A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.

Refractometry as an alternative to the biuret method for measuring total serum proteins in Podocnemis expansa (Podocnemididae) and Phrynops geoffroanus (Chelidae) / Refratometria como alternativa ao método do biureto para mensuração de proteínas séricas totais em Podocnemis expansa e Phrynops geoffroanus (Podocnemididae, Chelidae)

Total serum protein is a significant indicator of health condition in animals. The aim of this study was to analyze the precision of the portable refractometer in determining the concentration of total serum proteins in Podocnemis expansa and Phrynops geoffroanus. A total of 26 animals were used. The blood samples were collected from the supraoccipital sinus and stored in tubes without anticoagulant. Total serum protein was determined using both the biuret reaction and refractometry. The total serum protein mean concentration (g dL-1) with biuret method and refractometry for P. expansa were 3.16 and 3.2; and for P. geoffroanus were 3.56 and 2.72, respectively. These results indicate that total serum protein values can be determined with precision in P. expansa and P. geoffroanus using a portable refractometer.

A proteína sérica total é um indicador significativo do estado de saúde em animais. O objetivo desse estudo foi analisar a precisão do refratômetro portátil para determinar a concentração de proteínas séricas totais em Podocnemis expansa e Phrynops geoffroanus. Foram utilizados um total de 26 animais. As amostras de sangue foram coletadas por punção do seio supraoccipital e armazenadas em tubos sem anticoagulante. A concentração de proteína sérica total foi determinada utilizando tanto a reação de biureto como um refratômetro portátil. A média da proteína sérica total (g dL-1) pela reação de biureto e pela refratometria para P. expansa foram de 3,16 e 3,2; e para P. geoffroanus foram de 3,56 e 2,72, respectivamente. Estes resultados indicam que os valores de proteínas séricas totais podem ser determinados com precisão em P. expansa e P. geoffroanus usando o refratômetro portátil.

Comparison of refractometry and biuret assay for measurement of total protein concentration in canine abdominal and pleural fluid specimens.

OBJECTIVE: To compare total protein (TP) concentrations in canine pleural and abdominal fluid specimens as measured by refractometry and biuret assay. DESIGN: Diagnostic test evaluation. SAMPLE: Data regarding 92 pleural and 148 abdominal fluid specimens from dogs with various diseases. PROCEDURES: TP concentrations in fluid specimens as measured by refractometry and biuret assay were recorded. Strength of association between sets of measurements was assessed by Spearman rank correlations and Bland-Altman plots. Optimal concentration cutoff for diagnostic discrimination between exudate and nonexudate was identified by construction of receiver operating characteristic curves. RESULTS: Median TP concentration in pleural fluid specimens was 2.7 g/dL (range, 0.3 to 4.8 g/dL) for refractometry and 2.9 g/dL (range, 0.7 to 5.8 g/dL) for biuret assay. Median TP concentration in abdominal fluid specimens was 3.5 g/dL (range, 0.1 to 6.0 g/dL) for refractometry and 3.5 g/dL (range, 0.6 to 5.7 g/dL) for biuret assay. Correlation was significant between refractometric and biuret results for pleural (ρ = 0.921) and abdominal (ρ = 0.908) fluid. Bland-Altman plots revealed bias of -0.18 g/dL for pleural fluid and -0.03 g/dL for abdominal fluid for refractometry versus biuret assay. With a TP concentration of ≥ 3 g/dL used to distinguish exudate from nonexudate, sensitivity of refractometry was 77% for pleural fluid and 80% for abdominal fluid. Specificity was 100% and 94%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Refractometry yielded acceptable results for measurement of TP concentration in canine pleural and abdominal fluid specimens, providing a more rapid and convenient method than biuret assay.

Comparison of manual and laboratory PCV and total protein using EDTA and lithium heparin canine samples.

OBJECTIVES: To evaluate if lithium heparin (LiH) and potassium ethylenediaminetetraacetic acid (EDTA) can be used interchangeably to obtain packed cell volume (PCV) and total protein by refractometry (TPr), and to compare those values with laboratorywderived haematocrit (Hct) and total protein (TP) concentration, respectively, in canine blood samples. METHODS: Blood samples taken in LiH and EDTA were manually assessed for PCV and TPr. Results were correlated to Hct and TP. RESULTS: 238 EDTA and corresponding serum/LiH samples were obtained. There was excellent correlation but statistically significant difference between LiH and EDTA PCV (n=43). LiH and EDTA TPr (n=43) were excellently correlated without significant difference. PCV and Hct (n=176) were excellently correlated without significant difference. LiH (n=105) and serum (n=133) TP was respectively fairly or well correlated with TPr but with significant differences. An increase in cholesterol of 1 mmol/L was associated with a mean independent increase in TPr of approximately 1 g/L. CLINICAL SIGNIFICANCE: LiH and EDTA can be used interchangeably for TPr. Although TPr and serum/plasma TP were correlated, there were statistically significant differences that could impact on clinical decision making. TPr is increased by cholesterol but this alone could not account for the magnitude of the difference observed.

The colorimetric detection and quantitation of total protein.

Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.

Evaluation of four commercial biuret reagent kits of serum total protein by the American Association for Clinical Chemistry reference measurement procedure.

BACKGROUND: In China, the traceability of clinical chemistry methods is still immature. Therefore, it is necessary to establish a reference measurement procedure and evaluate commercial reagent kits using such established procedures. METHODS: We reproduced the reference measurement procedure for serum total protein, as recommended by the American Association for Clinical Chemistry (AACC). We evaluated the performance by Clinical Laboratory Standard Institute (CLSI) guidelines EP15-A and EP6-A. Subsequently, four commercial reagent kits were evaluated by the reproduced reference procedure following CLSI guideline EP9-A2. RESULTS: The performance of the reproduced reference procedure was as follows: CVs ranged from 0.47% to 0.85% at medical decision levels (X(c)) of 45 g/L, 60 g/L and 80 g/L. Linearity was Y=1.0022X-0.2121 (r=0.9999), and recovery ranged from 100.2% to 102.4%. The External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine (RELA) was applied, and the result was within the limit of equivalence. The linear relationships of four commercial reagent kits, Merit Choice, KHB, Leadman, and Olympus, were, respectively: Y=0.9922X+0.5776 (r=0.9961); Y=0.9936X+0.4316 (r=0.9992); Y=0.9949X+0.9129 (r=0.9987) and Y=0.9923X+0.8876 (r=0.9989). KHB showed slight negative bias, and the mean±SD was -0.03±0.60 g/L. Merit Choice, Leadman, and Olympus all showed positive bias, and the mean±SDs were 0.02±0.63 g/L, 0.55±0.77 g/L and 0.34±0.71 g/L, respectively. CONCLUSIONS: The correlation and bias of four commercial reagent kits for serum total protein were found to be acceptable. Thus, these reagent kits can be used reliably in China.

Albumin-based or albumin-linked calibrators cause a positive bias in serum proteins assayed by the biuret method.

BACKGROUND: Assay of total serum protein by the biuret method calibrated with albumin standards according to the reference method provides results with a positive bias approximately 3%-5% exceeding the total error of 3.4% allowable for total protein in serum analysis made by analysers using two-part reagents and short-term procedures. METHODS: We used two types of two-part biuret reagents utilised in a short-term measurement in analysers with albumin or serum calibrators, in which protein was attested by the Kjeldahl method. RESULTS: Tests with potentially interfering substances proved that serum blanking used in a short-term biuret procedure is not capable of sufficiently eliminating effects of serum interferents. A short-term blanking is evidently capable of suppressing only an absorbance caused by serum-present coloured and turbid interferents, but its capacity to transform them (oxidise, hydrolyse, saponify, etc.) to some other not-interfering substances is very low compared with a long-term blanking. Lipids and bilirubin are responsible for significant positive bias of total protein in normal serum samples (approximately 3%) and even a greater positive offset in lipaemic and icteric sera (approximately 5%). We verified that interference tests based on a normal serum spiked with endogenous lipids and bilirubin give quite false and misleading results in the biuret reaction. A pure albumin, not depending on its bovine/human origin, gives absorbance responding only to its copper complexes with protein with a biuret regent, while its absorbance with a serum also includes the absorbance of interferents present in serum. The simplest way to improve current short-term biuret procedures is the use of a human serum calibrator with total protein attested by the Kjeldahl method. A serum calibrator, behaving analogously to serum samples, compensates for a positive bias in most normal sera. Reagents with a greater concentration of active biuret components (copper and alkali, reference method included) seem to be unnecessarily aggressive to proteins and are responsible for a lower accuracy when used in short-term measurements. CONCLUSIONS: Standard Reference Material 927c based on pure bovine albumin is still recommended and used as the primary standard for assays of total protein by colourimetric methods. The albumin calibrator is responsible for a positive bias of approximately 3%-5% in serum total protein assayed by the biuret reaction both in the reference and in current methods. Its substitution by a serum calibrator attested by the Kjeldahl method could solve this drawback. Clin Chem Lab Med 2009;47:91-101.

A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking.

Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.

Comparison of five methods for determination of total plasma protein concentration.

Quantitation of exact total protein content is often a key step and is common to many applications in general biochemistry research and routine clinical laboratory practice. Before embarking on any type of protein analysis, particularly comparative techniques, it is important to accurately quantitate the amount of protein in the sample. In order to assess the quality of total protein estimation results, five methods were tested and were applied to the same pooled plasma sample. For this aim, Bradford (Coomassie Brilliant Blue), Lowry (Folin-Ciocalteau), Biüret, Pesce and Strande (Ponceau-S/TCA), and modified method of Schaffner-Weismann (Amido Black 10B) were used. The last two methods employ simultaneous precipitation of proteins with the acid containing dye solutions followed by dissolution of precipitate in a NaOH solution. It is shown that each assay has advantages and disadvantages relative to sensitivity, ease of performance, acceptance in literature, accuracy and reproducibility/coefficient of variation. All of the methods tested show a CV %<6. Besides pooled plasma, a known concentration of human serum albumin was also analyzed and discussed by means of standardization of plasma total protein content.

Effects of minimally invasive puncture and drainage of intracranial hematoma on the blood-brain barrier in patients with cerebral hemorrhage.

The effects of minimally invasive surgery on the blood-brain barrier (BBB) of 30 patients with cerebral hemorrhage were investigated. Difference of the BBB index and serum MBP concentration were assessed in 15 cases of conservative treatment group and 15 cases of minimally invasive surgery group. The BBB index in minimally invasive surgery group was significantly lower than in conservative treatment group (P<0.05), and the BBB index in the two treatment groups was significantly higher than in control group (P<0.01). Serum MBP concentration in minimally invasive surgery group was significantly lower than in conservative treatment group (P<0.05), and that in the two treatment groups was significantly higher than in control group (P<0.01). It was suggested the permeability of BBB in patients with cerebral hemorrhage was increased, and BBB index and serum MBP concentration in patients with cerebral hemorrhage were increased. Minimally invasive surgery can reduce the lesion of cytotoxicity to BBB and cerebral edema.

The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission.

Regulations recommend the routine application of biochemical tests, such as the Ninhydrin or Biuret tests, to confirm the efficacy of hospital sterile service department (SSD) washer-disinfector cycles in removing proteinaceous material, particularly with respect to prions. The effectiveness of these methods relies on both the effective sampling of the instruments and the sensitivity of the tests employed. Two commercially available contamination assessment tests were evaluated for their sensitivity to ME7 brain homogenate on surgical-grade stainless steel surfaces. Controls were visualized by the application of episcopic differential interference contrast/Epi-fluorecence microscopy (EDIC/EF) combined with the sensitive fluorescent reagent, SYPRO Ruby, which has been shown previously to rapidly visualize and assess low levels of contamination on medical devices. The Ninhydrin test displayed a minimum level of detection observed by 75% of volunteers (MLD(75)) of 9.25 microg [95% confidence interval (95% CI) 8.6-10.0 microg]. The Biuret test provided better sensitivity, with a MLD(75) of 6.7 microg (95% CI 5.4-8.2 microg). However, much lower concentrations of proteinaceous soiling (pg) were visualized using the EDIC/EF microscopy method. From these findings, it is clear that these approved colorimetric tests of cleaning are relatively insensitive. This investigation demonstrates how large amounts (up to 6.5 microg) of proteinaceous brain contamination could remain undetected and the instruments deemed clean using such methods. The application of more sensitive cleanliness evaluation methods should be applied to reduce the risk of iatrogenic transmission of prion disease in 'high-risk' instruments such as neurosurgical devices.

Cross-reactivity of amino acids and other compounds in the biuret reaction: interference with urinary peptide measurements.

BACKGROUND: Biuret assays for total protein measurement are considered to react with all peptides longer than 2 residues. Some studies using biuret assays of urine suggest that small peptides generally are more abundant than proteins in urine, but it is not clear whether this is a problem of assay specificity. METHODS: We analyzed the specificity and kinetics of a biuret reaction for solutions of amino acids, organic compounds, peptides, proteins, and ultrafiltered urine specimens and compared the results with standard clinical assays for protein measurement. RESULTS: The biuret assay cross-reacted with several amino acids, dipeptides, and other organic compounds able to form 5- or 6-member ring chelation complexes with copper. Reactions with amino acids and dipeptides had higher absorbance maxima (blue color) than with larger peptides and proteins (purple). Compounds forming potential 4-, 7-, 8-, or 9-member ring complexes with copper had low reactivity. Amino acid amides, dipeptides, and longer peptides had substantial reactivity, except those containing proline. Proteins and polypeptides had similar biuret reactivities per peptide bond, but reaction kinetics were slower for proteins than peptides. Urine specimens ultrafiltered through 3-kDa-cutoff membranes had substantial biuret reactivity, but absorbance maxima were consistent with cross-reactive amino acids rather than peptides. CONCLUSIONS: Many compounds, including amino acids, amino acid derivatives, and dipeptides, cross-react in biuret assays. Our studies improve understanding of the specificity of endpoint and kinetic biuret assays widely used in clinical laboratories. Amino acids, urea, and creatinine contribute to overestimation of urinary peptide content by biuret assays.

Interference of dextran in biuret-type assays of serum proteins.

Dextran interference in biuret-type assays of total serum proteins was investigated in a Belgian National External Quality Assurance Survey with 256 participants. In vitro supplementation of therapeutic (10% Gentran 70) dextran concentrations showed a broadly varying (from 0 to 20%) negative interference. The analytical interference was found to depend on both the sodium hydroxide and tartrate concentrations in the reagent formulation. The dry chemistry biuret method was not affected by the dextran interference. In a number of cases, the effects observed may be of clinical importance. Both clinicians and laboratory staff should be aware of the persistence of this analytical problem.

The level of some acute phase proteins, total protein, gamma-globulins and activity of lysozyme in blood plasma of rats supplemented with vitamin E and exposed to ozone.

In rats exposed for 28 days (5 hours a day) to ozone at a concentration of 0.5 ppm and receiving alpha-tocopherol at doses of 4.5 mg/rat and 15 mg/rat, levels of acute phase proteins (APP)--C-reactive protein (CRP), ceruloplasmin (Cp), total protein, gamma-globulins, and activity of lysozyme in blood serum were studied. The assays were performed in the presence of respective control groups, i.e. rats receiving the same doses of alpha-tocopherol but not exposed to ozone, a group of animals not supplemented with vitamin but exposed to ozone, a group of animals injected with physiological fluid and a control group not subjected to any of the treatments. The study revealed that the ozone-exposed animals had an increased lysozyme activity and a decreased total protein level. However, in rats protected by alpha-tocopherol and exposed to ozone, the concentration of APP, lysozyme activity and total protein were found to be decreased. Similar relationships also occurred in animals receiving alpha-tocopherol and not exposed to ozone.

Fabrication of microchannel structures in fluorinated ethylene propylene.

A new technique for fabrication of channel structures with diameters down to 13 microm in fluorinated ethylene propylene (also known as poly(tetrafluoroethylene-co-hexafluoropropylene), FEP) is described. The technique is based on the unique property of a dual-layer fluoropolymer tubing consisting of an outer layer of poly(tetrafluoroethylene) (PTFE) and an inner layer of FEP. When heated (>350 degrees C), the outer PTFE layer shrinks while the inner FEP layer melts, resulting in filling of all empty space inside the tubing with FEP. The channel structures are formed using tungsten wires as templates that are pulled out after completion of the shrinking and melting process. While several analytical devices have been reproducibly prepared and shown to function, this report describes a single example. A microreactor coupled to an electrochemical flow cell detects the biuret complex of the natively electroinactive peptide des-Tyr-Leu-enkephalin.

Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection.

The use of capillary electrophoresis (CE) with on-capillary Cu(II) complexation for the determination of angiotensin and its metabolites is described. The resulting copper-peptide complexes can be detected using either UV or electrochemical (EC) detection. Optimal reaction and separation conditions for the angiotensin peptides were first determined using CE with UV detection. With UV detection, the limit of detection (signal-to noise ratio S/N = 3) for native angiotensin II was 18 microM, while the limit of detection (LOD) obtained for the copper-angiotensin II complex is 2 microM. CE with EC detection was then evaluated, yielding significantly lower LODs--2 microM for native angiotensin II and 200 nM for the copper-angiotensin II complex. The addition of copper to the run buffer improved the separation and sensitivity for both CE-UV and CE-EC detection. The method was demonstrated by monitoring the conversion of angiotensin I to angiotensin II in plasma via angiotensin-converting enzyme (ACE) and subsequent inhibition of ACE by captopril.

Spectrophotometric determination of protein concentration.

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A(280)) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a(280)). Alternatively, absorbance measured at 205 nm (A(205)) is used to calculate the protein concentration. The A(280) and A(205) methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.

The colorimetric detection and quantitation of total protein.

Protein quantification is an important step for handling protein samples for isolation and characterization; it is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. Colorimetric methods are fast, simple, and not laborious. This unit describes a number of assays able to detect protein concentrations in the low microgram to milligram per milliliter ranges in a variety of formats.