[1] Preparation of monoclonal antibodies: Strategies and procedures

Galfrè, G.; Milstein, C.

doi:10.1016/0076-6879(81)73054-4

Cited by 1,610 publications

(711 citation statements)

References 30 publications

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“…Each mouse was boosted four times at 3-week intervals with the respective proteins in Incomplete Freund's Adjuvant (IFA) (Sigma). Animals with an anti-a-ECD serum response were killed, and their splenocytes were fused with NSO myeloma cells in the presence of 50% PEG 1500, HAT (Sigma) and HFCS (Hybridoma Fusion and Cloning Supplement, Sigma) using standard techniques [29].…”

Section: Immunizations and Fusionsmentioning

confidence: 99%

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Protopapadakis¹,

Kokla²,

Tzartos³

et al. 2005

Eur. J. Immunol.

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The isolation of human antibodies against muscle acetylcholine receptor (AChR), the autoantigen involved in myasthenia gravis (MG), is important for the development of therapeutically useful reagents. Monovalent antibody fragments from monoclonal antibodies against the main immunogenic region (MIR) of AChR protect the receptor from the destructive activity of MG autoantibodies. Human anti-AChR a-subunit antibody fragments with therapeutic potential have been isolated using phage display antibody libraries. An alternative approach for obtaining human mAb has been provided by the development of humanized mice. In this report, we show that immunization of transgenic mouse strains with the extracellular domain of the human AChR a-subunit results in antibody responses and isolation of hybridomas producing human mAb. Four specific IgM mAb were isolated and analyzed. mAb170 recognized the native receptor the best and was capable of inducing AChR antigenic modulation, suggesting its specificity for a pathogenic epitope. Moreover, the recombinant antigen-binding (Fab) fragment of this mAb competed with an anti-MIR mAb, revealing that its antigenic determinant lies in or near the MIR. Finally, Fab170 was able to compete with MG autoantibodies and protect the AChR against antigenic modulation induced by MG sera. This approach will be useful for isolating additional mAb with therapeutic potential against the other AChR subunits.

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Section: Immunizations and Fusionsmentioning

confidence: 99%

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Protopapadakis¹,

Kokla²,

Tzartos³

et al. 2005

Eur. J. Immunol.

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“…The spleen was removed aseptically and the splenocytes fused with Sp2/o myeloma cells (Celltech, UK) as described previously (Galfre and Milstein, 1987). After 7 days, the hybridoma supernatants were screened by ELISA using streptavidin plates as described above.…”

Section: Preparation Of Hybridomas and Ascitesmentioning

confidence: 99%

Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance

Desai

Luqmani²,

Walters³

et al. 1997

Br J Cancer

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Summary A variant form of the human oestrogen receptor (ER) mRNA lacking sequences encoded within exon 5 has been described (Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malloy BW, McGuire WL 1991, Cancer Res 51: 105-1 09).We have examined the expression of the exon 5-deleted ER (HEA5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT -PCR). HEA5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% Cl, P=0.05). Presence of the HEA5 mRNA was associated with the presence of immunohistochemically detected ER (P=0.015) and progesterone receptor (PR) (P=0.02). There was a positive correlation between the presence of HEA5 and disease-free survival (P=0.05), suggesting that the presence of HEA5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HEA5. This antibody recognized the variant but not the wild-type ER protein. We show that HEA5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HEA5 in mammalian cells and showed that, in MCF-7 cells, HEA5 competes with wild-type ER to inhibit ERE-dependent trans-activation. Our results indicate that this variant is unlikely to be responsible for endocrine resistance of breast cancer, but its presence at both the mRNA and protein level suggest that it may, nevertheless, be involved in regulating the expression of oestrogen-responsive genes in breast cancer.Keywords: breast neoplasm; exon; polymerase chain reaction; oestrogen receptor; transcription Two-thirds of human breast carcinomas are characterized by the presence of appreciable amounts of oestrogen receptor (ER) protein.A proportion of these tumours also contain progesterone receptor (PR) and it is generally accepted that ER regulates PR gene expression. The presence of ER is correlated with a better prognosis and ER+/PR+ tumours are much more likely to respond to endocrine therapy than ER-/PR-tumours. Interestingly, ER-/PR+ tumours are twice as likely to respond as ER+/PR-tumours. A significant proportion of ER+/PR+ tumours, however, fail to respond to endocrine therapy and those that do so eventually become resistant to such therapy. The mechanisms leading to endocrine resistance are not yet clear (for reviews see McGuire, 1978;McGuire et al, 1991;Fuqua, 1994;Horwitz, 1994;Sluyser, 1994).The human oestrogen receptor cDNA and its gene (Ponglikitmongkol et al, 1988) have been cloned and the molecular mechanisms by which it acts are well understood. Alignment of the predicted ER amino acid sequences from different species shows that it can be divided into six regions A to F on the basis of differing amino acid sequence homology . Functional studies have shown that region C encodes the DNA- Kumar et al, 1987). Regions A/B and E contain trans-activation functions 1 (AFI) and 2 (AF-2) respectively (Kumar et al, , 1987Webster et al, 1988; Lees et al, 1989; Tora et al, 1989a;Berry et ...

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“…The peptides were coupled to a purified protein derivative of tuberculin (PPD) (Morrison et al, 1987) and were then injected into female Balb/c mice. The spleen, from a selected mouse, was removed and the splenocytes fused with Sp2/O myeloma cells as previously described (Galfre and Milstein, 1981). Hybridoma supematants were screened by ELISA before selection for recloning, after which the antibodies were isotyped and total IgG concentrations were evaluated.…”

mentioning

confidence: 99%

The location of acidic fibroblast growth factor in the breast is dependent on the activity of proteases present in breast cancer tissue

Coope

Browne²,

Yiangou³

et al. 1997

Br J Cancer

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Summary Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 370C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.Keywords: breast cancer; fibroblast growth factor 1; protease; immunohistochemistry Fibroblast growth factor 1 (FGF1) belongs to a family of multifunctional polypeptides that are involved in a wide array of biological processes, which include cellular proliferation and differentiation, angiogenesis, chemotaxis, embryonal development and tissue repair (Basilico and Moscatelli, 1992). To date, the FGFs consist of a family of nine homologous polypeptide growth factors that include FGF1 (acidic FGF), FGF2 (basic FGF), FGF3 (int-2), FGF4 (hst-l/Kaposi FGF), FGF5, FGF6 (hst-2), FGF7 (keratinocyte growth factor), FGF8 (androgen-induced growth factor) and FGF9 (glial-activating factor) (Basilico and Moscatelli, 1992;Tanaka et al, 1992;Miyamoto et al, 1993). These proteins share 35-50% overall homology of their amino acid sequences (Basilico and Moscatelli, 1992;Givol and Yayon, 1992).Unlike other members of the family, FGF1, FGF2 and FGF9 are synthesized without a signal peptide sequence and thus may remain sequestered in the cell (Basilico and Moscatelli, 1992;Cao and Pettersson, 1993). However, release of FGF may occur through leakage from damaged cells or from viable cells via a novel mechanism (Mignatti et al, 1992;Cao and Pettersson, 1993). Yeoman (1993) has postulated that proteoglycan-bound FGF may be released from the cell surface or extracellular matrix by the action of proteases, and Briozzo et al (1991) have shown that Received 10 June 1996 Revised 26 November 1996 Accepted 3 December 1996 Correspondence to: J Gomm MCF7 breast cancer cells secrete cathepsin D, which is able to digest the extracellular matrix and releas...

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[1] Preparation of monoclonal antibodies: Strategies and procedures

Cited by 1,610 publications

References 30 publications

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Isolation and characterization of human anti-acetylcholine receptor monoclonal antibodies from transgenic mice expressing human immunoglobulin loci

Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance

The location of acidic fibroblast growth factor in the breast is dependent on the activity of proteases present in breast cancer tissue

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