A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli

Zhu, Darning; Saul, Allan; Miles, Aaron P.

doi:10.1016/j.jim.2005.07.021

Cited by 28 publications

(27 citation statements)

References 11 publications

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“…To determine the intensity of the interaction between the LigA fragments and equine anti-Leptospira antibodies, we used a quantitative slot assay as previously described (30). The membrane to be analyzed was scanned immediately following the blotting procedure.…”

Section: Matmentioning

confidence: 99%

Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

Yan

Saleem

McDonough

et al. 2013

Clin Vaccine Immunol

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Leptospira immunoglobulin (Ig)-like (Lig) proteins are a novel family of surface-associated proteins in which the N-terminal 630 amino acids are conserved. In this study, we truncated the LigA conserved region into 7 fragments comprising the 1st to 3rd (LigACon1-3), 4th to 7.5th (LigACon4-7.5), 4th (LigACon4), 4.5th to 5.5th (LigACon4.5-5.5), 5.5th to 6.5th (LigACon5.5-6.5), 4th to 5th (LigACon4-5), and 6th to 7.5th (LigACon6-7.5) repeat domains. All 7 recombinant Lig proteins were screened using a slot-shaped dot blot assay for the diagnosis of equine leptospirosis. Our results showed that LigACon4-7.5 is the best candidate diagnostic antigen in a slot-shaped dot blot assay. LigACon4-7.5 was further evaluated as an indirect enzyme-linked immunosorbent assay (ELISA) antigen for the detection of Leptospira antibodies in equine sera. This assay was evaluated with equine sera (n ‫؍‬ 60) that were microscopic agglutination test (MAT) negative and sera (n ‫؍‬ 220) that were MAT positive to the 5 serovars that most commonly cause equine leptospirosis. The indirect ELISA results showed that at a single serum dilution of 1:250, the sensitivity and specificity of ELISA were 80.0% and 87.2%, respectively, compared to those of MAT. In conclusion, an indirect ELISA was developed utilizing a recombinant LigA fragment comprising the 4th to 7.5th repeat domain (LigACon4-7.5) as a diagnostic antigen for equine leptospirosis. This ELISA was found to be sensitive and specific, and it yielded results that concurred with those of the standard MAT.

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Section: Matmentioning

confidence: 99%

Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

Yan

Saleem

McDonough

et al. 2013

Clin Vaccine Immunol

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“…Capsids were incubated for 30 min at 22°C with the purified feline TfR ectodomain at molar ratios of 1:15, 1:30, and 1:60 and then passed through a 60-ϫ 0.6-cm Sepharose CL2B column in Pipes-HCl (pH 7.5) and 0.15 M NaCl. Fractions corresponding to aggregation (void volume), CPV-TfR complex, and control fractions to establish background were probed for CPV and TfR by using quantitative Western analysis of slot blots on nitrocellulose filters (34). The band intensity of each sample was determined, and the quantities of the unknown samples were estimated with respect to a calibration curve produced by using predetermined amounts of CPV and TfR.…”

Section: Biochemical Determination Of Stoichiometry Of Receptor and Cmentioning

confidence: 99%

Asymmetric binding of transferrin receptor to parvovirus capsids

Hafenstein

Palermo

Kostyuchenko

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Although many viruses are icosahedral when they initially bind to one or more receptor molecules on the cell surface, such an interaction is asymmetric, probably causing a breakdown in the symmetry and conformation of the original infecting virion in preparation for membrane penetration and release of the viral genome. Cryoelectron microscopy and biochemical analyses show that transferrin receptor, the cellular receptor for canine parvovirus, can bind to only one or a few of the 60 icosahedrally equivalent sites on the virion, indicating that either canine parvovirus has inherent asymmetry or binding of receptor induces asymmetry. The asymmetry of receptor binding to canine parvovirus is reminiscent of the special portal in tailed bacteriophages and some large, icosahedral viruses. Asymmetric interactions of icosahedral viruses with their hosts might be a more common phenomenon than previously thought and may have been obscured by averaging in previous crystallographic and electron microscopic structure determinations.asymmetric reconstruction ͉ canine parvovirus ͉ receptor-virus interaction ͉ unique binding

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“…There have been several reports on the generation of anti-HCP antibodies and development of immunoassays (Anicetti et al, 1986;Dagouassat et al, 2001;Thalhamer and Freund, 1984;Zhu et al, 2005). It is worth noting that a high quality preparation of the HCPs to be used in anti-HCP antibody generation is critical to the success of the HCP program.…”

Section: Development Of Immunoassays For Hcp Quantitationmentioning

confidence: 99%

Host cell proteins in biologics development: Identification, quantitation and risk assessment

Wang

Hunter

Mozier

2009

Biotech & Bioengineering

196

184

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Host cell proteins (HCPs) are those produced or encoded by the organisms and unrelated to the intended recombinant product. Some are necessary for growth, survival, and normal cellular processing whereas others may be non-essential, simply carried along as baggage. Like the recombinant product, HCPs may also be modified by the host with a number of post-translational modifications. Regardless of the utility, or lack thereof, HCPs are undesirable in the final drug substance. Though commonly present in small quantities (parts per million expressed as nanograms per milligrams of the intended recombinant protein) much effort and cost is expended by industry to remove them. The purpose of this review is to summarize what is of relevance in regards to the biology, the impact of genomics and proteomics on HCP evaluation, the regulatory expectations, analytical approaches, and various methodologies to remove HCPs with bioprocessing. Historical data, bioinformatics approaches and industrial case study examples are provided. Finally, a proposal for a risk assessment tool is provided which brings these facets together and proposes a means for manufacturers to classify and organize a control strategy leading to meaningful product specifications.

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A quantitative slot blot assay for host cell protein impurities in recombinant proteins expressed in E. coli

Cited by 28 publications

References 11 publications

Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

Development of an Enzyme-Linked Immunosorbent Assay Using a Recombinant LigA Fragment Comprising Repeat Domains 4 to 7.5 as an Antigen for Diagnosis of Equine Leptospirosis

Asymmetric binding of transferrin receptor to parvovirus capsids

Host cell proteins in biologics development: Identification, quantitation and risk assessment

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