Raising Antibodies by Coupling Peptides to PPD and Immunizing BCG‐Sensitized Animals

Lachmann, P. J.; Strangeways, L; Vyakarnam, Annapurna; Evan, Gérard I.

doi:10.1002/9780470513286.ch3

Cited by 33 publications

(7 citation statements)

References 36 publications

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“…To generate sufficient CD4 T cell help, Mycobacterium tuberculosis PPD was coupled to the peptide:MHC complexes used as the immunogen, and an M. bovis BCG-vaccinated host was used. This use of PPD as an atypical "carrier" has been reported to facilitate Ab responses against other difficult antigens, and has the added advantage of not eliciting anti-carrier Abs (Lachmann et al, 1986).…”

Section: Immunization Strategymentioning

confidence: 99%

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand α-galactosylceramide bound to mouse CD1d

Yu¹,

Im²,

Illarionov³

et al. 2007

Journal of Immunological Methods

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The alpha-galactosylceramide (alpha-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of alpha-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with alpha-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other alpha-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells.

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Section: Immunization Strategymentioning

confidence: 99%

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand α-galactosylceramide bound to mouse CD1d

Yu¹,

Im²,

Illarionov³

et al. 2007

Journal of Immunological Methods

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“…4 In the present study, good antibody responses to tuberculoproteins were achieved by coupling them to KLH and coadministering the conjugates with Gram-negative endotoxin-containing adjuvant as B-lymphocyte stimulants. The anti-PPD IgG was prepared primarily against a commercial PPD preparation.…”

Section: Discussionmentioning

confidence: 79%

“…Whereas PPD proteins are potent T lymphocyte haptens and elicit strong T lymphocyte responses in vivo, their abilities to elicit antibody production are negligible. 4 Although monoclonal antibodies have been produced to various mycobacterial antigens, including some tuberculin components, [5][6][7] and are potentially useful as the basis of assays for specific components or individual epitopes, 7 they are too narrow in specificity for the characterisation of complex tuberculin preparations.…”

Section: Introductionmentioning

confidence: 99%

Tuberculin Purified Protein Derivative (PPD) Immunoassay as an In Vitro Alternative Assay for Identity and Confirmation of Potency

Ho¹,

Kairo²,

Corbel³

2006

Human Vaccines

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KEY WORDS ABSTRACTTuberculin purified protein derivative (PPD) currently can only be standardised by delayed hypersensitivity skin reactions in sensitised guinea pigs. An in vitro dot blot immunoassay was developed for both identity and confirmation of potency estimation of PPD. Polyclonal antibodies (mainly IgG) were generated and immunoreacted with human, bovine and, to lesser extent, avian PPD preparations. Combining size exclusion chromatography (FPLC-SEC) and dot blot immunoassay, the results showed that PPD preparations were mixtures of very heterogeneous tuberculoproteins ranging in size from very large aggregates to very small degraded molecules. All individual fractions of PPD separated by size were immunoreactive, although those of the largest molecular sizes appeared the most immunoreactive in this in vitro dot blot immunoassay. This method is very sensitive and specific to tuberculoproteins and can be an in vitro alternative for the in vivo intradermal skin assay which uses guinea pigs for identity of PPD preparations. Although the capacity of PPD to elicit cell-mediated immune responses on intradermal testing has to be confirmed by in vivo assay, the dot blot immunoassay offers a rapid, sensitive and animal-free alternative to in vivo testing for confirming the identity of PPD preparations with appropriate potencies. This alternative assay would be particularly useful for national regulatory laboratories for confirming the data of manufacturers and thus reducing the use of animals.

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“…The abundance of GLUT5 protein in the BBMV from the intestine of either adult or fetal horses was determined by Western blotting as described previously (Dyer et al 1997a(Dyer et al , 2002. In order to detect the equine GLUT5 protein, a polyclonal antibody was raised in-house against a synthetic peptide corresponding to the horse GLUT5 carboxy-terminus sequence (ELKDIPLSAVEL), according to the procedure described previously (Lachmann et al 1986;Lescale-Matys et al 1993). The signal was detected using an enhanced chemiluminescence (ECL) system 6 , according to the manufacturer's instructions.…”

Section: Western Blot Analysismentioning

confidence: 99%

Molecular characterisation of fructose transport in equine small intestine

Merediz

Dyer

Salmon

et al. 2004

Equine Veterinary Journal

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Summary Reasons for performing study: Fructose can be a suitable carbohydrate supplement for horses before and/or during endurance exercise. In comparison to glucose, the ingestion of fructose results in a lower insulin peak and less marked fluctuations in blood glucose during exercise, potentially avoiding hypoglycaemia‐induced exhaustion. Objectives: To assess the capacity of the equine small intestine to absorb fructose and to determine the mechanism, molecular structure and properties of equine intestinal fructose transport. Methods: Using PCR‐based strategies, RNA isolated from equine small intestine and primers designed to homologous regions of the fructose transporter, GLUT5, cDNA of other species, we cloned and sequenced equine GLUT5 (eGLUT5). Northern and western blot analyses, in conjunction with immunohistochemistry, utilising eGLUT5 cDNA and antibodies, assessed expression of eGLUT5 along the longitudinal and radial axes of the small intestine. Functional properties of fructose transport in intestinal brush‐border membrane vesicles were measured using the rapid‐filtration technique. Results: eGLUT5 is expressed in the villus enterocytes with highest levels in duodenum>jejunum and lowest in the ileum. Kinetic studies indicate eGLUT5 is a low affinity, high capacity transporter. Conclusions: Equine small intestine has the capacity to absorb fructose. Potential relevance: The molecular probes produced in these studies can be used as diagnostic aids to determine equine intestinal monosaccharide malabsorption.

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Raising Antibodies by Coupling Peptides to PPD and Immunizing BCG‐Sensitized Animals

Cited by 33 publications

References 36 publications

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand α-galactosylceramide bound to mouse CD1d

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand α-galactosylceramide bound to mouse CD1d

Tuberculin Purified Protein Derivative (PPD) Immunoassay as an In Vitro Alternative Assay for Identity and Confirmation of Potency

Molecular characterisation of fructose transport in equine small intestine

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