Production and characterisation of monoclonal antibodies directed against different epitopes of human thyroid stimulating hormone.

We have produced monoclonal antibodies (mAbs) to human thyroid stimulating hormone (hTSH) and selected five that specifically recognize hTSH and do not cross-react with the other human glycoprotein hormones such as luteinizing hormone (LH), chorionic gonadotropin (CG), follicle stimulating hormone (FSH). All of the antibodies were of the IgG1 subclass with affinities ranging from 5.3 X 10(8) to 1.9 X 10(10) mol-1.l; they could be assigned to two subgroups on the basis of their epitope specificity.     

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 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 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 characterisation of monoclonal antibodies to parathyroid hormone-related protein.

The production and characterisation of 17 monoclonal antibodies to human parathyroid hormone-related protein (PTH-rP) 1-34 is described. Five of the antibodies were shown to be of high avidity (Ka 4 X 10(10)-1.9 X 10(11) L/M) and able to detect 15-100 pg PTH-rP 1-34 per tube by RIA. None cross-reacted with PTH 1-34, and inhibition studies with peptide subfragments of PTH-rP 1-34 indicated that all recognise a central region extending from residues 9-18 to between residues 23 and 34. All antibodies tested cross-reacted with native PTH-rP in culture fluids from keratinocytes and squamous cancer cell lines and in human and bovine milk. The concentrations of PTH-rP 1-34 (ng/ml) in these fluids as determined by RIA were: keratinocytes 1-3, squamous cancer 0.2-2.5, human milk, up to 80. Selected antibodies coupled to Sepharose 4B were used to extract PTH-rP from biological fluids with high yields.     

Production and characterisation of monoclonal antibodies against a very small hapten, 3-methylindole

Monoclonal antibodies were produced against a very small (131.2 Da) hapten, 3-methylindole. Nine derivatives of 3-methylindole were synthesised with spacers ending in a carboxyl group, and coupled to immunogenic carriers and europium chelate labels. Almost all the antigens elicited an antihapten response, but the majority of the mAbs produced strongly recognised the spacer group and did not bind free 3-methylindole. However, specific antibodies were obtained with five immunogens. Specificity could be directed against the pyrrole ring by locating the bridging group to the aromatic moiety of the indole ring system. Any modification in the position 3 of the indole ring strongly hindered mAb binding to the compound, and the cross-reactivity of physiologically important compounds, such as tryptophan and tryptamine, was negligible for all of the mAbs. The developed hapten structures successfully focused antibody recognition to the important sub-determinants in the indole ring system. Similar constructs could also be useful in the development of antibodies against other indolic compounds.     

抗人巨细胞病毒单克隆抗体的建立及初步临床应用

应用人巨细胞病毒AD169株(HcMV-AD169)免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,获得两株(1F9 2H10)分泌抗HCMV单克隆抗体(McAb)的杂交瘤细胞系。经鉴定,两株McAb的Ig亚类为IgG1,腹水效价间接ELISA法为10~(-5)和10~(-6)。两株McAb仅与HCMV反应,而与其他疱疹病毒无反应,2H10有中和病毒作用而1F9则无。用HCMV-McAb建立抗体捕获ELISA法测定150例孕妇血清中HCMV-IgM抗体,阴阳性总符合率与间接ELISA法相比较,为99.3％(149/150)。文中尚对HCMV-McAb用途作了讨论。     

Production and characterisation of anti-theophylline monoclonal antibodies suitable for immunoassay

Spleen cells from BALB/c mice immunised with KLH-theophylline conjugate were fused with a mouse myeloma cell line P3-X63-Ag8.653, and antibody-producing hybrids were identified by enzyme immunoassay. Three cell clones were obtained, each capable of producing a unique monoclonal antibody to theophylline. Using these monoclonal antibodies, an immunoassay system for theophylline was developed.     

Development of human monoclonal antibodies against human cytomegalovirus.

Human monoclonal antibodies (HMAbs) against human cytomegalovirus (HCMV) have been developed by fusion of human spleen cells and human lymphoblastoid cell lines (NP101 and NP197). The cell line NP101 had great advantages in its high fusion frequency and the stability of the resultant hybridomas. The specificity of HMAbs was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Two of the six HMAbs obtained, which were IgG3 subclass, neutralized viral infectivity in the absence of complement. The neutralizing activity of one of these two HMAbs was enhanced in the presence of human complement, whereas the other was not. Another IgG1 subclass HMAb neutralized viral infection only in the presence of complement. The remaining three HMAbs showed no neutralizing activity. Those HMAbs may provide an important approach to studying human immune responses to HCMV. HMAbs having neutralizing activity may prove to be useful for passive immunotherapy of HCMV diseases.     

Production of human monoclonal antibodies to myeloperoxidase.

Two mouse-human heterohybridomas secreting human antibodies to myeloperoxidase (MPO) were derived from the peripheral blood of a patient who developed microscopic polyarteritis as the result of long-term treatment with hydralazine. Forty-five immunoglobulin-secreting lines were obtained from the fusion of patient lymphocytes with the CB-F7 heteromyeloma cell line. Of these, two antibodies, one IgG and one IgM, bound to myeloperoxidase in solid phase ELISA and gave a perinuclear staining pattern on ethanol-fixed human neutrophil cytospin preparations. The staining patterns were similar to those seen with serum from the patient. Antigen-inhibition studies revealed that the affinity of the IgG monoclonal antibody was 28 times higher (k = 1.4 x 10(-7)) than the IgM antibody (k = 5 x 10(-5)). Cross-inhibition studies further suggested that the two monoclonal antibodies recognized the same epitope on MPO. Of the other secreting cell lines, none produced antibody which reacted with the panel of autoantigens used for testing. Neither mononuclear antibody reacted with this panel indicating that they were not simply polyreactive natural autoantibodies. These are the first human monoclonal antibodies to native myeloperoxidase to be reported.     

Production and characterisation of a set of monoclonal antibodies against tissue-type plasminogen activator (t-PA)

To generate bispecific monoclonal antibodies, reactive to both fibrin and tissue-type plasminogen activator (t-PA), we planned to generate anti-t-PA monoclonal antibodies (mAb) which eliminate negative aspects of t-PA such as the inhibition by plasminogen activator inhibitor-type 1 (PAI-1) and the rapid clearance of t-PA. Here we report on the isolation and characterisation of a set of 13 mAb against t-PA, some of which meet the above requirements. Apart from their potential in the production of bispecific antibodies, these and the other mAb can be useful in structure-function analysis and a variety of other applications.Experiments involving PAI-1 showed that one mAB (12-5-3) reacts only with free t-PA, and prevents the subsequent binding of PAI-1 to mAb-bound t-PA. In vitro studies on the receptor mediated uptake of t-PA by hepatic cells, showed that one mAb (1-3-1) specifically inhibited the association of t-PA with liver endothelial cells. Other tests showed that mAb 7-8-4 and 12-5-3, but not 1-3-1, inhibited in vitro the enzymatic activity of t-PA.On the basis of these and other observations, we conclude that especially mAb 1-3-1, and in vivo possibly 7-8-4 and 12-5-3 may be good candidates for incorporation in bispecific monoclonal antibodies.     

Production and characterisation of a human monoclonal thyroid peroxidase autoantibody.

A human-mouse hybridoma has been produced by fusion of Hashimoto thyroid lymphocytes with the mouse myeloma line X63-Ag8.653. The cloned hybridoma secreted 2.5 micrograms per 10(6) cells per day of an IgG kappa thyroid peroxidase (TPO) autoantibody (2G4) with high affinity (2.5 x 10(9) molar-1) and specificity for human TPO. 2G4 did not react with lactoperoxidase, horseradish peroxidase or human myeloperoxidase or with porcine TPO or with human thyroglobulin. Plastic tubes coated with 2G4 bound about 50% of 125I-labelled human TPO added and the binding was inhibited by IgGs prepared from 18/18 TPO autoantibody-positive sera. This indicated that all 18 sera contained autoantibodies which recognised the same (or closely related) epitope as 2G4. Plastic tubes coated with IgGs from different TPO autoantibody-positive patient sera also bound 125I-labelled TPO but inhibition by 2G4 in this system was not complete. This suggested that the sera contained at least 2 types of TPO autoantibodies, with only one type of autoantibody reactive with the same epitope as 2G4.     

Production and characterisation of monoclonal antibodies specific for staphylococcal enterotoxin B

We have generated monoclonal antibodies (MABs) to staphylococcal enterotoxin B (SEB) in BALB/c mice. Five out of 20 clones which produce anti-SEB MABs have been characterised. Among them, three produce IgG1/kappa, one produces IgM/lambda, and one apparently produces both IgG1/lambda and IgM/lambda MABs. The anti-SEB titres of ascites fluids range from 3200 to greater than 819200 by ELISA. All of the MABs analysed thus far neutralise the mitogenic response of BALB/c splenocytes to a suboptimal dose of SEB. Also, the induction of suppressor cells by SEB in vitro is reversed by pre-incubating SEB with these MABs. Limited digestion with chymotrypsin, trypsin or Staphylococcus aureus V8 protease yields peptide fragments which have been tested by Western-blot analysis. MABs 1FD7 and 2GD9 are specific for the carboxy-terminal end of SEB, and have a similar, but not identical, binding epitope. MABs 2DA3 and 2HA10 bind to intact SEB but not to cleaved products, and are probably specific for antigenic determinants altered by the cleavage or by the denaturing conditions of the electrophoresis, or by both.     

Production and characterization of monoclonal antibodies against amino-terminus of human alpha-atrial natriuretic polypeptide

Monoclonal antibodies directed against human, alpha-atrial natriuretic polypeptide (alpha-ANP; Human, 1-28) were obtained by somatic cell fusion between P3-X63-Ag8.653 myeloma cells and spleen cells from a BALB/c mouse immunized with human, alpha-ANP selectively coupled to keyhole limpet hemocyanin. From the analysis of polyclonal sera with respect to determinant specificity before the fusion, the strategy was primarily used to pick up monoclonal antibody specific for the N-terminal residues of human, alpha-ANP. Screening of antibodies in the hybridoma culture supernatants were performed by binding to iodinated synthetic human, alpha-ANP. Two stable clones producing anti-human, alpha-ANP antibodies, designated 13A1 and 10B1, were obtained by the limiting dilution technique. The ability of ANP(Rat, 1-28) to inhibit binding of 125I-human, alpha-ANP to these antibodies was almost equipotent to ANP(human, 1-28). However, ANP fragments (Human, 7-28) and (18-28) did not compete the binding completely. These results suggest that both 13A1 and 10B1 monoclonal antibodies can specifically recognized N-terminus of human, alpha-ANP, and may be a useful tool to investigate receptor binding of human, alpha-ANP by the antagonizing effect.     

Production of monoclonal antibodies against gastric parietal cell antigens.

Two mice DBA/1 were each immunized with a single injection of one million enriched parietal cells in the hind foot pads. Monoclonal antibodies to be used as research tools in studies on regulatory mechanisms in gastric parietal cells were obtained after fusion of mouse myeloma cells (SP2) with cells from the popliteal lymph nodes of the mice. Twelve hybridomas produced antibodies reactive with structures only present in parietal cells as assessed by immunohistochemistry of oxyntic mucosa sections. Three hybridomas were subcloned and the antibodies produced by them, designated as PC4, PC8, and PC117, were characterized. In an enzyme-linked immunosorbent assay, all antibodies reacted with H,K-ATPase-containing vesicles. The antibody PC8 recognized a 94 kDa protein after immunoblotting of H,K-ATPase-containing vesicles and all antibodies precipitated a 94 kDa protein from [125I]H,K-ATPase-containing vesicles. The antibodies PC4 and PC117 recognized extracellular structures with a polarized distribution in viable, purified parietal cells. The results suggest that the structure recognized by all three antibodies is the alpha-subunit of the H,K-ATPase. The antibodies produced by another hybridoma, PC43, recognized a structure present in parietal and surface epithelial cells of the oxyntic mucosa. In an enzyme-linked immunosorbent assay, they reacted with a high-activity carbonic anhydrase which had been affinity-purified from pig oxyntic mucosa and they recognized a 30 kDa protein after immunoblotting. Thus, monoclonal antibodies against both intracellular and extracellular parietal cell structures were obtained after immunization with a small number of parietal cells.     

Production of human allotype-specific anti-CD45 monoclonal antibodies.

The aim of this work was to raise allotype specific monoclonal antibodies to human CD45, with the long-term objective of producing a reagent which could be used to prolong graft survival in renal transplantation through removal of passenger leukocytes from the graft. At present there are no anti-CD45 monoclonal antibodies able to distinguish between host and donor leukocytes. An in vitro immunisation technique has been developed through which donated human leukocytes are sensitised to CD45 prior to fusion with a myeloma cell line. IgM was produced by all the anti-CD45-positive clones. Flow cytometric analysis using these antibodies showed their ability to differentiate between blood from individual donors, indicating the existence of allotypic forms of human CD45, in conformity with the findings in rats and pigs. Therefore, a reagent which could be used in renal transplantation is a technical possibility.     

Production and characterisation of antibodies against isolated placental retroviral-like particles

The mammalian genomic DNA is known to contain a variety o f endogenous proviruses but their expression is usually restricted to reproductive tissues such as the placenta and a variety of human tumour cells. More definitive characterization of retroviral gene products has been hampered by unavailability of specific biological reagents. In this study, polyclonal and a total of six monoclonal antibodies (mAbs) were raised against endogenous (intact) retroviral particles isolated from human placental villous tissue. These antibodies were characterized using immunohistochemical and biochemical methods. Five polyclonal antibodies and one mAb (RV1-17) showed strong specific immunohistochemical and immunogold staining with submembraneous structures within placental syncytiotropblast. The reactivity of these antibodies was consistent with the pattern of apical syncytiotrophoblastic budding of retroviral particles previously reported in ultrastructural analyses.     

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.