Production and characterization of monoclonal antibodies against human thyroglobulin

Spleen cells of Biozzi-HR mice immunized with human thyroglobulin (hTg) were fused with P3-X63-Ag8.653 mouse myeloma cells. Twenty monoclonal antibodies (MAbs) selected by an enzyme immunoassay (indirect ELISA) were produced, purified and characterized. The equilibrium association constant (Ka) of one of the MAbs, determined by Scatchard analysis of the ELISA data, was found to be 2 X 10(9) M-1; the Ka of the other MAb, estimated from titration curves by comparison with the aforementioned MAb, ranged from 8 X 10(9) M-1 to 6 X 10(7) M-1. The reaction between the MAb and hTg was not inhibited by thyroxin (T4), triiodothyronine (T3) and triiodothyropropionic acid (DT3). Species specificity of the MAb was studied using bovine and porcine Tgb. The topology of the MAb was investigated by competitive inhibition immunoassays. Seven distinct antigenic regions were identified.     

Production and characterization of high-affinity monoclonal antibodies against morphine

Twelve hybridoma cell lines producing MAbs against morphine were established by using morphine hemisuccinate-conjugated bovine serum albumin as an immunogen. The MAbs belonged to the IgG1 subclass with kappa- or lambda-chains. The association constants of the antibodies ranged from 4.6 x 10(8) to 4.7 x 10(10) (M-1). These antibodies revealed slightly different cross-reactivities with various agonistic opiates and antagonists. In general, the antibodies were strongly cross-reactive with the opiate agonists, codeine, ethylmorphine, dihydromorphine and dihydrocodeine, while their cross-reactivities with norcodeine and the opiate antagonists, naloxone and naltrexone, were weak. The cross-reactivities with dihydromorphinone, dihydrocodeinone, naloxone, naltrexone, dextromethorphan and homatropine varied from clone to clone. Interestingly, certain MAbs displayed weak but significant cross-reactivities with the synthetic opiate, meperidine. However, none of the antibodies was cross-reactive with the opioid peptides, beta-endorphin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalinamide. Radioimmunoassay for morphine using one of the antibodies (MOR 131.5.13) was shown to be sufficiently sensitive (IC50 = 0.1 nM) for the purposes of forensic analysis of morphine. This set of monoclonal anti-opiate antibodies is assumed to be suitable for analyzing the structure-function relationship in the hapten-antibody interaction, since the antibodies revealed similar but not identical cross-reactivities with various morphine related compounds.     

Production and partial characterization of monoclonal antibodies to Mycobacterium leprae.

Monoclonal antibodies to Mycobacterium leprae were produced by the fusion of BALB/c splenocytes and lymph node cells to BALB/c myeloma (NSI/1) cells. Eleven monoclonal antibodies were characterized as to their reactivity with M. leprae and 18 other mycobacterial species by enzyme-linked immunosorbent assay and immunofluorescence. Two monoclonal antibodies reacted only with M. leprae, and the other nine showed unique patterns of reactivity by enzyme-linked immunosorbent assay. One monoclonal antibody (IIH9) reacted with a 68,000-dalton protein present in extracts from M. leprae, M. tuberculosis H37Rv, M. gastri, and M. smegmatis. Potential uses for these antibodies in serological tests and immunochemical analyses are discussed.     

Production and characterization of murine monoclonal antibodies against human podocalyxin

Podocalyxin (podxl) is a protein with a peptide bone of approximately 55.5 kDa that undergoes a post-translational glycosylation, yielding a final molecular mass from approximately 145 to approximately 200 kDa. This protein is normally found covering the vascular side of the epithelial glomerular cells, the podocytes, and its presence is essential to maintain a normal renal function. It has also been reported in other cells and tissues although its function has not been yet clarified. The carboxy-terminal intracellular domain of podxl is nearly 100% identical in most species; however, the ectodomain shows considerable variations although the cysteine residues are conserved. Detection of this protein is elusive, most likely due to differences in post-translational modifications. We aimed at producing murine monoclonal antibodies against human podxl. Immunization with Chinese hamster ovarian -hpodxl-green fluorescence protein live cells yielded five different monoclonal antibodies that were characterized by enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis/western blot, flow cytometry, immunohistochemistry, and immunoprecipitation. The different behavior of these antibodies suggests that some of them may react against epitopes masked by different glycosylated protein moieties.     

Production and characterization of monoclonal antibodies against human airway mucins

The objective of this study was to generate and characterize monoclonal antibodies (MAbs) against human airway mucins, and therefore, should serve as a useful tool in studying the regulation of airway mucins in various physiological or pathological situations of human airway. As an antigen, we used a high molecular mass mucin preparation purified from the sputum of normal human subjects. Two monoclonal hybridomas, namely MAbs HM02 and HM03 were obtained and they showed strong immunoreactivity against purified or crude mucin in sputum or bronchial washing of normal human subject. With the high immunoreactivity of these MAbs, mucin contents could be analyzed with more than 100-fold dilution of human airway secretion. The antibodies recognized carbohydrate epitopes because their immunoreactivity was completely abolished by treatment of the mucin with 5 mM periodate. Further characterization of MAbs HM02 and HM03 showed that: (1) they belong to the IgM type; (2) they bind to high molecular mass mucins based on Western blot; (3) they could indirectly immunoprecipitate human airway mucin and as we know, this is the first to demonstrate immunoprecipitation of human airway mucin with anti-human mucin antibodies; and (4) they bind to the goblet cell in airway epithelium as well as some submucosal glands based on immunohistochemistry. Therefore, MAbs HM02 and HM03 should be able to serve as an invaluable tool in studying the regulation of airway mucins in various physiological and pathological situations of human airway.     

Production and characterization of monoclonal antibodies directed against pseudorabies virus

Hybridoma cell lines producing monoclonal antibodies to pseudorabies virus (PRV) were established. The monoclonal antibodies were characterized with respect to their antigenic specifications and biological activities. Two monoclonal antibodies immunoprecipitated the 50 kDa PRV glycoprotein (gp50) and two immunoprecipitated the 82 kDa glycoprotein (gp82). The monoclonal antibodies were used to analyze the biological roles of these two glycoproteins. One monoclonal antibody directed against each glycoprotein did not require complement for in vitro viral neutralization while the other monoclonal antibody directed against the glycoprotein required complement for neutralization. The monoclonal antibodies against gp50 were shown to be directed against different epitopes within the glycoprotein. In contrast, the monoclonal antibodies against gp82 were shown to be directed against the same antigenic site on the glycoprotein. In vivo passive immunity studies in mice showed that monoclonal antibodies directed against either gp50 or gp82 could be protective.     

阪崎肠杆菌单克隆抗体的制备与特性鉴定

目的:制备抗阪崎肠杆菌的单克隆抗体并对其生物学特性进行鉴定。方法:采用灭活的阪崎肠杆菌菌体为抗原,免疫BALB/c小鼠,取血清效价高的小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,间接ELISA法筛选阳性杂交瘤细胞,mAb亚类检测试剂盒鉴定单克隆抗体的亚型,通过Western blot和间接ELISA法鉴定该单克隆抗体的特性、效价及mAb相对亲和力。结果:获得2株能稳定分泌抗阪崎肠杆菌mAb的杂交瘤细胞株,分别命名为1H7、2B12,抗体Ig亚类分别为IgG1和IgG2b;交叉反应显示单抗具有良好的特异性;ELISA分析表明制备的单抗效价在1×107～2×107,相对亲和常数达109L/mol,染色体鉴定分别为104和106条,符合杂交瘤细胞的特性。结论:抗阪崎肠杆菌单克隆抗体的成功制备为其快速检测方法的建立奠定了基础。     

Production, characterization, and epitope mapping of monoclonal antibodies against human polydeoxyribonucleotide kinase

Polydeoxyribonucleotide kinase (PNK) is a mammalian DNA repair enzyme that has the capacity to phosphorylate 5' DNA termini and dephosphorylate 3' DNA termini. A series of murine monoclonal antibodies (MAbs) was raised against the full-length recombinant human PNK. Seven of these antibodies were selected and characterized by enzyme immunoassay, Western blot analysis, and their capacity to immunoprecipitate PNK. The epitope location was defined by cyanogen bromide digestion and by using a truncated PNK for Western blot analysis. All of the MAbs recognize a single 60-kDa protein in human cell extracts. PNKs from calf, monkey, and Chinese hamster cell and tissue extracts were also detected by some or all of the MAbs. These antibodies can be successfully used for the cellular, biochemical, and functional analysis of PNK in different mammalian cell lines.     

Production and characterization of monoclonal antibodies against sterigmatocystin o‐methyltransferase

Monoclonal antibodies (MAbs) against sterigmatocystin (ST) O‐methyltransferase (OMTase), an enzyme responsible for the conversion of ST to O‐methylsterigmatocystin (OMST) in the late stage of aflatoxin biosynthesis, were produced by fusion of P3/NS‐1/1‐AG4–1 myeloma cells with spleen cells isolated from Balb/c mice that had been immunized with a purified ST‐OMTase preparation. Two clones, 1D9 and 8F11, which produced antibodies showing highest affinity toward ST‐OMTase, were chosen for antibody production and characterization. Enzyme‐linked immunosorbent assay (ELISA) analysis of various fungal extracts showed that the MAbs obtained from either ID9 or 8F11 were highly specific for the ST‐OMTase. Results of ELISA analysis using MAb obtained from clone 8F11 correlated well with those of ST‐OMTase activity in fungal extracts determined by spectrofluorometry, and only MAb 8F11 was capable of neutralizing part of the ST‐OMTase activity. Immunochemical analysis of various fungal extracts with MAb 1D9 revealed that the antibodies primarily reacted with the 40‐kDa ST‐OMTase purified with DEAE‐Sephadex chromatography, but reacted with the 46‐kDa ST‐OMTase species in the crude protein extracts of Aspergillus parasiticus.     

Production, characterization, and uses of monoclonal antibodies against recombinant nucleoprotein of elk coronavirus

This is the first report of the production of monoclonal antibodies against elk coronavirus. The nucleoprotein gene of elk coronavirus was amplified by PCR and was cloned and expressed in a prokaryotic expression vector. Recombinant nucleocapsid protein was used to immunize mice for the production of hybridomas. Twelve hybridomas that produced monoclonal antibodies against the nucleocapsid protein of elk coronavirus were selected by an indirect fluorescent-antibody test, an enzyme-linked immunosorbent assay, and a Western blot assay. Ten of the monoclonal antibodies were of the immunoglobulin G1 (IgG1) isotype, one was IgG2a, and one was IgM. All had kappa light chains. By immunohistochemistry four monoclonal antibodies detected bovine coronavirus and elk coronavirus in formalin-fixed intestinal tissues. Antinucleoprotein monoclonal antibodies were found to be better at ruminant coronavirus detection than the anti-spike protein monoclonal antibodies. Because nucleoprotein is a more abundant antigen than spike protein in infected cells, this was not an unexpected finding.     

Production and characterization of monoclonal antibodies against major allergens of American cockroach

From several fusion experiments between spleen cells obtained from BALB/c mice immunized with partially purified Cr-PI of American cockroach and NS-1 cells, growth was observed in many wells. Seven stable subclones secreting monoclonal antibodies (mAbs) against Cr-PI, as determined by enzyme-linked immunosorbent assay (ELISA) with high absorbance values and immunoblot analysis, were obtained. All seven mAbs were characterized as IgG1 subclass by immunodiffusion, and reacted strongly with 72 kilodaltons (kD) of Cr-PI which have been identified as a major allergen of American cockroach. Six mAbs were found to have similar epitope specificities against Cr-PI by ELISA. The remaining mAb was found to have different epitope specificities with others. Interestingly, all mAbs did not react with any components of crude extracts of Oriental and German cockroaches as determined by immunoblot analysis and ELISA. A mAb-based double-antibody sandwich ELISA was developed, and the ELISA was dose-dependent and capable of detecting as little as 140 ng of Cr-PI allergen.     

Production and partial characterization of monoclonal antibodies to four Chlamydia psittaci isolates.

Monoclonal antibodies (MAbs) were produced to four Chlamydia psittaci isolates: NJ1 and TT3 from turkeys, VS1 from a parakeet, and B577 from an ovine abortion. MAbs were tested for reactivity with each isolate by the indirect immunofluorescent antibody technique and for neutralization by an inclusion reduction neutralization technique in tissue culture. Two genus-specific and 14 serovar-specific MAbs were produced. Genus-specific MAbs reacted with all avian and mammalian isolates; however, each failed to neutralize its homologous chlamydial isolate. Turkey isolates NJ1 and TT3 were antigenically similar; serovar-specific MAbs produced to each reacted equally with both isolates yet showed little or no reaction with other serovars. Serovar-specific MAbs to the parakeet and abortion isolates were distinct; no cross-reactions were seen with other serovars. None of the serovar-specific MAbs reacted with an ovine arthritis isolate. Of the 14 serovar-specific MAbs, 13 partially neutralized homologous strains with or without the addition of complement. Because of the high specificity, the serovar-specific MAbs used with the immunofluorescence technique provided a rapid and precise method to identify three serovars of C. psittaci.     

Production and characterization of monoclonal antibodies against serotype strains of Pseudomonas aeruginosa

Monoclonal antibodies against 12 of the 17 IATS serotype strains of Pseudomonas aeruginosa were produced. Eighty-seven hybridoma clones were isolated, and the antibodies secreted were found to be reactive with both Formalin-fixed whole cells and purified lipopolysaccharide of homologous strains in enzyme-linked immunosorbent assays. Among these monoclonal antibodies, the predominant antibody class was immunoglobulin M (IgM) (76%), although antibodies of the IgG2a and IgG3 isotypes were also produced. The monoclonal antibodies could further be divided into two groups based on their ability to agglutinate whole cells of homologous strains. The agglutinating monoclonal antibodies were found to immunoblot with the O side chains of homologous lipopolysaccharide, while the nonagglutinating monoclonal antibodies were found to be reactive with outer membrane protein-associated lipopolysaccharide. The applicability of monoclonal antibodies for serotyping was examined, and several antibodies were found to agglutinate whole cells and immunoblot with the O antigen of corresponding serotypes of clinical isolates from cystic fibrosis patients. In conclusion, a set of monoclonal antibodies against the IATS serotype strains of P. aeruginosa have been produced. These antibodies represent a bank of invaluable immunological reagents which may have application in serotyping, epitope mapping, lipopolysaccharide structural determination, and studies of protection against P. aeruginosa.     

Production and characterization of monoclonal antibodies against binary ethylenimine inactivated Nipah virus

Nipah virus, a zoonotic paramyxovirus which emerged recently was chemically inactivated using binary ethylenimine (BEI). The inactivated virus was concentrated and purified by sucrose gradient centrifugation. The gradient fractions were examined by electron microscopy and Western immunoblot, and gradient fraction containing mainly Nipah matrix (M) and nucleocapsid (N) proteins was used for immunizing BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones identified five strongly positive clones producing IgG monoclonal antibodies (mAbs) reactive to the Nipah virus antigen. The protein specificity of these mAbs was determined by Western immunoblot using Nipah virus and recombinant Nipah virus proteins expressed in mammalian cells. Four mAbs reacted with Nipah N protein and one reacted with Nipah M protein. None of the mAbs neutralized Nipah virus infectivity in vitro. However, all mAbs recognized Nipah virus in ELISA and immunofluorescence assay. F45G2 mAb was most suitable for immunohistochemistry on long term formalin-fixed Nipah virus infected swine tissues. Three of the anti-nucleocapsid mAbs (F45G2, F45G3 and F45G6) showed cross-reactivity with closely related Hendra virus N protein in both immunofluorescence and Western Immunoblot assays. Two of the mAbs were specific for the Nipah virus only, F45G4 (anti-N) and F45G5 (anti-M), and could be used in the primary identification of Nipah virus. The use of these immunoreagents to develop new diagnostic assays is discussed.     

Production and characterization of monoclonal antibodies against DNA single ring diamines adducts

The synthetic conjugate of the genotoxic compound 2,4 diaminotoluene (2,4 DAT) with gelatin (2,4 DAT-GEL) was employed to elicit specific antibodies directed against a restricted class of aromatic diamines. Using this immunogen, mouse monoclonal antibodies (MAbs) have been produced. These MAbs have been characterized and used in ELISA to detect 2,4 DAT covalently linked to biopolymers. The MAbs could bind to different synthetic 2,4 DAT-biopolymer adducts as well as to DNA from rats treated in vivo with the aromatic diamine, but they did not react with gelatin or biopolymers alone. The use of these MAbs has been investigated in order to develop a highly sensitive test to detect adducts of this genotoxic compound with nuclear DNA.     

Production and characterization of monoclonal antibodies directed against Bordetella pertussis lipopolysaccharide.

Hybrid cell lines producing monoclonal antibodies against Bordetella pertussis lipopolysaccharide (LPS) were established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA) and ELISA-inhibition experiments with LPS and delipidated polysaccharide fragments (PS-1 and PS-2) prepared from B. pertussis LPS. Monoclonal antibody 9-1-H5 reacted with B. pertussis LPS only, whereas monoclonal antibodies 6-4-H6 and 9-2-A8 reacted with PS-1 and PS-2 as well as B. pertussis LPS. The antibodies did not react with LPS prepared from B. parapertussis and B. bronchiseptica in an LPS-specific ELISA. A monoclonal antibody-based sandwich ELISA was developed for detection of B. pertussis LPS. This assay had a detection limit of B. pertussis LPS in concentrations ranging from 0.16 to 0.32 microgram/ml. The assay was also shown to be specific for the detection of whole B. pertussis bacteria. No cross-reactions were observed with strains of Branhamella catarrhalis, Neisseria meningitidis, Streptococcus miteor, Haemophilus influenzae, or Legionella pneumophila. The monoclonal antibodies might be useful for the detection of soluble antigens and whole bacteria in clinical samples and for studies of the immunochemical structure of B. pertussis LPS.     

Production and characterization of monoclonal antibodies against non-A1c glycated hemoglobin

Hybridomas secreting monoclonal antibodies specific for hemoglobin nonenzymatically glycated in the non-A_1c position were produced by fusion of SP 2/0 myeloma cells with spleen cells from BALB/c mice immunized with nonenzymatically glycated hemoglobin prepared from human erythrocytes. Wells containing hydridomas secreting antibodies against glycohemoglobin were identified by binding, in an enzyme-linked immunosorbent assay, to purified glycated hemoglobin. The colony designated E85, which secreted antibodies discriminating between glycated versus unglycated hemoglobin, was cloned at least four times by limiting dilution and used for further study, performed with purified monoclonal antibody. Specificity of E85 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted with glycated hemoglobin but not with hemoglobin A_1c or with unglycated hemoglobin. Immunoblotting of human plasma with E85 on nitrocellulose yielded no reactive proteins, indicating site specificity for glycated epitopes residing in hemoglobin but not in other nonenzymatically glycated proteins present in plasma. E85 differs from other antibodies raised against glycated hemoglobin and other glycated proteins, which recognize hemoglobin glycated at the N terminal valine of the β chain (HbA_1c) or which recognize glycated residues only after reductive conversion to glucitollysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against a physiologic (unreduced) form of non-A_1c glycohemoglobin, and for the glycated epitope when it resides in glycohemoglobin but not in other proteins or in hemoglobin A_1c.     

Production and characterization of monoclonal antibodies directed against guinea pig IgG subclasses

Monoclonal antibodies directed against guinea pig IgG subclass-specific epitopes have been generated and characterized. Three monoclonal antibodies designated GP1, GP2 and GP3 have been compared to rabbit polyclonal reagents for specificity against purified IgG subclass antigens and for the detection of herpes simplex-specific IgG subclasses, from infected guinea pig, by an indirect ELISA. The monoclonal antibodies were shown to have greater specificity than the corresponding polyclonal reagents.     

Production and characterization of monoclonal antibodies against pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPP-A) was found to be a good first trimester maternal serum marker, together with free beta-human chorionic gonadotrophin (HCG) subunits, for the biochemical screening of fetal trisomy 21 (Down's syndrome). We have raised monoclonal antibodies (mAbs) against PAPP-A purified from human pregnancy serum. The different antibodies were characterized biochemically by Western blot analysis and in terms of specificity (reaction with non-pregnant and male serum). Their performance in Down's syndrome screening was assessed in comparison with an existing enzyme-linked immunosorbent assay method after labelling of the different mAbs with biotin or horseradish peroxidase. A pair of mAbs was eventually chosen for a double-antibody sandwich protocol. The selected combination was found to have a significantly increased specificity (P = 0.0116) over the method using (purified) polyclonal antibodies, together with slightly increased sensitivity. In our limited number of Down's syndrome pregnancy samples (n = 17) and controls (n = 18), the medians as well as the multiples of the median values (for the affected cases) were comparable between the two methods described.     

Production and characterization of monoclonal antibodies against 3, 4, 15-triacetylnivalenol and 3,15-diacetyldeoxynivalenol

Mouse monoclonal antibodies against analogues of two mycotoxins (deoxynivalenol (DON) and nivalenol (NIV)) commonly found in cereal crops were isolated and used to develop an immunological detection assay. We immunized BALB/c mice with 4,15-diacetylnivalenol-3-O-hemisuccinate conjugated to keyhole limpet hemocyanin and isolated 21 hybridomas secreting monoclonal antibody (MAB) which reacted with 3,4,15-triacetylnivalenol (3,4,15-TANIV). These MABs were classified into two distinct types of reactivity against analogues of NIV and DON. Two monoclonal antibodies, KTM-205 and 208, were highly specific for 3,4,15-TANIV, whereas KTM-233, 239 and 240, cross-reacted with both 3,15-diacetyl deoxynivalenol (3,15-DADON) and 3,4,15-TANIV to the same extent. Acetylation under mild conditions can derivatize nivalenol and deoxynivalenol to 3,4,15-TANIV and 3,15-DADON, respectively. Thus the competitive indirect ELISAs for measuring nivalenol and deoxynivalenol were developed using these monoclonal antibodies. The detectable level of 3,4,15-triacetylnivalenol and 3,15-diacetyldeoxynivalenol was about 0.3 to 1000 pg/mL in buffer by the indirect competitive ELISA with KTM-240. Thus we have successfully generated useful MABs for an ELISA for the detection of nivalenol and deoxynivalenol in agricultural crops.