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Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein.

Scheffer GL, Reurs AW, Jutten B, Beiboer SH, van Amerongen R, Schoester M, Wiemer EA, Hoogenboom HR, Scheper RJ - Br. J. Cancer (2002)

Bottom Line: BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs.The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies.Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Free University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.

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Related in: MedlinePlus

BstNI fingerprinting of six MVP-reactive clones from selection rounds 3 and 4 and a randomly selected non-reactive clone. cDNA of individual clones was PCR amplified, BstNI digested and loaded on an agarose gel. The MVP-reactive clones all show a very similar banding pattern, strikingly different from the irrelevant clone.
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fig1: BstNI fingerprinting of six MVP-reactive clones from selection rounds 3 and 4 and a randomly selected non-reactive clone. cDNA of individual clones was PCR amplified, BstNI digested and loaded on an agarose gel. The MVP-reactive clones all show a very similar banding pattern, strikingly different from the irrelevant clone.

Mentions: The successive rounds to select anti-MVP human Fabs on immunotubes coated with recombinant MVP, showed a gradual increase in the amount of antigen bound phages (output number). When the output number of round one was set to 1, output numbers of 50, 500 and 10 000 were noted for round 2, 3 and 4, respectively (Table 1Table 1Enrichment of MVP binding phages per selection round). Apparently, the cycles of selection and re-amplication yielded increasing numbers of antigen-binding phages. ELISA screening of approximately 750 individual clones from round 2, 3 and 4, resulted in the identification of several MVP-reactive phages. The percentage of antigen binding phages per selection round is depicted in Table 1. Twenty-two individual MVP-reactive clones from round 3 and 4 were selected for BstNI fingerprinting. These clones all showed a very similar banding pattern, that was strikingly different from a randomly selected non-reactive clone (Figure 1Figure 1


Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein.

Scheffer GL, Reurs AW, Jutten B, Beiboer SH, van Amerongen R, Schoester M, Wiemer EA, Hoogenboom HR, Scheper RJ - Br. J. Cancer (2002)

BstNI fingerprinting of six MVP-reactive clones from selection rounds 3 and 4 and a randomly selected non-reactive clone. cDNA of individual clones was PCR amplified, BstNI digested and loaded on an agarose gel. The MVP-reactive clones all show a very similar banding pattern, strikingly different from the irrelevant clone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2364164&req=5

fig1: BstNI fingerprinting of six MVP-reactive clones from selection rounds 3 and 4 and a randomly selected non-reactive clone. cDNA of individual clones was PCR amplified, BstNI digested and loaded on an agarose gel. The MVP-reactive clones all show a very similar banding pattern, strikingly different from the irrelevant clone.
Mentions: The successive rounds to select anti-MVP human Fabs on immunotubes coated with recombinant MVP, showed a gradual increase in the amount of antigen bound phages (output number). When the output number of round one was set to 1, output numbers of 50, 500 and 10 000 were noted for round 2, 3 and 4, respectively (Table 1Table 1Enrichment of MVP binding phages per selection round). Apparently, the cycles of selection and re-amplication yielded increasing numbers of antigen-binding phages. ELISA screening of approximately 750 individual clones from round 2, 3 and 4, resulted in the identification of several MVP-reactive phages. The percentage of antigen binding phages per selection round is depicted in Table 1. Twenty-two individual MVP-reactive clones from round 3 and 4 were selected for BstNI fingerprinting. These clones all showed a very similar banding pattern, that was strikingly different from a randomly selected non-reactive clone (Figure 1Figure 1

Bottom Line: BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs.The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies.Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Free University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.

Show MeSH
Related in: MedlinePlus