摘要:

Recombinant single chain Fv (scFv) antibodies offer many advantages over mouse monoclonal antibodies (MAbs) such as faster clearance from blood, improved tumor localization, reduced human anti-mouse antibody (HAMA) response, and the availability to manipulate the scFv through genetic approaches. The scFv antibody (designated RK10.2) was generated using anti-CEA T84.66 hybridoma cells as a source of genetic starting material and the Pharmacia Recombinant Phage Antibody System (RPAS).Escherichia coliclones expressing antigen-positive soluble scFv were identified using a modified colony-life selection procedure and antigen-coated filters. The resultant anti-CEA scFv (designated RK10.2) had a molecular weight of approximately 33.6 kDa and an isoelectric point of 5.2 at 15°C. The RK10.2 scFv interacted with LS174 T cells bearing the CEA antigen and inhibited the anti-CEA MAb/CEA antigen interaction in ELISA and the anti-CEA MAb/LS174 T cell interaction in a RIA. The modified colony-lift approach circumvented the more time-consuming phage-display approach that is normally taken to affinity select for antigen-positive scFv clones

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.1

年代:1998

数据来源: MAL

摘要:

The exact mechanism by which the human T cell leukemia viruses (HTLV) infects target cells remains unclear; although some molecules have been identified to be important in viral infection and entry. To investigate these phenomena, we generated a panel of monoclonal antibodies (MAb) against a B cell line (BJAB-WH) which is highly permissive for infection with HTLV. These MAb have been used to further characterize the membrane molecules important for HTLV infection. Three of these MAb designated 4.2.3, 3.3.10, and 11.2.3 were capable of inhibiting syncytium formation induced in human B and T cell lines (i.e., BJAB-WH and SupT-1, respectively) by co-culture with HTLV-I infected MT-2 cells. All of these MAbs immunoprecipitated a 80-85 kDa antigen from the lysates of metabolically labeled BJAB-WH but not from BJAB-CC/84, a noninfectible target cell. The binding of these MAb with different HTLV target cells was analyzed and compared with binding of polyclonal monospecific antisera to the same cell lines. A 80-85 kDa membrane glycoprotein was isolated with an immunoaffinity Chromatographic column constructed with MAbs 4.2.3 and 3.3.10. This cellular antigen was capable of inhibiting HTLV I/MT-2 induced fusion. This is the first direct demonstration that a 80-85 kDa cellular glycoprotein is directly involved in HTLV I/II infection and syncytium formation.

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.9

年代:1998

数据来源: MAL

摘要:

The development of specific antibody probes for characterizing the expression of the family of 4 fibroblast growth factor receptor (FGFR) types has been difficult because of their close homology to each other and high degree of evolutionary conservation. Of the existing anti-FGFR monoclonal antibodies (MAbs), there are few that are useful for staining paraffin-embedded tissues. We have raised MAbs against human FGFR1 and FGFR2 in both rats and mice using bacterial recombinant receptor fusion proteins as immunogens. We used peptide epitope mapping to characterize the immune sera and the selected MAbs. Immunized animals were selected that displayed the broadest reactivity against epitopes unique to the immunizing receptor type. We produced FGFR1 specific MAbs that bind epitopes in immunoglobulin domain I (Ig-I) and FGFR2 specific MAbs that bind epitopes in Ig-I, Ig-II, and the acid box. The specificity of the antibodies was demonstrated by ELISA and immunoblot analysis of purified recombinant FGFR1 and FGFR2 extracellular domains produced both inE.coliand in eucaryotic cells. Based on the lack of epitope homology, these MAbs would not be expected to cross-react with FGFR3 or FGFR4. We isolated MAbs that bound to paraffin embedded tissue and immunoblots of recombinant receptor. These epitope-defined MAbs can distinguish between members of the FGF receptor family and should be useful as tools for assessing FGF receptor expression in a variety of normal and diseased tissues.

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.21

年代:1998

数据来源: MAL

摘要:

We have previously described the isolation andin vitrobinding properties of eight anti-DNA monoclonal antibodies (MAbs) from an MRL-lprmouse. In light of recent reports that have indicated it is possible to isolate multiple MAbs from a single hybridoma, our pathogenic hybridoma, 1118, was examined for evidence of similar phenomena. Chromosome counting suggested that 11F8 cells are unusual and might indeed be expressing multiple heavy and/or light chains. PCR, cloning, and sequencing of immunoglobulin heavy and light chains indicate that 11F8 displays expression of both γ2a and γ3 heavy chains at the DNA level. Flow cytometry and amino acid sequencing reveals that expression of multiple isotypes also occurs at the protein level but only a single heavy- and light-chain sequence is able to bind DNA. Based on these results, we conclude that 11F8 is an unusual hybridoma that secretes two distinct heavy and at least one light chain from a single cell, and may represent a trioma, a stable three-cell fusio

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.33

年代:1998

数据来源: MAL

摘要:

SCID mice were inoculated with five human-mouse heterohybridomas derived by fusion of human lymph node lymphocytes from lung cancer patients with murine myeloma cells or human-mouse heteromyeloma cells, and the production of their human monoclonal antibodies (MAb) in the mouse ascites was investigated. In a comparison of the effects of pretreatment by IP (intraperitoneal) injection of pristane and anti-asialo GM1 serum on the antibody production of three of the hybridomas, pristane pretreatment resulted in substantial antibody production by all three, and pretreatment with anti-asialo GM1 serum resulted in similar or slightly lower levels of antibody production by two of the hybridomas but none by the third. In a second series of experiments using four of the hybridomas with pristane pretreatment, the co-injection of either penicillin G and streptomycin or kanamycin together with the hybridoma at the time of IP inoculation resulted in enhanced MAb production by the two heterohybridomas that had been propagated in medium containing hypoxanthine-aminopterin-thymidine (HAT) but not by the two that had been propagated in HAT-free medium.

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.41

年代:1998

数据来源: MAL

摘要:

We report the production and characterization of two PPARα subtype-specific monoclonal antibodies raised against the N-terminal domain of PPARα. Pαbll.80A is a Western-reactive antibody, whereas Pαb32.51 is useful for immunohistochemistry. Both antibodies exhibited high affinity against the immunogen based on BIAcore analysis, recognized full-length PPARα protein in PPARα-transfected CV-1 cells, and displayed no cross-reactivity against the N-terminal domains of PPARγ or PPARδ proteins as demonstrated by various immunoassays. The application of these antibodies to a panel of normal human tissues revealed that PPARα protein expression is highest in skeletal muscle, liver, and kidney, consistent with previously reported mRNA expression data. These antibodies provide us with valuable tools to further explore the function

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.47

年代:1998

数据来源: MAL

摘要:

To study heterophile blood antigens on erythrocytes between human and experimental or domestic animals, we have produced 295 monoclonal antibodies (MAbs) to human erythrocyte membrane protein. According to the affinity, reactivity, and titre of the MAbs, we selected 40 clones to study the heterophile blood antigens between human and bovine, chicken, guinea pig, horse, rabbit, sheep, and swine. Five MAbs commonly reacted with human type A, type B, and type O erythrocytes and reacted with bovine erythrocytes as well but did not react with erythrocytes from other species. Other MAbs did not react with erythrocytes from all the tested animals. These five MAbs reacted with the same erythrocyte membrane protein, 90 KD glycophorin A (GPA) of human or 200 KD major glycoprotein and other two components of bovine by immunoblotting and GPA competitive inhibition assay. Furthermore, by enzyme treatment and monosaccharide competitive inhibition assay, it was confirmed that these five MAbs recognized antigen epitope of glycosylation free amino acid portion but not glycosylation portion of GPA of erythrocyte membrane.

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.55

年代:1998

数据来源: MAL

摘要:

We have generated murine monoclonal antibodies (MAb) againstVibrio choleraemannose-sensitive hemagglutinin (MSHA) using conventional hybridoma procedures. Seven hybridomas were obtained and one characterized. Hybridoma 2F12/F1 secreted an antibody of the IgG3type that reacted with a 17-kDa antigen corresponding to the product of themshAgene. This MAb inhibited mannose-sensitive agglutination of chicken erythrocytes by EL tor and O139 vibrios. Vibrios expressing MSHA activity inhibited binding of the antibody secreted by 2F12/F1 to MSHA-coated microtiter plates.

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.63

年代:1998

数据来源: MAL

摘要:

Traditional hybridoma fusion technology requires complete medium with serum supplements to support the growth of hybridoma cells. Serum is also required for subcloning of hybridoma cells to support low density cell growth. IL-6 has been shown to enhance the growth of hybridomas and stimulate antibody production by B cells. We found that the serum requirement in media used for generation of hybridomas can be totally eliminated by substituting with 300 units/ml of IL-6. Stable hybridoma cell lines were generated to peptide and protein antigens using serum-free adapted P3.653 myelomas as the fusion partner and medium containing IL-6. Our results indicate that, in general, the fusion efficiencies of serum-free IL-6 supplemented fusions are lower than the fusions employing serum containing media (40%–60% vs. 80%–100%). However, in spite of the lower fusion efficiency, the number of antigen-specific clones generated using IL-6 was equal to or greater than fusions using serum supplements. The use of IL-6 instead of serum in the generation of monoclonal antibodies (MAbs) has several advantages. We are able to eliminate the costly need for serum in media by using IL-6 that is prepared in house. In addition, we eliminate the need for time-consuming serumfree adaptation of hybridoma cell lines prior to transfer to hollow fiber bioreact

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.69

年代:1998

数据来源: MAL

摘要:

Twenty monoclonal antibodies (MAbs) were obtained by immunizing Balb/c mice with recombinant pl7 (rpl7) of HIV-1. Epitope specificity of each MAb was determined using six peptides that cover the entire region of pl7. We found that each MAb reacts with only one of the peptides, residues 12–29, 30–52, 53–87, and 87–115 (P12–29, P30–52, P53–87, P87–115) of pl7 with the exception of one MAb. Three kinds of MAbs that recognize P30–52, P87–115, and a conformational epitope, suppressed the infectivity of HIV-1 (JMH-1) when they added in the culture of MT-4 cells infected by HIV-1 within 24

ISSN:0272-457X    

DOI:10.1089/hyb.1998.17.73

年代:1998

数据来源: MAL