Affinity of the monoclonal antibody M1 directed against the FLAG peptide

Einhauer, A.; Jungbauer, Alois

doi:10.1016/s0021-9673(01)00831-7

Cited by 43 publications

(22 citation statements)

References 26 publications

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“…Based on these findings, we chose SPR to quantify the aMptD-receptor interactions. The difference in the association constants revealed an explicitly higher affinity ) are comparable to those described for high-affinity antibodies or peptides (11,35). These results demonstrate that the MptD peptide binds to M. avium subsp.…”

Section: Discussionsupporting

confidence: 74%

Peptide aMptD-Mediated Capture PCR for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bulk Milk Samples

Stratmann

Dohmann

Heinzmann

et al. 2006

Appl Environ Microbiol

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A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. Paratuberculosis (Johne's disease) is a chronic and incurable granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (29). The disease occurs worldwide with increasing frequency (32) and has a considerable economic impact on the livestock industry (21). Calves are mostly infected in early life, with high shedding and clinical disease commonly occurring at 2 to 5 years of age (37). Previous attempts to eradicate the disease from infected herds have frequently failed, despite a high degree of organizational and financial efforts (3,26). This is most likely due to the ubiquitous presence of the pathogen in the environments of infected herds (39) and its very great tenacity (7,20,52).An economically feasible alternative to eradication would be a control program aiming at the early identification and removal of high shedders, thereby reducing environmental contamination and infectious pressure on the herd. High shedders are more likely to secrete M. avium subsp. paratuberculosis in milk (50), and in addition, in herds with high shedders the pathogen is more likely to enter the milk by fecal contamination (8). Therefore, bulk milk might be a suitable diagnostic substrate for such an approach. Since the general infrastructure for testing of bulk milk from farms (i.e., untreated raw milk) is established (25), regular testing of this milk for the presence of M. avium subsp. paratuberculosis could allow early detection of herds with high shedders. The most convenient and amenable methods for such detection of M. avium subsp. paratuberculosis DNA in milk are enrichment via immunomagnetic separation (17,28,38) and peptide-mediated capture (47) followed by PCR, as these methods can be adapted to high-throughput testing using standard laboratory automation.In the study presented here, we followed this approach, using the phage display-derived peptide aMptD (46) for the capture of M. avium subsp. paratuberculosis in milk samples. The aMptD peptide was shown to bind to the surface-exposed MptD protein of M. avium subsp. paratuberculosis (ORF 3733C) (31), which is part of an M. avium subsp. paratuberculosis-specific pathogenicity island (46), and therefore this method, in contrast to the previously described peptide-mediated capture assay (47), is based on a defined receptor-ligand interaction. Furthermore, we thoroughly elucidated the strain and species cross-specificity of peptide aMptD for M. avium subsp. paratuberculosis by performing competitive capture assays, and we determined the kinetics and affinity of the receptor-ligand interaction by surface plasmon resonance (SPR) (27) in BIAcore biosensor experiments. Finally, in order to

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Section: Discussionsupporting

confidence: 74%

Peptide aMptD-Mediated Capture PCR for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bulk Milk Samples

Stratmann

Dohmann

Heinzmann

et al. 2006

Appl Environ Microbiol

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“…For example, the tandem-affinity purification (TAP) tag [1] enabled the analysis of protein complexes in yeast including a large scale qualitative analysis of protein complexes from S. cerevisiae cells grown in YPD media [2]. Another notable report of a high-throughput protein complex identification is the study by Ho et al, which also analyzed protein complexes from S. cerevisiae grown in YPD media [3] using the FLAG epitope tag [4]. However, neither method proved sufficiently comprehensive to provide full coverage, and 50% of the data reported in these two high throughput proteomic analyses [2,3] were predicted to be spurious [5].…”

Section: Introductionmentioning

confidence: 99%

Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors

Florens

Carozza

Swanson

et al. 2006

Methods

314

279

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Mass spectrometry based approaches are commonly used to identify proteins from multiprotein complexes, typically with the goal of identifying new complex members or identifying post translational modifications. However, with the recent demonstration that spectral counting is a powerful quantitative proteomic approach, the analysis of multiprotein complexes by mass spectrometry can be reconsidered in certain cases. Using the chromatography based approach named multidimensional protein identification technology, multiprotein complexes may be analyzed quantitatively using the normalized spectral abundance factor that allows comparison of multiple independent analyses of samples. This study describes an approach to visualize multiprotein complex datasets that provides structure function information that is superior to tabular lists of data. In this method review, we describe a reanalysis of the Rpd3/Sin3 small and large histone deacetylase complexes previously described in a tabular form to demonstrate the normalized spectral abundance factor approach.

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“…The FLAG/anti-FLAG system is a comparable commercial system (Sigma Aldrich) that is frequently used for the purification of recombinant proteins by immunoaffinity chromatography [17].…”

Section: Characterization Of the Different Tag Versionsmentioning

confidence: 99%

One-Step Protein Purification: Use of a Novel Epitope Tag for Highly Efficient Detection and Purification of Recombinant Proteins

Rasche¹

2011

TOBIOTJ

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We used the interaction between an epitope of the Tobacco mosaic virus 54K replicase (tag54) and its corresponding monoclonal antibody 54 (mAb54) for the design of a new epitope-tagging system. We fused the DNA sequence of tag54 and two elongated derivates thereof to the C-terminus of the chloramphenicol acetyltransferase (CAT) gene and produced the tagged proteins in tobacco. Immunoblot and ELISA analysis demonstrated that the tagged proteins were detected with high sensitivity in plant extracts. Moreover, the tag54 system enabled the full recovery of the recombinant CAT with excellent purity through immunoaffinity chromatography.We consider this novel epitope-tag system to be a valuable tool for both the detection and purification of recombinant proteins.

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Affinity of the monoclonal antibody M1 directed against the FLAG peptide

Cited by 43 publications

References 26 publications

Peptide aMptD-Mediated Capture PCR for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bulk Milk Samples

Peptide aMptD-Mediated Capture PCR for Detection ofMycobacterium aviumsubsp.paratuberculosisin Bulk Milk Samples

Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors

One-Step Protein Purification: Use of a Novel Epitope Tag for Highly Efficient Detection and Purification of Recombinant Proteins

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