Production Of Specific Monoclonal Antibodies Research Articles

Production and characterization of a new specific monoclonal antibody against A-isoform of SALL4: A novel emerging testicular cancer marker.

SALL4 transcription factor plays an important role to maintain the pluripotent and self-renewal of embryonic stem cells. It contributes to the growth of many cancers and embryonic development. With the exception of spermatogonia, SALL4 expression is silenced in most adult tissues after birth; nevertheless, it is re-expressed in a subset of different solid malignancies. SALL4 is a new, precise biomarker for testicular germ cell cancers that was just introduced. The whole isoform of SALL4 is called SALL4-A. Regarding the lack of antibody against human SALL4 isoforms, the pattern of expression, the role of each isoform remain unknown. Furthermore, in isoform specific evaluations, we aimed, for the first time, to produce and characterize mAb against human SALL4-A. Immunization of mice were performed with a selected 33-mer synthetic peptide of SALL4-A conjugated with KLH. Hybridoma cells were screened by ELISA for positive reactivity with SALL4-A peptide. From the ascites fluid of mice that had been injected with hybridoma cells, anti-SALL4-A mAbs were isolated using a protein G column. Reactivity of the mAbs was evaluated using the peptide and SALL4-A recombinant protein by ELISA and IHC on testicular cancer tissue as positive control, and normal kidney, stomach and prostate tissues as negative control. The produced mAb could well detect SALL4-A in testicular cancer tissues using IHC, while the reactivity was negative in normal kidney, stomach and prostate tissues. Using ELISA, the mAb affinity for the peptide and SALL4-A recombinant protein was assessed, and it was shown to be reasonably high. The mAb detected SALL4-A in nucleus and cytoplasm of several cancer cells and spermatogonia in testicular cancer tissue. In addition, it could recognize SALL4-A recombinant protein. Our produced monoclonal antibody against isoform-A of human SALL4 can specifically recognize SALL4-A using either IHC or ELISA. We hope that this mAb could help researchers in isoform-specific study of human SALL4.

Production of a common epitope specific anti-ankyrin monoclonal antibody

Background: Ankyrin (Ank) is a protein family with crucial roles in retaining normal cellular physiology. In addition, ankyrin offers the potential as a non-antibody binder against various biomolecules. The designed ankyrin repeat protein (DARPin) selected from phage display libraries is useful for molecular detection and therapy. Monoclonal antibodies (mAbs) specific to the common epitope of DARPin are required to detect protein-protein interaction. Objectives: This study aimed to establish mAbs against common antigenic determinant of ankyrins for further application in immunological techniques. Materials and methods: Ank1D4 monomer and dimer were generated in the Escherichia coli expression system for immunogen preparation and validation of established mAbs. The binding activity of anti-Ank mAb obtained from different hybridoma clones was characterized using Ank1D4 by indirect ELISA. Candidate anti-Ank mAbs were validated for their cross-reactivity against irrelevant ankyrin (Ank2D3). The binding kinetic of mAbs from three candidate hybridoma clones (Ank-54, Ank-59, and Ank-94) was evaluated using bio-layer interferometry (BLI). The highest affinity clone (Ank-94 mAb ) was further validated for its specificity against Ank1D4 and dimeric Ank1D4 using indirect ELISA. The interaction of three anti-Ank mAbs and ankyrins was compared by western immunoblotting analysis. The specificity of Ank-94 mAb was determined using a closely related scaffold, i.e., alpha-helicoidal HEAT-like repeat protein scaffold (αRep) by indirect ELISA. Ankyrins were detected by sandwich ELISA using Ank-94 mAb. Results: The culture supernatant from hybridoma clones were characterized for their anti-ankyrin binding properties. Using indirect ELISA, three clones exhibited positive reactivity against the immunized ankyrin antigen (Ank1D4). The interactive epitope was found to rely on common antigenic determinants found in Ank1D4, dimeric Ank1D4, and an irrelevant ankyrin, Ank2D3. The immunoblotting results suggest that all mAbs interact with the sequential epitope of ankyrins. The cross-reactivity of Ank-94 mAb was not observed with αRep. Ank-94 mAb was selected for further purification and evaluation of binding properties due to its highest degree of binding affinity against Ank1D4. Conclusion: The establishment of a novel Ank-94 mAb could be a valuable research tool in tracing the target of DARPins or developing immunoassays. Ank-94 mAb is superior over formerly produced Ank mAbs since it recognizes a common epitope on DARPins and relies on sequential epitope. Ank-94 mAb has no cross-reactivity with another scaffold, αRep.

Production of a common epitope specific anti-ankyrin monoclonal antibody

Background: Neck pain is associated with scapular dyskinesis and impaired axio-scapular and shoulder muscle activity and function. However, there is little research on force steadiness, specifically during shoulder motion in patients with neck pain with scapular dyskinesis. Its relationship with characteristics of neck pain is also unknown. Objectives: to investigate force steadiness at 20% and 50% of MVC of shoulder abduction (30 degrees) in persons with neck pain with scapular dyskinesis compared to asymptomatic controls and to determine its relationships with characteristics of neck pain. Materials and methods: Fifty-two women and men (26 neck pain with scapular dyskinesis and 26 asymptomatic controls) were recruited to the study. Force steadiness of 30 degrees of shoulder abduction was measured at 20% and 50% of maximal voluntary contraction (MVC) and coefficient of variation (CV) of force values were calculated. Characteristics of neck pain included neck pain intensity, duration and disability and upper limb disability. Results: Participants in neck pain had significantly reduced force steadiness of isometric shoulder abduction at 20% and 50% of MVC compared to the control group (p<0.05, η2p = 0.10 and p<0.001, η2p = 0.26, respectively). No between-group differences in shoulder abduction MVC were observed. There were no correlations between force steadiness and characteristics of neck pain (p>0.05). Conclusion: Patients with neck pain with scapular dyskinesis had reduced shoulder abduction force steadiness. This may reflect impaired shoulder/scapular sensory-motor control associated with neck pain. The relationships between force steadiness and characteristics of neck pain are still needed to be explored in further studies.

Engineering of Chimeric Proteins to Enhance Immunogenicity for the Production of High-Affinity Specific Monoclonal Antibodies

A wide range of biomedical research and diagnostic applications utilize high-affinity antibodies, the production of which depends on effective immunogens. However, some proteins are intrinsically weak immunogens incapable of eliciting a strong immune response for the production of useful antibodies. To remedy this problem, weak immunogens are conjugated to exogenous carrier proteins to increase immunogenicity and provide an adjuvant effect. In the present study, we tested an alternative strategy of using chimeric fusion protein constructs to enhance immunogenicity. We first applied the N-terminal domain of glucose-regulated protein 96 (GRP-NTD), a protein with newly identified activities in autoimmune diseases, as a carrier to present several experimentally identified weak immunogens: cardiac troponin C (cTnC), the N-terminal peptide of cardiac troponin T (N69), and the C-terminal peptide of calponin 2 (C85). Antigen peptides were engineered to the N-terminus of the carrier, expressed in E. coli, large scale purified, and injected intramuscularly into adult mice without adjuvants. The effects of immunization were assessed by ELISA titration of serum samples after three immunizations and/or the outcome of high-affinity specific monoclonal antibodies (mAb) in hybridoma development. The results demonstrate significantly enhanced immunogenicity for cTnC and N69, but not C85. Based on an observation that cloned mouse ApoE protein served as an effective carrier for the production of mAb against a small peptide, we constructed a C85-ApoE fusion protein for hybridoma development. Surprisingly, C85-ApoE immunization was not effective for C85 but yielded high-affinity mAbs against mouse ApoE, an endogenous protein abundant in host plasma. The results suggest a potentially new strategy of using chimeric immunogens to improve the production of specific antibodies.

Production of specific monoclonal antibody against CD3ε in olive flounder

T cell activation is initiated by the binding of antigen to the specific T cell receptor (TCR) that triggers the formation of CD3-signal transduction complex and ends in proliferation and differentiation of antigenspecific T cells. CD3, an essential component of the CD3-TCR complex, has been classified into γ, δ, e, and ζ chains. The CD3γ, CD3δ, and CD3e chains are highly related to cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. In mammals, T cells were characterized by detecting one of the CD3 molecules through the use of specific antibody for CD3e. This suggests that CD3 molecules can be used as markers in identifying T cells in teleost fish; hence, it is essential to produce CD3-specific antibodies that could serve as T cell markers in fish. Here, we produced a CD3e-specific monoclonal antibody. Western blot result showed a distinct band at approximately 15 kDa detecting the CD3e antibody while MALDI-TOF analysis, clearly identified the band as olive flounder CD3e. Once a monoclonal antibody has been produced, it can be used to detect the presence of a substance specific to it. The results from this study revealed that the CD3e monoclonal antibody produced could detect CD3e in olive flounder. However, cell markers for T cell differentiation in olive flounder were not fully investigated yet so we hope that this antibody could be useful in elucidating T cell differentiation in teleosts.

Development of a Stable Cell Line, Overexpressing Human T-cell Immunoglobulin Mucin 1.

Recent researches have demonstrated that human T-cell immunoglobulin mucin 1 (TIM-1) glycoprotein plays important roles in regulation of autoimmune and allergic diseases, as well as in tumor immunity and response to viral infections. Therefore, targeting TIM-1 could be a potential therapeutic approach against such diseases. In this study, we aimed to express TIM-1 protein on Human Embryonic kidney (HEK) 293T cell line in order to have an available source of the TIM-1 antigen. The cDNA was synthesized after RNA extraction from peripheral blood mononuclear cells (PBMC) and TIM-1 cDNA was amplified by PCR with specific primers. The PCR product was cloned in pcDNA™3.1/Hygro (+) and transformed in Escherichia coli TOP 10 F'. After cloning, authenticity of DNA sequence was checked and expressed in HEK 293T cells. Finally, expression of TIM-1 was analyzed by flow cytometry and real-time PCR. The result of DNA sequencing demonstrated correctness of TIM-1 DNA sequence. The flow cytometry results indicated that TIM-1 was expressed in about 90% of transfected HEK 293T cells. The real-time PCR analysis showed TIM-1 mRNA expression increased 195-fold in transfected cells compared with un-transfected cells. Findings of present study demonstrated the successful cloning and expression of TIM-1 on HEK 293T cells. These cells could be used as an immunogenic source for production of specific monoclonal antibodies, nanobodies and aptamers against human TIM-1.

Production of a Monoclonal Antibody for C/EBPβ: The Subnuclear Localization of C/EBPβ in Mouse L929 Cells

The CCAAT/enhancer-binding protein (C/EBP)β belongs to the C/EBP family of proteins that possesses a basic leucine zipper DNA-binding domain. These proteins bind DNA by dimerization and play a role in the transcriptional regulation of various cells. There are six different types of C/EBPs, and some form isoforms through the use of alternative translation initiation sites. The functional analysis of the C/EBP family is therefore difficult to achieve. Here we report on the production of specific monoclonal antibodies against mouse C/EBPβ using a rat medial iliac lymph node method. Immunoblotting using C/EBPβ monoclonal antibodies identified two types of isoforms, while immunostaining revealed a subnuclear localization for C/EBPβ. Use of this antibody should contribute to the further elucidation of the transcriptional regulatory function of C/EBPβ.

A Murine Monoclonal Antibody with Broad Specificity for Occupationally Relevant Diisocyanates

Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 μg of 4,4′-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4′-MDI– human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4′-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4′-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers.

Biological assays on the effects of Acra3 peptide from Turkish scorpion Androctonus crassicauda venom on a mouse brain tumor cell line (BC3H1) and production of specific monoclonal antibodies

Constitutes of the venom scorpion are a rich source of low molecular mass peptides which are toxic to various organisms, including man. Androctonus crassicauda is one of the scorpions from the Southeastern Anatolia of Turkey with public health importance. This work is focused on the investigation of biological effects of Acra3 peptide from Androctonus crassicauda. For this purpose, Acra3 isolated from crude venoms was tested for its cytotoxicity on BC3H1 mouse brain tumor cells using tetrazolium salt cleavage and lactate dehydrogenase activity assays. To determine whether the cytotoxic effects of Acra3 was related to the induction of apoptosis, the morphology of the cells and the nuclear fragmentation was examined by using Acridin Orange staining and DNA fragmentation assay, respectively. Caspase 3 and caspase 9 activities were measured spectrophotometrically and flow cytometric assay was performed using Annexin-V FITC and Propidium Iodide staining. Furthermore toxic peptide Acra3 was used as an antigen for immunological studies.Results showed that Acra3 exerted very strong cytotoxic effect on BC3H1 cells with an IC50 value of 5 μg/ml. Exposure of the cells to 0.1 and 0.5 μg/ml was resulted in very strong appearance of the apoptotic morphology in a dose dependent manner. On the other side, not any DNA fragmentation was observed after treatment of the cells. Caspase 3 and 9 activities were slightly decreased with Acra3. Results from flow cytometry and lactate dehydrogenase activity assays indicate that Acra3 exerts its effects by inducing a stronger necrosis than apoptosis in BC3H1 cells. To evaluate its immunogenicity, monoclonal antibody (MAb) specific for Acra3 antigen (5B9) was developed by hybridoma technology using spleen and lymph nodes of mice and immunoglobulin type of antibody was found to be IgM.We suggest that Acra3 may exert its effects by inducing both necrotic and apoptotic pathway in some way on mouse brain tumor cells. These findings will be useful for understanding the mechanism of cell death caused by venom in vitro. Anti-Acra3 monoclonal antibody can be further used as a bioactive tools for exploring the structure/function relationship and the pharmacological mechanism of scorpion peptide neurotoxins.

Production of a Methylenediphenyl Diisocyanate Hapten-Specific Monoclonal Antibody

4,4’-Methylenediphenyl diisocyanate (MDI) is used in the manufacturing of rigid polyurethane foams, coatings, adhesives, sealants and elastomers. MDI is a low molecular weight allergen and occupational exposure is associated with allergic sensitization, dermatitis, and asthma. Here we describe the production and initial characterization of an MDI-hapten specific monoclonal antibody (mAb).

Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat

The objective of this study was to produce a generic antibody for immunoassay of fluoroquinolone drugs in meat. Two novel haptens of sarafloxacin were synthesized that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 12 fluoroquinolone drugs (sarafloxacin, diflocaxin, marbofloxacin, ofloxacin, ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, lomefloxacin, amifloxacin, enofloxacin and danofloxacin). After evaluation of different coating antigen/antibody combinations, a heterologous competitive indirect enzyme linked immunosorbent assay (ELISA) was developed to determine the 12 drugs. The crossreactivities to these analytes were in the range of 18%–113% and the limits of detection were in the range of 0.8–6.5 ng/mL depending on the compound. Eight fluoroquinolones licensed as veterinary drugs in China were fortified into blank chicken for ELISA analysis. The recoveries were in the range of 67.6%–94.6% with coefficients of variation lower than 12.4%. Therefore, this method could be used as a screen tool for routine monitoring of the residues of these fluoroquinolone drugs in animal derived foods.

Production, characterization and applications of a tetanus toxin specific monoclonal antibody T-62

Tetanus neurotoxin (TeNT) represents a potent toxin that binds to its receptors on neurons and inhibits the release of neurotransmitters. Additionally, its fragments are used to transport pharmacological substances to neuronal cell bodies. The main objective of this study was the development of a suitable model system to study internalization of the TeNT. We have produced a monoclonal antibody (MoAb) specific for TeNT by hybridoma technology, after immunization of BALB/c mice with tetanus toxoid, and have named it T-62. The immunochemical characteristics of MoAb T-62 were tested using ELISA, PAGE and immunoblotting. Finally, we have used an immunohistochemical method to detect specific binding of MoAb T-62 to TeNT bound to PC 12 cells. Our results show that MoAb T-62 is highly specific for TeNT, even when it is bound to its receptor, and that it could be of considerable importance in studies regarding fundamental research on TeNT receptors, intracellular transport of TeNT, as well as retrograde transport of pharmaceutical substances and non-invasive delivery of polypeptides through the blood brain barrier. In addition, MoAb T-62 is an invaluable tool in TeNT vaccine production as it can be used for the detection of reverse toxicity, which could drastically reduce the need to use animals in these experiments.

Production of Group Specific Monoclonal Antibody to Aflatoxins and its Application to Enzyme-linked Immunosorbent Assay

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1- carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with λ-type light chains. The IC50s of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml (R2 > 0.99) and from 1 to 100 ng/ml (R2 > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.

Rapid drug haptenization procedure: application to gentamicin and quinidine.

A rapid and simple drug conjugation procedure which utilizes the heterobifunctional reagent N-hydroxysuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate is described. With this protocol two drugs of diverse structure and solubility have been successfully haptenized. Rabbits immunized with these conjugates yielded drug-specific antibodies as judged by a solid phase radioimmunoassay. Furthermore, using this technique, no cross-reactivity could be detected. Such haptens also elicited an immune response in mice thus further extending their potential for the production of drug specific monoclonal antibodies.

Characterization of rickettsial adhesin Adr2 belonging to a new group of adhesins in α-proteobacteria

Background Rickettsia prowazekii is the etiological agent of epidemic typhus and is an obligate intracellular bacterium that grows as a parasite freely within the cytoplasm of a eukaryotic host cell. Previous studies have shown that rOmpA and rOmpB which belong to the family of rickettsial cell surface antigens are involved in vitro in the adhesion of Rickettsiae to epithelial cells. Recently, two putative rickettsial adhesins have been identified using high resolution 2D-PAGE coupled with mass spectrometry. In this study, we further characterize and describe the adhesin Adr2 from R. prowazekii. Methodology/Principal findings Using an overlay assay coupled with mass spectrometry two adhesins, Adr1 (RP827) and Adr2 (RP828), were identified from the R. prowazekii proteome Recombinant R. prowazekii Adr2 was expressed through fusion with Dsbc in Escherichia coli, purified and concentrated, thus allowing production of specific monoclonal antibodies, as confirmed by western blot assays. Finally, inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-Adr2 antibody has showed a greatest impact on bacterial cell entry at 8 h post-infection (ca50%) and then decreased progressively to attempt 18% of inhibition at day 7. These, correlated to the inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-rOmpB antibody. Thus, Adr2 is sufficient to mediate R. prowazekii entry into the cell at early stage of mammalian cell infection. Conclusions Our results suggest that R. prowazekii Adr2 could be the main actor promoting the entry of rickettsiae into the host cells. The present study opens the framework for future investigations for better understanding of the Adr2 -mediated mechanisms involved in adhesion/invasion or intracellular survival of R. prowazekii.

Monoclonal antibodies against human CD34 antigens do not cross-react with ovine umbilical cord blood cells

CD34 is a cell surface glycoprotein expressed by hematopoietic progenitors and endothelial cells. It is widely used in the clinic for isolation of human hematopoietic stem cells. In recent years large animals are gaining increasing importance in biomedical research for the study and therapy of human diseases. Sheep has proved to be an useful experimental model for preclinical trials in transplantation procedures. Unfortunately, the lack of specie-specific monoclonal antibodies (MABS) recognizing hemopoietic progenitor cells hampers the use of this animal in experimental hematology. The aim of this paper was to determine whether commercial monoclonal antibodies specific for human CD34 molecule could cross-react with hematopoietic progenitor cells (HPC) present in sheep umbilical cord blood (UCB). Six antihuman CD34 MABS, recognizing the three different epitope classes, were tested in flow cytometry on purified mononuclear cells (MNC) isolated from cord blood of both species. None of the MABS used in this trial seemed to be able to identify HPC from sheep UCB. These data suggest that the panel of monoclonal antibodies used for cross reactivity detection has to be expanded with recently produced reagents. Further studies should be directed towards the production of ovine specific anti CD34 MABS.

Characterization of monoclonal antibodies specific to the transcription factor ETS-2 protein

ETS-2 is a member of the ETS family of transcription factors. ETS-2 was initially characterized as a nuclear oncogene and has been shown to play a role in regulation of apoptosis and cell cycle progression. Members of the ETS family display high sequence homology, thus, there is considerable controversy concerning the specificity of existing ETS-2 polyclonal antibodies that have been used to define ETS-2 function. We therefore embarked on the production of ETS-2 specific monoclonal antibodies. In this report, we describe the production and characterization of six antibodies and the localization of their target epitopes to distinct domains of the ETS-2 protein. Four antibodies are ETS-2 specific and two antibodies cross-react with ETS-1, an ETS family member with the highest amino acid sequence homology to ETS-2. This report provides a comprehensive evaluation of ETS-2 specific monoclonal antibodies verified using ETS-2 null cells. These antibodies can be used for EMSA, Western blotting, immunoprecipitation and immunofluorescence staining experiments. Collectively, these reagents are invaluable molecular tools that should help better understand the biological function of ETS-2.

Production of a species specific monoclonal antibody against a 56-kDa Mycobacterium marinum antigen which is potentially useful in culture identification.

A monoclonal antibody (MoAb) designated 1H11 has been produced against a 56-kDa antigen from Mycobacterium marinum. This MoAb has been shown to be species specific by both enzyme-linked immunosorbent assay and Western blot. The relationship of this 56 kDa to other known mycobacterial antigens is currently under investigation. MoAb 1H11 recognised bacilli taken from cultures by both immunofluorescence and immunoperoxidase staining. This MoAb 1H11 may provide a simple method for rapid identification of M. marinum from cultures of clinical isolates.

Production of a species specific monoclonal antibody against a 56-kDa Mycobacterium marinum antigen which is potentially useful in culture identification

A monoclonal antibody (MoAb) designated 1H11 has been produced against a 56-kDa antigen from Mycobacterium marinum. This MoAb has been shown to be species specific by both enzyme-linked immunosorbent assay and Western blot. The relationship of this 56 kDa to other known mycobacterial antigens is currently under investigation. MoAb 1H11 recognised bacilli taken from cultures by both immunofluorescence and immunoperoxidase staining. This MoAb 1H11 may provide a simple method for rapid identification of M. marinum from cultures of clinical isolates.

Production and characterization of specific monoclonal antibodies of the human thyroid stimulating hormone.

Spleen cells from BALB/c mice immunized with human thyroid stimulating hormone (beta-subunit) were fused with mouse myeloma cells (P3/X63-Ag8) and five hybridomas secreting monoclonal antibodies (MAbs) were obtained. These hybridomas specifically recognize (hTSH) and do not cross-react with the other human glycoprotein hormones such as: luteinizing hormone (LH), follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hcG). The MAbs were of the IgG1 subclass and ascitic fluid from these hybridomas was purified by affinity chromatography on Protein A-sepharose CL-4B column to isolate the IgG1 active fraction. The affinity constant of these MAbs ranged from 3.2 x 10(10) to 1.5 x 10(11) M(-1).