A modified method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells

Lacy, Michael J.; Voss, Edward W.

doi:10.1016/0022-1759(86)90527-2

Cited by 65 publications

(30 citation statements)

References 21 publications

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“…The relative guinea pig potency assay also requires the use of a laboratory with biosafety containment levels that meet safety regulations to conduct research using virulent strains of B. anthracis. Standards for the quantitative anti-PA IgG ELISA and TNA assay were prepared from ascitic fluids prepared in Balb/c mice based upon the protocol published by Lacy and Voss [23]. Using this method, we were able to collect a large amount of ascites from fewer animals as compared to traditional methods of collecting serum.…”

Section: Discussionmentioning

confidence: 99%

“…Standards for the quantitative anti-PA IgG ELISA and TNA assay were obtained from ascitic fluids prepared in Balb/c mice based upon the protocol published by Lacy and Voss [23]. The procedure used called for scheduled injections of pristine-primed Balb/c mice with antigen either emulsified with Freund's complete adjuvant (for preparation of the ELISA standard) or adsorbed to Alhydrogel (for the TNA assay standard) followed by injection of Sp2/0-Ag14 myeloma cells.…”

Section: Preparation Of Standards For Elisa and Tna Assaymentioning

confidence: 99%

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Development of an in vitro-based potency assay for anthrax vaccine

Little

Webster

Ivins

et al. 2004

Vaccine

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Section: Discussionmentioning

confidence: 99%

Section: Preparation Of Standards For Elisa and Tna Assaymentioning

confidence: 99%

Development of an in vitro-based potency assay for anthrax vaccine

Little

Webster

Ivins

et al. 2004

Vaccine

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“…mAbs to rsG1 and rsG2 were generated in the facility of the University of Illinois (Urbana-Champaign, IL) using standard techniques (24,25). Tenweek-old female BALB/c mice were immunized by i.p.…”

Section: Mab Generation and Characterizationmentioning

confidence: 99%

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

Morales¹,

Pace

Platt³

et al. 2003

The Journal of Immunology

106

101

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The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with β2-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8α expression on PBMC without changing CD3δ expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.

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“…The method for the production of ascites fluid has been described previously (Lacy and Voss, 1986). Preimmune and immune serum or ascites fluid were collected by personnel at the hybridoma facility at the University of Illinois and screened in our laboratory by Western blot analysis (see below).…”

Section: Immunological Proceduresmentioning

confidence: 99%

Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF

Cieslewicz

Vimr

1997

Molecular Microbiology

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SummaryNeuroinvasive Escherichia coli K1 synthesizes and assembles a polysialic acid capsule virulence factor on the external leaflet of the outer membrane. This capsule functions in pathogenesis by blocking nonimmune host defence mechanisms and acting as a relatively non-immunogenic molecular mimic of the polysialic acid chains found in high concentrations on neural cell adhesion molecules of the human embryo and neonate. The synthetic, regulatory and export components for capsule expression are encoded in three functionally distinct gene blocks or regions of the 20 kb kps 'pathogenicity island'. These regions are organized as two convergently transcribed operons inserted into the monocistronic tRNA gene, pheV. The six genes of the so-called region 1 operon are transcribed in the same direction as pheV, and at least four of these genes are required for polysialic acid export. Expression of this operon is thermoregulated by transcriptional control of its first gene, kpsF. To investigate the function of region 1 further, two independent chromosomal disruptions were engineered by inserting promoterless, terminatorless kanamycin or chloramphenicol resistance cassettes into the HindIII site of the kpsF coding sequence. The chromosomal insertions were regulated by temperature in the same way as the wild-type operon, demonstrating that this control mechanism remained intact in these mutants. Chemical, immunological and ultrastructural microscopical methods demonstrated that full-length polysialic acid chains were synthesized but not exported by the kpsF mutants. This phenotype was correlated with decreased plaque diameter when the mutants were infected with the capsule-specific bacteriophage K1F. The export defect could not be complemented in trans with kpsF þ containing its cis-regulatory region because of titration of an apparent positive regulator of region 1 expression, whereas complementation was observed with a plasmid expressing kpsF from a physiologically irrelevant promoter. An N-terminal polyhistidine peptide was attached to KpsF and used to purify the overproduced polypeptide. Antibodies raised against KpsF identified at least one of its paralogues in E. coli, GutQ, suggesting that KpsF and its homologues are membrane associated. The results indicate the requirement for a precise balance between region 1 components of the capsule export machinery, and that KpsF plays a positive role in the assembly, operation or regulation of this apparatus.

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A modified method to induce immune polyclonal ascites fluid in BALB/c mice using Sp2/0-Ag14 cells

Cited by 65 publications

References 21 publications

Development of an in vitro-based potency assay for anthrax vaccine

Development of an in vitro-based potency assay for anthrax vaccine

Placental Cell Expression of HLA-G2 Isoforms Is Limited to the Invasive Trophoblast Phenotype

Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF

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