Monoclonal Hybridoma Cell Lines Research Articles

Development of a highly sensitive and specific monoclonal antibody-based immunoassay for detection of okadaic acid in oysters and green mussels

ABSTRACT Okadaic acid (OA), one of marine biotoxins produced by several species of dinoflagellates, can accumulate in marine animals. The consumption of OA-contaminated seafood can cause diarrhoeic shellfish poisoning. Many countries have established regulatory restriction to limit the level of OA in seafood. In the present study, we report a highly sensitive monoclonal antibody (mAb) against OA produced by a new immunogen. Two monoclonal hybridoma cell lines (M8 and M12) that producing mAb with the best sensitivity and specificity were obtained. The obtained mAb-M12 was used to develop an indirect competitive ELISA (icELISA) with 50% inhibition concentrations of 0.15 ng mL−1 and a working range of 0.02–2.74 ng mL−1. The developed icELISA was more sensitive than the previous reports. HPLC-MS confirmed the accuracy and the working range of icELISA, suggesting that the developed icELISA is suitable for the rapid detection of OA in oysters and green mussels.

Development of a Competitive Cystatin C-Specific Bioassay Suitable for Repetitive Measurements.

Human cystatin C (hCC), a cysteine protease inhibitor, has been proposed as a diagnostic marker because its serum levels correlate with certain cardiovascular and kidney diseases. All current hCC assays are based on ex vivo detection. Here we describe the generation and evaluation of antibodies that allow the repetitive binding and release of hCC and hCC-fusion proteins, a prerequisite for long-term measurement, which is required for compatibility with implantable biochip devices and for the development of innovative antibody-based assays suitable for continuous in vivo and in vitro monitoring. Recombinant hCC and hCC-fusion proteins were produced in Escherichia coli and HEK293T cells and were used to generate antibodies by hybridoma technology. After screening by indirect and sandwich ELISAs, 12 monoclonal hybridoma cell lines producing hCC-specific monoclonal antibodies were identified. To determine their hCC association and dissociation properties, the antibodies were analysed by surface plasmon resonance spectroscopy, revealing three with the desired fast binding and moderate-to-fast release characteristics. The analysis of binding and dissociation in the presence of hCC and hCC-fusion proteins using fluorescence-based replacement assays showed that mAb CyDI-4 was the most suitable for further analysis. The results showed that repetitive replacement on mAb CyDI-4 was possible and that most of the change in signal intensity occurred after 20–30 min. Furthermore, the suitability of mAb CyDI-4 for serum hCC measurement was confirmed by a fluorescence-based replacement assay using serially-diluted reference serum from the Institute for Reference Materials and Measurements (ERM-DA471/IFCC). Our results suggest that the assay covers the physiological and pathological ranges of hCC.

Development of Monoclonal Hybridoma Cell Lines and Extracting Antibody Against Fummonisin B1

Objective:To acquire monoclonal hybridoma cell lines against fummonisin B1(FB1) and extract monoclonal antibody against FB1.Methods: Coupling antigens of FB1-KLH and FB1-BSA with chemical methods and immune 6-8 weeks old femaleBALB/c mice with FB1-KLH. Integrating spleen cells with sp2/0 myeloma cells to acquire hybridoma cell lines secreting McAb against FB1. The method of multiple subclones was used to select cell lines stably secreting McAb. McAbs was got from ascites and purified. The subclass of antibody was measured and the molecular weight was identified. The specificity and sensitivity of McAb were identified with indirect competitive inhibition ELISA.Results:The results of serum from immuned mice showed that after five times of immunization the titer stables at 1×10-6, and the McAb belongs to IgG1 subclass, the light chain was ?, the molecular weight of heavy and light chain were 55kDa and 32kDa, respectively. ELISA results showed that McAb could react with FB1. The linear range indirect competitive inhibition ELISA is 10-500ng/ml.Conclusion:The monoclonal hybridoma cell lines and the high specificity,high sensitivity of FB1-McAb was got.

Development of monoclonal antibodies and quantitative ELISAs targeting insulin-degrading enzyme

BackgroundInsulin-degrading enzyme (IDE) is a widely studied zinc-metalloprotease implicated in the pathogenesis of type 2 diabetes mellitus, Alzheimer disease (AD) and varicella zoster virus infection. Despite more than six decades of research on IDE, progress has been hampered by the lack of well-characterized reagents targeting this biomedically important protease. To address this important need, we generated and characterized new mouse monoclonal antibodies (mAbs) targeting natively folded human and rodent IDE.ResultsEight monoclonal hybridoma cell lines were derived in house from mice immunized with full-length, natively folded, recombinant human IDE. The mAbs derived from these lines were shown to detect IDE selectively and sensitively by a wide range of methods. Two mAbs in particular—designated 6A1 and 6H9—proved especially selective for IDE in immunocytochemical and immunohistochemical applications. Using a variety of methods, we show that 6A1 selectively detects both human and rodent IDE, while 6H9 selectively detects human, but not rodent, IDE, with both mAbs showing essentially no cross reactivity with other proteins in these applications. Using these novel anti-IDE mAbs, we also developed sensitive and quantitative sandwich ELISAs capable of quantifying IDE levels present in human brain extracts.ConclusionWe succeeded in developing novel mAbs that selectively detect rodent and/or human IDE, which we have shown to be suitable for a wide range of applications, including western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, and quantitative sandwich ELISAs. These novel anti-IDE mAbs and the assays derived from them constitute important new tools for addressing many unresolved questions about the basic biology of IDE and its role in multiple highly prevalent human diseases.

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Enhancement of the enzymatic activity of activated coagulation factor IX by anti-factor IX antibodies

Functional Comparison of Human and Drosophila Hop Reveals Novel Role in Steroid Receptor Maturation

Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes. Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear. Human Hop and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting the efficient maturation of glucocorticoid receptor (GR) function. To gain a better understanding of Hop structure/function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1. Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop failed to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding. Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function.

Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity

Degenerate primers were designed for PCR amplification of unknown mouse immunoglobulin (Ig) light (L) and heavy (H) chain variable (V) genes. Each subgroup of mouse Ig gene sequences [Kabat, E.A., Wu, T.T., Perry, H.H., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunological Interest, 5th edn. US Department of Health and Human Services, Public Health Service, NIH.] was analyzed, and highly degenerate primers in the framework one (FR1) region were designed. A single highly degenerate FR1 primer sufficed for the amplification of light chains; for heavy chains, a series of FR1 primers was used. At the same time, we assessed the effect of 3′ to 5′ exonuclease activity of DNA polymerase on the utilization of these degenerate primers. Using Taq polymerase, which lacks 3′ to 5′ exonuclease activity, we successfully amplified the Ig VL and VH genes expressed in more than a hundred monoclonal hybridoma cell lines reactive against a phosphonamidate hapten. Sequence analysis of the cloned VL and VH genes, 52 of each, showed that they are derived from multiple germline families (10 of the 17 VL families and 9 of the 14 VH families) as recently defined [Martinez, C., Lefranc, M., 1998. The mouse ( Mus musculus) immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp. Clin. Immunogenet. 15, 184.]. The universality of our primers was also demonstrated by successful amplification of other mouse hybridoma cell lines that are specific to different antigens.

Characterization and epitope mapping of several new anti-P-glycoprotein monoclonal antibodies.

Monoclonal antibodies (MAbs) were raised against partially purified Class I P-glycoprotein from multidrug-resistant Chinese hamster ovary CHRB30 cells. Fifteen stable monoclonal hybridoma cell lines were established, and the secreted antibodies were classified into 8 groups on the basis of banding pattern on immunoblots of P-glycoprotein digested with cyanogen bromide or partially digested with proteases. One representative of each group was tested further for several activities. Six of the 8 recognized human P-glycoprotein in the multidrug-resistant SKVLBI cell line. None of the antibodies recognized P-glycoprotein in unfixed cells, suggesting that all recognize cytoplasmic epitopes or extracellular epitopes not accessible in native P-glycoprotein. All 8 antibodies were able to immunoprecipitate P-glycoprotein from non-denaturing detergent solution. The linear epitopes of the antibodies were mapped to 11-27 amino acids. Two of the antibodies had epitopes in the linker region, 3 in the N-terminal nucleotide binding domain, 2 in the C-terminal nucleotide binding domain and 1 in the predicted cytoplasmic loop between predicted transmembrane helices 8 and 9. These antibodies, with known epitopes, could have uses for P-glycoprotein detection, structure/function studies, purification and quantitation.

Development of monoclonal antibodies against the fungi of the ‘Ascochyta complex’

Two monoclonal antibodies (MAbs) were produced against soluble antigens from the ‘Ascochyta complex’ fungi. Specificity of MAbs was tested by ELISA using antigen‐coated wells. MAbs secreted by the monoclonal hybridoma cell line JIM 44 recognized epitopes present in the antigen preparations from Mycosphaerella pinodes and Phoma medicaginis var. pinodella, but not those present in preparations from Ascochyta pisi. At high tissue culture supernatant concentration, MAbs produced by the monoclonal line JIM 45 recognized epitopes from all three fungi, however, on dilution of MAb the antigens from A. pisi were recognized preferentially to those from M. pinodes and P. medicaginis var. pinodella. On the basis of heat and periodate treatment of the antigens from the three fungi it can be concluded that the epitope recognized by JIM 44 is carbohydrate in nature, whereas that recognized by JIM 45 is proteinaceous in nature, carried on a glycoprotein antigen. Antigen preparations from other fungi, including other pea pathogens, non‐pathogens associated with pea and other fungi closely related to the ‘Ascochyta complex’, were not detected with either of the two MAbs. Antigen preparations from peas could be used to differentiate healthy and infected seeds in a dot‐blot assay, therefore indicating the potential of using the MAbs in the development of a diagnostic test for infection of Pisum seeds by the ‘Ascochyta complex’ fungi.

Synthesis of a functional anti-phytochrome single-chain Fv protein in transgenic tobacco.

We have expressed a synthetic gene that encodes an antigen-binding single-chain FV protein (scFV) in transgenic tobacco plants. The scFV gene was created by polymerase chain reaction (PCR) amplification of the variable domain coding regions from a mouse monoclonal hybridoma cell line. The monoclonal cell line secretes an IgG1 antibody that binds to the plant regulatory photoreceptor protein, phytochrome. The cloned scFV gene was expressed initially in Escherichia coli and shown to produce a 28 kD, phytochrome-binding binding scFV protein. Transgenic tobacco plants expressing the scFV gene were also found to produce a functional scFV protein, and seeds from transgenic R1 progeny displayed aberrant phytochrome-dependent germination. The scFV from transgenic tobacco could be isolated, to near homogeneity, by a single phytochrome-Sepharose affinity chromatography step.

Enhancing monoclonal antibodies and hybridoma cell lines.

We are interested in determining the range of variants present in a cell population that can actually be isolated. We have used subcloning and sublining to search for variants with increased antibody stability, increased cell line stability to freezing and defrosting, increased cell population viability, increased antibody production and the ability to grow in simpler media. This paper presents the case histories of several different hybridoma cell lines which required some property changed before they became production ready clones. We found that switching the class of an antibody from IgG3 to IgG1 did increase its stability, decrease its tendency to aggregate and allowed it to be used in a commercial diagnostic kit. We could isolate subclones that produced twice the level of antibody with a frequency of 1-3%. It was straight forward to isolate clones that were stable to freezing and defrosting or grew in a simpler media. We were not successful in increasing the maximum viability of a cell line. In conclusion, we have found that any population of hybridoma cells has natural variants with significantly enhanced properties that can be isolated.

Immunobiological properties of a recombinant simian retro virus-1 envelope protein and a neutralizing monoclonal antibody directed against it

We previously reported that an area encompassing amino acids 147–162 of the envelope region of the simian (type D) retrovirus serotype 1 (SRV-1) constitutes an antigenic site for the binding of murine and rhesus neutralizing antibodies. Neutralizing antibodies to SRV-2 are directed to a different area, encompassing residues 96–102 of SRV-2. This paper presents data on the activity of an SRV-1 recombinant envelope protein (rEP) and of monoclonal hybridoma cell line, C11B8, produced from murine spleen cells immunized with SRV-1 rEP. Purified monoclonal antibodies from C11B8 bind to the SRV-1 rEP and to both SRV-1 and SRV-2. However, the monoclonal antibody exhibits strain specificity in the capacity to neutralize SRV-1 infection in vitro. Thus, C11B8 neutralizes SRV-1 infection but fails to neutralize four other known serotypes of the virus. C11B8 also binds to an SRV-1 synthetic peptide representing residues 142–167, which encompasses the previously defined antigenic site of recognition for neutralizing antibodies to SRV-1. This paper also contains evidence that the SRV-1 rEP construct binds the site for SRV-1 attachment to the cell receptor. This is indicated by the ability of SRV-1 rEP to compete with SRV-1 (but not with SRV-2) and inhibit its infectivity in vitro. In addition, SRV-1 rEP inhibits the neutralizing activity of C11B8 against SRV-1 infection in vitro. SRV-1 rEP has no inhibitory effect on rhesus neutralizing antibodies to SRV-2. Taken together, the above findings indicate that immunity conferred at the level of neutralizing antibodies during SRV infection is strain-specific and involves the recognition of envelope sequences unique to each strain.

Production of a murine monoclonal antibody with anti‐hiv‐1 neutralizing properties

A mouse monoclonal hybridoma cell line producing IgG 1k to human immunodeficiency virus (HIV-1) gp120 envelope protein was cultured in several systems. A small-scale flask culture was essential for characterizing the culture variables of the hybridoma. A dialysis tubing culture appeared to be an excellent alternative to in vivo cultures of ascitic fluid, and gave high mouse monoclonal antibody (Mab) concentrations. Two continuous culture systems were both very effective in producing large amounts of Mabs. The hollow fiber system has the advantage of giving a concentrated product in the harvest. The ceramic core system, on the other hand, allows excellent monitoring of the cellular growth and production phases and gave the highest HIV antigen reactivity/micrograms of the produced IgG. Twelve grams of HIV-1 neutralizing Mabs were produced. The Mab was purified with a yield of 61%. The neutralizing capacity of the Mab was studied in vitro and shown to be excellent with 50% neutralizing titers using 5 ng Mab. The biological half-life of the Mab given intravenously to an HIV-infected individual was shown to be around 30 h.

Efficient hybridoma production using previously frozen splenocytes

Splenocytes from immunized mice and rats can be frozen, thawed, and fused with myeloma cells to generate monoclonal antibody-secreting hybridoma cell lines. The hybridoma yeild per splenocyte is comparable to, and often better than, the yield when fresh splenocytes are used. Practical advantages of the technique include the ability to utilize all splenocytes from a given mouse or rat, greater scheduling flexibility, and the opportunity to perform small-scale ‘test fusions’ to monitor the immune responses of a number of immunized animals.

A monoclonal antibody toBabesia divergenswhich inhibits merozoite invasion

A monoclonal antibody-producing hybridoma cell line has been raised against merozoites of Babesia divergens. This antibody is strongly reactive against merozoites in an enzyme-linked immunosorbent assay (ELISA) and is also positive in an indirect fluorescent antibody test (IFAT) on preparations of live merozoites. The antibody recognizes an antigen, of molecular weight (Mr) 50-60 K by Western blot analysis on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Merozoite neutralization assays show that the antibody significantly inhibits merozoite invasion of bovine erythrocytes.

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the calcium channel structure

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.

High-affinity monoclonal antibodies specific for human prostatic acid phosphatase.

We produced two monoclonal hybridoma cell lines that secrete IgG immunoglobulins with high affinities (Kd = 7.3 to 8.0 X 10(-11) mol/L, Ka = 1.25 to 1.37 X 10(10) L/mol) for 125I-labeled human prostatic acid phosphatase (PAP), and that specifically bind this enzyme from human serum. The antibodies were produced in high titers in murine ascitic fluid (700 mg/L) and in cell-culture media (30 mg/L) and were further purified to homogeneity by affinity chromatography on PAP- and Protein A-Sepharose CL-4B. After purification they were shown to be homogeneous by liquid chromatography. Both of these monoclonal antibodies exhibit strict specificity for PAP as determined by radioimmunoassay and by immunofluorescence studies of human pancreas, kidney, prostate, and leukocytes. The antibodies react only with the native form of the enzyme, as shown by the slot-immunoblotting method.

Monoclonal antibodies to the major capsid protein of human papillomavirus type 1.

Two stable monoclonal hybridoma cell lines secreting type-specific antibodies against the human papillomavirus type 1 (HPV-1) were isolated. The monoclonal antibodies detected HPV-1 antigens in frozen sections of HPV-1-induced warts, using immunofluorescence or immunoperoxidase techniques, and they reacted with HPV-1 particles in an immunodiffusion test. The two monoclonal antibodies recognized the major structural viral polypeptide, with a molecular weight of 54 000, and a minor polypeptide, with molecular weight of 76 000, in both the dissociated viral particles and in the wart extracts.

Purification of hog kidney renin with immobilized monoclonal antirenin.

Spleen cells from mice immunized with partially purified hog kidney renin were fused with mouse myeloma cells to produce a stable monoclonal hybridoma cell line that synthesizes an antibody against renin. A single monoclonal antibody was chosen for study and has been produced in large quantity and purified by affinity chromatography on protein A-Sepharose. The antirenin, which belongs to the IgG1 subclass, exhibits anticatalytic activity against both hog and rabbit renin. An immunoaffinity column prepared from antibody coupled to Sepharose has been used in the purification of renin from hog kidney. Although renin is quantitatively adsorbed from solution, it can be eluted from the column under gentle conditions. The highly purified renin, with specific activity of 2122 Goldblatt Units/mg protein, exhibits both charge (pH 4.1 to 5.1) and size (38,000 to 42,700) heterogeneity. Hog kidney renin dissociates in the presence of sodium dodecyl sulfate (SDS) and mercaptoethanol to heavy and light chains with molecular weights of 33,700 and 5,800, respectively. In the presence of SDS, a small amount of a nw form of renin is observed with a molecular weight of 19,500 which retains activity on renaturation. The monoclonal antibody should be a useful tool for the study of the renin-angiotensin system and especially for the purification of renin. The hybridoma cell line used in this study (F-32 VIII C4) has been donated to the American Type Culture Collection.

Characterization of a monoclonal antibody to human sex hormone binding globulin

We have produced a monoclonal hybridoma cell line (S 1B 5) that secretes an IgG 2α immunoglobulin with a high affinity ( K d 0.38 × 10 −11 M) for 125I-labelled sex hormone binding globulin (SHBG), and which will specifically immunoprecipitate SHBG from serum. The antibody is produced in high titre in culture medium and ascites fluid, and can be purified to apparent homogeneity by protein A affinity chromatography. When examined by isoelectrofocussing, a characteristic series of bands, which bind 125I-SHBG, are observed at pH 8.5–9. Competitive, superimposable displacement of 125I-SHBG from the antibody is achieved with dilutions of human, chimpanzee and gorilla sera at comparable SHBG concentrations. Partial cross-reaction is observed with sera from other Old World primates, but not with sera from New World monkeys or any other vertebrate species studied.