblastoid cell lines, and immunoprecipitates alpha- and beta-chains characteristic of DR antigens from them. Cytotoxic activity of the antibody segregates with the DR5-bearing haplotype in a family. The antibody reacted with the cells of 16 of 17 DR5 individuals and was negative on all DR5-negative cells tested. SFR3-DR5 reacted weakly with PWM-activated cells of the single DR5 individual whose B lymphocytes were unreactive with the monoclonal antibody by cytotoxicity. Possible interpretations of these results are discussed.     

Muscle cell differentiation in ascidian embryos analysed with a tissue-specific monoclonal antibody

Utilizing a muscle-specific monoclonal antibody (Mu-2) as a probe, we analysed developmental mechanisms involved in muscle cell differentiation in ascidian embryos. The antigen recognized by Mu-2 was a single polypeptide with a relative molecular mass of about 220 X 10(3). It first appeared at the early tailbud stage and continued to be expressed until the swimming larva stage. There were distinct and separate puromycin and actinomycin D sensitivity periods during the occurrence of the antigen, suggesting the new synthesis of the polypeptide by developing muscle cells. Embryos that had been permanently arrested with aphidicolin in the early cleavage stages up to the 32-cell stage did not express the antigen. DNA replications may be required for the antigen expression. Embryos that had been arrested with cytochalasin B in the 8-cell and later stages developed the antigen, and the number and position of the arrested blastomeres exhibiting the differentiation marker almost corresponded to those of the B4.1-line muscle lineage. Furthermore, in quarter embryos developed from each blastomere pair isolated from the 8-cell embryo, all the B4.1 as well as a part of b4.2 partial embryos expressed the antigen, while the a4.2 and A4.1 partial embryos did not show the antigen expression. These results may provide further support for the existence of cytoplasmic determinants for muscle cell differentiation in this mosaic egg.     

A chicken monoclonal antibody with specificity for the N-terminal of human prion protein

Chickens were immunized with human prion protein (PrP) peptide H25 (amino acid residues 25-49) coupled to keyhole limpet hemocyanin. From a fusion experiment using the chicken fusion partner cell line MuH1 and immune spleen cells, one mAb, HUC2-13, was generated which reacted with the peptide. HUC2-13 was specific for a pentapeptide (RPKPG) of the N-terminal of the peptide H25. In Western blotting analysis, the mAb reacted with PrP materials from a human Creutzfeldt-Jakob disease (CJD) case and the membrane fraction from normal murine brain, but not with the same materials pretreated with proteinase K. When compared with the HUC2-13 and the conventional mouse mAb 3F4, the background stainings using the HUC2-13 were minimal. In immunohistochemistry, the HUC2-13 stained positively with kuru plaques in brain sections from patients with Gerstmann-Straussler syndrome (GSS), and also reacted with synaptic structures of the CJD patients. However, any immunolabelings using the HUC2-13 were not observed in the section from a patient with amyotrophic lateral sclerosis (ALS) as CJD-negative control. These results indicate that the mAb HUC2-13 is a suitable tool for immunological and diagnostic analyses of prion disease in humans and other mammals.     

庆大霉素单克隆抗体的制备及试剂盒的配制

目的建立庆大霉素直接竞争酶联免疫吸附分析方法。方法应用戊二醛法制备庆大霉素完全抗原,通过杂交瘤技术筛选分泌特异性庆大霉素抗体的杂交瘤细胞株,并建立庆大霉素竞争酶联免疫吸附分析检测方法。结果获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,建立了庆大霉素竞争酶联免疫吸附分析检测方法,该方法操作简单具有良好的线性、特异性和精密度;庆大霉素质量浓度在1.5625～50.0000 ng/mL范围内,呈现良好的线性,r2=0.9913,50%抑制浓度为(IC50)为7.37 ng/mL,检测限(LOD)为1.54 ng/mL,该试剂盒与链霉素等8种药物无交叉反应。结论获得3株能稳定分泌庆大霉素单克隆抗体的杂交瘤细胞株,研制的庆大霉素竞争ELISA检测试剂盒具有良好的线性、特异性和精密度。     

Diversity in the fine specificity and idiotypic profile of mouse anti-HLA-DR monoclonal antibody elicited with the syngeneic anti-idiotypic monoclonal antibody F5-830.

Four hundred and sixty-six hybridomas were generated from a BALB/c mouse immunized with the syngeneic anti-idiotypic mAb F5-830 that recognizes an idiotope in the Ag-combining site of mAb AC1.59. At an appropriate concentration, the latter reacts with a determinant expressed by HLA-DR1, DRw8, and DRw9 Ag and subtypes of HLA-DR4 and DRw6 allospecificities. Serologic and immunochemical assays identified eight anti-HLA-DR anti-anti-idiotypic mAb. They are heterogeneous in their reactivity with a panel of HLA-typed B lymphoid cells: like mAb AC1.59, the anti-anti-idiotypic mAb MA1/38, MA1/40, MA1/47, and MA1/98 recognize the determinant shared by HLA-DR1, DRw8, and DRw9 Ag and subtypes of HLA-DR4 Ag. On the other hand, the anti-anti-idiotypic mAb MA1/52, MA1/157, MA1/281, and MA1/285 have a more restricted reactivity, inasmuch as the corresponding determinant(s) is detectable on only some of the allospecificities recognized by mAb AC1.59. Each anti-anti-idiotypic mAb varies in its extent of reactivity with HLA-DR allospecificities. These results suggest differences in the fine specificity of anti-HLA-DR anti-anti-idiotypic mAb and in the structural characteristics of the mAb AC1.59 defined determinant shared by HLA-DR1, DRw8, and DRw9 Ag and subtypes of DR4 allospecificities. Furthermore, the anti-anti-idiotypic mAb are heterogeneous in terms of expression of idiotopes and of their spatial relationship with their Ag-combining site. The heterogeneity in the characteristics of anti-HLA-DR antibodies elicited with anti-idiotypic mAb F5-830 suggests that the Id cascade triggered by immunization with incompatible HLA allospecificities may account for the changes in the anti-HLA antibody specificity that have been observed in the course of an immune response to mismatched HLA alloantigens.     

金霉素单克隆抗体的制备及检测方法的建立

采用羰基二咪唑法,将半抗原金霉素(AM)分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制备金霉素免疫抗原AM-BSA和检测抗原AM-OVA,通过紫外光谱扫描检测偶联产物。采用细胞杂交瘤技术,制备抗金霉素单克隆抗体杂交瘤细胞株,建立了金霉素竞争ELISA检测方法,其灵敏度达到50ng/ml,且呈现良好的线性关系(r=0.9812),并且与其他抗生素无交叉反应。     

Detection of bluetongue virus group-specific antigen using monoclonal antibody based sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.     

A monoclonal antibody (HNo-G7) with specificity for a human nucleolar protein

A murine monoclonal antibody which reacts strongly with the nucieoli of human epithelial cells has been isolated. The antibody is of the IgM class and the antigen has a molecular weight of 45 000. The antibody appears to react with interphase chromatin only and to have specificity for epithelial cells.     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。     

抗CD52单克隆抗体中残留蛋白AELISA定量检测方法的验证

目的验证抗CD52单克隆抗体(单抗)中残留蛋白A(Protein A,Pro A)ELISA定量检测方法。方法使用Cygnus公司的Mix-N-Go Protein A检测试剂盒,对抗CD52单抗中的Pro A残留量进行检测,并验证该方法的专属性、准确度、精密度、线性及定量限。结果亲和填料(Mab Select SuRe)稀释1 000倍后仍检测到含有Pro A 1.9 ng/m L;制剂及抗CD52单抗细胞发酵液中未检出Pro A;3种不同Pro A加标质量浓度6次试验的加标回收率均在80%~120%之间;3种不同Pro A加标质量浓度重复性检测结果及中间精密度检测结果 RSD均<20%;分别对3次Pro A残余检测结果绘制的四参数标准曲线,R2均>0.990;定量限为0.16 ng/m L。结论经验证,抗CD52单抗中Pro A残留量的ELISA定量检测方法专属性好,准确度、精密度高且线性良好。     

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.     

High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody

A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed.     

A protective monoclonal antibody with dual specificity for Plasmodium falciparum and Plasmodium berghei circumsporozoite proteins.

An IgM monoclonal antibody (Mab 36) which reacts with the circumsporozoite (CS) proteins of both P. falciparum and P. berghei was isolated from Plasmodium falciparum sporozoite-immunized mice. In assays of biological activity, Mab 36 induces the CS precipitation reaction with live sporozoites and blocks the invasion of hepatoma cells by sporozoites in vitro at concentrations much lower than those observed for previously reported CS protein-specific monoclonal antibodies. Mab 36 also provided complete protection against P. berghei sporozoite challenge in mice at low doses. Linear epitope mapping revealed that the epitope specificities recognized by Mab 36 are completely encompassed by other monoclonals previously shown to be associated in vivo with protection against P. falciparum or P. berghei sporozoite infection. These results suggest that the ability to make high-affinity IgM antibody to specific CS protein repeat epitopes may be important for eliciting protection against malarial infection.