Simple Immunization Protocol for High Frequency Production of Soluble Antigen-Specific Hybridomas

Cianfriglia, Maurizio; Armellini, D.; Massone, A.; Mariani, Massimo A.

doi:10.1089/hyb.1983.2.451

Cited by 74 publications

(19 citation statements)

References 9 publications

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“…In order to establish specific MAbs to M. penetrans LAMP, BALB/c mice were immunized by the method of Cianfriglia et al (6) with 10 8 cells of M. penetrans 75 per immunization. The splenocytes were fused with myeloma cells as previously described (20).…”

Section: Methodsmentioning

confidence: 99%

Phase Variation among Major Surface Antigens of Mycoplasma penetrans

et al. 2001

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The pathogenicity and prevalence of Mycoplasma penetrans, a Mycoplasma species recently isolated from humans, are still debated. A major P35 antigen, which is used as target epitope in serological assays, was shown to be a phase-variable lipid-associated membrane protein (LAMP). In this study, we performed a comparative analysis of the LAMP patterns from five M. penetrans clinical isolates and from the type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and immunoblots with sera serially collected from an M. penetrans-infected patient indicated that these strains expressed different LAMP repertoires. Furthermore, the intraclonal variation in the expression of LAMPs (P34A, P34B, P35, and P38) was monitored by immunoblot analysis with three specific monoclonal antibodies (MAbs) developed in this study and MAb 7 to P35. The phase variation of these LAMPs occurs in an independent manner, with frequencies of variation ranging from 10 ؊2 to 10 ؊4 per cell per generation. Consistent with their amphipathic nature, the P34B and P38 antigens were found exposed at the cell surface. The DNA sequence encoding the P38 antigen was defined and found to be related to those of the P35 gene and other putative LAMP-encoding genes, suggesting that these variable antigens are encoded by a family of related genes. Finally, the serum samples from an M. penetrans-infected patient contained antibodies that reacted with a P36 antigen expressed in different M. penetrans strains but not in the isolate recovered from this patient. This result suggested that in vivo phase variation of P36 occurred, which would support a role for these LAMP variations in avoiding the host's immune vigilance.

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Section: Methodsmentioning

confidence: 99%

Phase Variation among Major Surface Antigens of Mycoplasma penetrans

et al. 2001

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“…A monospecific mouse polyclonal antibody (pAb 20.8) was generated against the native 20.8-kDa polypeptide following the immunization protocol described by Cianfriglia et al (19). The 20.8-kDa antigen was obtained from worm lysates by immunoaffinity chromatography using mAb 709A2/2 as outlined by Harlow and Lane (20), followed by SDS-PAGE separation.…”

Section: Methodsmentioning

confidence: 99%

Molecular Characterization of a 20.8-kDaSchistosoma mansoni Antigen

Hoffmann¹,

Strand

1997

Journal of Biological Chemistry

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“…Balb/c mice were im-munized with the purified membrane-bound enzyme according to the procedure proposed in [16]. Hybridomas were cloned by the method of limited dilutions [17].…”

Section: Methodsmentioning

confidence: 99%

Topology of Na⁺,K⁺‐ATPase Identification of the extra‐ and intracellular hydrophilic loops of the catalytic subunit by specific antibodies

et al. 1988

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To study the topology of Na+,K+‐ATPase monoclonal antibodies (MAbs) specific for membrane‐bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra‐ or intracellular portions of the α‐subunit. The extracellular location of peptide loop 804–841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween‐20), was shown to bind to the hydrophilic loop 868–945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887–904 and 810–825, respectively. The C‐terminus of the α‐subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity‐purified antibodies to the synthetic peptide corresponding to fragment 999–1008.

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Simple Immunization Protocol for High Frequency Production of Soluble Antigen-Specific Hybridomas

Cited by 74 publications

References 9 publications

Phase Variation among Major Surface Antigens of Mycoplasma penetrans

Phase Variation among Major Surface Antigens of Mycoplasma penetrans

Molecular Characterization of a 20.8-kDaSchistosoma mansoni Antigen

Topology of Na⁺,K⁺‐ATPase Identification of the extra‐ and intracellular hydrophilic loops of the catalytic subunit by specific antibodies

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Simple Immunization Protocol for High Frequency Production of Soluble Antigen-Specific Hybridomas

Cited by 74 publications

References 9 publications

Phase Variation among Major Surface Antigens of Mycoplasma penetrans

Phase Variation among Major Surface Antigens of Mycoplasma penetrans

Molecular Characterization of a 20.8-kDaSchistosoma mansoni Antigen

Topology of Na+,K+‐ATPase Identification of the extra‐ and intracellular hydrophilic loops of the catalytic subunit by specific antibodies

Contact Info

Product

Resources

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Topology of Na⁺,K⁺‐ATPase Identification of the extra‐ and intracellular hydrophilic loops of the catalytic subunit by specific antibodies