Estimation of Lactose Interference in Vaccines and a Proposal of Methodological Adjustment of Total Protein Determination by the Lowry Method

Kusunoki, Hideki; Okuma, Kazu; Hamaguchi, Isao

doi:10.7883/yoken.65.489

Cited by 6 publications

(3 citation statements)

References 15 publications

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“…The Lowry assay is also somewhat more sensitive to protein quality. Thiol‐containing compounds such as 2‐mercaptoethanol and DTT can interfere with the Lowry assay ; lactose, a reducing sugar, has also been shown to interfere with the Lowry protein assay .…”

Section: Methods For Total Protein Assay and Protein Qualitymentioning

confidence: 99%

Review of methods for determination of total protein and peptide concentration in biological samples

Sapan¹,

Lundblad

2015

Proteomics Clinical Apps

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Clinical proteomics can be defined as the use of proteomic technologies to identify and measure biomarkers in fluids and tissues. The current work is intended to review various methods used for the determination of the total concentration of protein or peptide in fluids and tissues and the application of such methods to clinical proteomics. Specifically, this article considers the approaches to the measurement of total protein concentration, not the measurement of the concentration of a specific protein or group of proteins in a larger mixture of proteins. The necessity of understanding various concepts such as fit-for-use, quality-by-design, and other regulatory elements is discussed, as is the significance of using suitable standards for the protein quality of various samples.

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Section: Methods For Total Protein Assay and Protein Qualitymentioning

confidence: 99%

Review of methods for determination of total protein and peptide concentration in biological samples

Sapan¹,

Lundblad

2015

Proteomics Clinical Apps

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“…Cells were washed twice with cold phosphate-buffered saline (PBS), cell lysis buffer was added (25 mmol/l Tris-HCl, 10 mmol/l EDTA, a volume fraction of 0.01 NP-40, 150 mmol/l sodium chloride and 1 g/l phenylmethylsulfonyl fluoride), and the solution was placed in an ice bath for 1 h. The solution was centrifuged at 4˚C and 16,000 g for 25 min. The protein concentration of the supernatant was measured by the Lowry method (17). Cell lysates (50 µg) were extracted and electro-transferred to a polyvinylidene fluoride (PVDF) membrane by SDS-PAGE gel electrophoresis.…”

Section: Reverse Transcription-quantitative Polymerase Chain Reactionmentioning

confidence: 99%

Effects of suppressor of cytokine signaling 1 silencing on human melanoma cell proliferation and interferon-γ sensitivity

Xu¹,

Wang

Gou³

et al. 2014

Molecular Medicine Reports

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The aim of the current study was to observe the effects of suppressor of cytokine signaling 1 (SOCS1) silencing in human melanoma cells on cell biological behavior and interferon-γ (IFN-γ) sensitivity, and to investigate the use of SOCS1 as a therapeutic target in the treatment of melanoma. Western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to verify that SOCS1 interference effectively silenced the expression of SOCS1 in the Mel526 human melanoma cell line. For IFN-γ stimulation, western blot analysis was used to observe changes in expression levels of signal transduction and transcription activator (STAT) 1 and phosphorylated STAT (pSTAT) 1. Changes in the expression levels of IFN-γ regulatory factor 1 (IRF-1) were measured with RT-qPCR. Changes in the sensitivity of melanoma cells to IFN-γ were detected using an MTT assay. The cell proliferation rate was observed by cell counting and changes in the cell cycle were detected with flow cytometry. The results revealed that SOCS1 interference effectively silences SOCS1 expression in Mel526 cells. However, the S stage of the cell cycle was markedly extended. Following the inhibition of SOCS1 expression, the proliferation experiment demonstrated that the proliferation ability of Mel526 cells was decreased. Following IFN-γ stimulation, the expression levels of pSTAT and IRF-1 increased significantly compared with those in the controls. The MTT experiment showed that SOCS1 interference caused the median inhibitory concentration (IC50) of oxaliplatin in Mel526 cells to decrease significantly. In conclusion, SOCS1 interference reduced the proliferation ability of Mel526 human melanoma cells and increased their sensitivity to IFN-γ.

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“…However, various substances used for vaccine production, such as sucrose, Triton X-100, EDTA, Tris-HCl, lactose, and amino acid derivatives, may interfere with protein determinations [3][4][5][6][7][8][9][10]. Therefore, several modifications, including such as sodium deoxycholate (DOC) treatment, trichloroacetic acid (TCA) precipitation, or heat treatment, have been investigated for removal of interfering substances [13,14]; these modifications are Q4 included in the method referred to as the "conventional Lowry method."…”

Section: Introductionmentioning

confidence: 99%

Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method

Lee

Shin

Chung

et al. 2015

Vaccine

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Please cite this article in press as: Lee N, et al. Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method. Vaccine (2015), http://dx.a b s t r a c t Aluminum (Al) components in vaccines are known to act as adsorbents that interfere with accurate protein quantification by the Lowry method. Therefore, certain modifications based on the characteristics and compositions of the vaccine are required for determination of protein contents.We investigated the effects of an additional centrifugal separation and found that protein contents were overestimated by up to 238% without centrifugation through a collaborative study performed with hepatitis B vaccines containing Al. However, addition of a centrifugation step yielded protein concentrations that were similar to the actual values, with small coefficients of variation (CVs). Proficiency testing performed in 11 laboratories showed that four laboratories did not have satisfactory results for vaccines containing aluminum hydroxide, although all laboratories were proficient in protein analysis when samples did not contain aluminum hydroxide. Incomplete resuspension of aluminum hydroxide solution with alkaline copper solution was the major cause of insufficient proficiency in these laboratories.

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Estimation of Lactose Interference in Vaccines and a Proposal of Methodological Adjustment of Total Protein Determination by the Lowry Method

Cited by 6 publications

References 15 publications

Review of methods for determination of total protein and peptide concentration in biological samples

Review of methods for determination of total protein and peptide concentration in biological samples

Effects of suppressor of cytokine signaling 1 silencing on human melanoma cell proliferation and interferon-γ sensitivity

Improved quantification of protein in vaccines containing aluminum hydroxide by simple modification of the Lowry method

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