摘要:

The haptoglobin-related gene product (HGRP) having>90% sequence homology with plasma haptoglobin, has recently been implicated as a tumor maker of recurrent breast cancer. Therefore, availability of a monoclonal antibody which is specific to the HGRP and ensuing ELISAs to quantitate the HGRP without cross-reactivity with plasma haptoglobin will allow determination of the clinical value of the HGRP in human plasma under various pathophysiological conditions.A peptide representing the first 34 residues of the N-terminal portion of the HRGP was synthesized and conjugated to keyhole limpet hemocyanin (KLH) to immunize Balb/c mice. Hybrid clones that produced specific antibodies to HRGP were screened with the same peptide but conjugated to bovine serum albumin (HRGP-BSA). From 12 hybridoma clones that recognized HGRP-peptide, we obtained one IgM producing clone (27.5) by limiting dilution technique that selectively reacted with HGRP-peptide with minimal cross-reactivity with haptoglobin. Using a separate monoclonal antibody, clone 21.7, which is directed to the alpha subunit of haptoglobin, we developed an enzyme-linked immunosorbent assay (ELISA) to determine the quantities of the HGRP in human plasma, which will allow assessment of HGRP as a clinical marker of malignancy.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.1

年代:1992

数据来源: MAL

摘要:

We have obtained 41 monoclonal antibodies directed against type III group B streptococci by immunizing Balb/c mice with formalin-killed bacteria. All of these antibodies reacted with purified type-specific carbohydrate by enzyme-linked immunosorbent assay and immunoprecipitation tests. The epitope recognized by all of these antibodies was associated with terminal sialic acid residues, as indicated by abrogation of immune reactions by treatment of the type-specific carbohydrate with neuraminidase. Two purified monoclonal antibodies (the IgM P9D8 and the IgG3 P4F12) were further characterized for their protective activity in a neonatal rat model of infection. P9D8 and P4F12 antibodies were significantly protective when administered in a dose of 0.5 and 2.5 mg/kg, respectively, at the same time as 3x105colony forming units of type III streptococci. Protection was still observed when the antibodies were given up to 9h after challenge. No protection was afforded against infections with type la/c and II streptococci. Similarly, both antibodies effectively opsonized type III, but not la, lb or II bacteria, in anin vitroassay. These and similar, previously described, monoclonal antibodies may be useful, possibly after "humanization" by genetic engineering, for the therapy of neonatal group B streptococcal infections.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.13

年代:1992

数据来源: MAL

摘要:

The isolation of the rearranged immunoglobulin genes from a hybridoma cell line, which is a prerequisite for the construction of a recombinant antibody, can easily be achieved by polymerase chain reaction. Here we demonstrate that this method, which was originally described for cloning murine immunoglobulin genes from cDNA, is also applicable for rat genes. We show that the procedure also works with crude cell lysates as starting material, thereby gready reducing the time required for sample preparation. In addition we have sequenced the nonfunctional heavy chain variable gene of the fusion partner X63Ag8.653, which was readily amplified from our hybridoma cells, and whose sequence has been so far unknown.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.23

年代:1992

数据来源: MAL

摘要:

Human anti-murine antibodies (HAMA) can be found in serum of many patients who have received murine monoclonal antibodies for diagnosis or therapy. These antibodies are known to give false positive results in sandwich-type assays (e.g. ELISA or RIA). This interference problem will increase in the future as more patients are treated with murine monoclonal antibodies in vivo. HAMA can also be found in sera from patients that has not been treated with monoclonal antibodies.In this work we have studied the interference of HAMA in sandwich ELISAs containing antibodies from different species. HAMA, present in the sample, may react both with the capture antibody and the detection antibody in these assays to give a false positive reaction. HAMA did not react with chicken IgG, and if one of (or both) the capture and detection antibody was of avian origin, the interference of HAMA in sandwich assays could be avoided.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.33

年代:1992

数据来源: MAL

摘要:

A procedure was investigated to transduce a bacterial plasmid containing a specific drug resistance marker (pSV2-neo), into a hybridoma cell line using electroporation. The effect of several buffers and the form of plasmid DNA (circular or linearized) on the stable transfection frequency were examined.When complete cell culture medium (DMEM) was used as electroporation buffer, we observed a two-fold increase in post-pulse viability and a ten- to thirty-fold increase in the transfection frequency of pSV2-neo, as compared with HEPES buffered 0.15 M sodium chloride. Supplementing DMEM with fetal bovine serum (DMEM+FBS) had some beneficial effect on post-pulse viability of the cells after electroporation, but did not markedly increase stable transfection frequency as compared with DMEM alone.Furthermore, with DMEM+FBS, the intact plasmid was transfected as effectively as linearized PSV2-neo. However, when using HEPES buffered saline, the transfection frequency of pSV2-neo increased two-fold after linearization as compared with intact plasmid.The drug resistance was used successfully as a marker for the selection of hybrid hybridoma (quadroma) cell lines after fusing two different hybridoma cell lines, producing anti-fibrin and anti-plasminogen activator antibodies respectively. The quadroma cells produced bispecific antibodies that are capable of accumulating plasminogen activator on a fibrin surface.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.41

年代:1992

数据来源: MAL

摘要:

A monoclonal antibody was produced against the recombinant human α subunit of CK-2. The antibody was of the IgGl subclass and it was isolated fromRserum-free cell culture media and purified by affinity chromatography on Protein G Sepharose. The antibody can be used to detect specifically the CK-2 α subunit in immunoblots from tissue extracts. An ELISA detection test was also established which also allows the identification of the CK-2 α subun

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.53

年代:1992

数据来源: MAL

摘要:

Ten hybridomas producing monoclonal antibodies (Mabs) against rabbit platelet factor 4 (PF4) were obtained from the fusion of splenocytes from mice immunized with purified rabbit PF4 and NSO mouse myeloma cells. When the reactivities of these monoclonal antibodies were determined by enzyme-linked immunosorbent assay and immunoblotting with human and rabbit PF4, they showed a high degree of specificity. Only one Mab recognized an epitope common to the human and rabbit molecules, the other nine reacted only with the rabbit protein. All the antibodies recognized, in crude platelet lysates, a band that comigrates with the purified PF4 protein. None of these antibodies cross-reacted with major rabbit or human platelet-poor plasma proteins. The significance of the Mabs in immunological and physiological studies is discussed.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.61

年代:1992

数据来源: MAL

摘要:

Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels offurgene encoded proteins appeared to be very low. In cells transfected with human or mousefurcDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.71

年代:1992

数据来源: MAL

摘要:

In addition to the bispecific antibody, the hybrid-hybridoma 28.19.8 secretes antibodies monospecific for carcinoembryonic antigen (CEA) and antibodies monospecific for vinca alkaloids. By exhaustive affinity purification, the immunoglobulins isolated by Protein A chromatography from ascitic fluid have been fractionated into four populations, three of which are immunoreactive. Studies on these fractions by FPLC, SDS-PAGE, and a variety of ELISAs have shown that seven of the ten possible combinations of heavy and light chains exist as immunoreactive forms. The results suggest that the other three inactive combinations are also secreted by the hybrid-hybridoma.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.87

年代:1992

数据来源: MAL

摘要:

We describe an approach that allows the generation of stable hybridomas secreting antigen specific human IgG antibodies with an efficiency comparable to that of the generation of IgM and IgA secreting hybridomas. This was achieved by evaluating means to increase the frequency of human hybridoma formation and the stability of the generated hybridoma cells when subjected to conditions for large scale growth. To this end, we generated new fusion lines with an increased human DNA content and modified the culture system. However, the application of these new fusion lines primarily resulted in unstable giant cells. As a consequence, we evaluated whether the viability of newly formed hybrids between existing fusion lines and lymphoblastoid cell lines might be improved. In an attempt to provide as many components necessary for the growth of antibody secreting hybridomas as possible, we propagated fused cells in medium supplemented with human serum. Our results show that with this approach the frequency of initially growing hybrids was significantly increased. Furthermore, only in culture medium supplemented with human serum was it possible to obtain stable IgG secreting clones.

ISSN:0272-457X    

DOI:10.1089/hyb.1992.11.99

年代:1992

数据来源: MAL