Standards for total serum protein assays--a collaborative study.
(1/13)
We have studied the standardization of total serum protein assay with the biuret reaction. Standard solutions were prepared from lyophilized preparations of human serum albumin and bovine serum albumin, with corrections made for volatile material and ash contents. These solutions and a solution of crystalline albumin standard were analyzed with a new stable biuret reagent, to establish absorptivity values (values for the absorbance of a 1 g/liter final reaction mixture). The mean values obtained were 0.302, 0.292, and 0.290 for human serum albumin, bovine serum albumin, and the crystalline albumin, respectively. We believe that the established absorptivity value will improve the accuracy of serum protein determinations. We studied the linearity of the relation between color produced and protein concentration, with use of the solutions described above and a serum pool. The color adheres to Beer's law up to the highest concentration tested: 3 g/liter for HSA and BSA, and 2.8 g/liter for serum in the final reaction mixture. The new biuret reagent has been stable for one year at room temperature. We recommend the use of bovine serum albumin as a primary standard for serum protein assays. It is inexpensive, easily available, and exhibits the best linearity in the biuret reaction. (+info)
Fabrication of microchannel structures in fluorinated ethylene propylene.
(2/13)
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. (+info)
Cross-reactivity of amino acids and other compounds in the biuret reaction: interference with urinary peptide measurements.
(3/13)
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. (+info)
A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking.
(4/13)
The Coomassie Brilliant Blue method underestimates drug-induced tubular proteinuria.
(5/13)
The sensitivity of the Coomassie Brilliant Blue (CBB) procedure differs for different proteins. To investigate the impact of this variability on clinical measurements of proteinuria, we collected urine specimens from patients with glomerular disease or drug-induced tubular nephrotoxicity and compared urinary protein concentrations as determined by the CBB method with those obtained by the biuret method. The CBB method underestimated the protein concentration predicted by the biuret method by as much as eightfold in specimens with low proportions of albumin. Determinations of protein by the CBB method in serially diluted urine specimens from patients with tubular damage also deviated from linearity by as much as twofold for protein concentrations less than 1 g/L. We conclude that CBB binding is relatively insensitive for measuring proteins specifically associated with tubular damage and hence may be inappropriate for clinical use. (+info)
Molar absorptivity and the blank correction factor.
(6/13)
In photometry, where both the product formed and one or several reactants absorb light at the same wavelength, the absorbance of the "blank" of the sample at the end of the reaction may be less than that measured at the beginning of the reaction, because of consumption of reactant(s). The blank correction factor for the determined result with one light-absorbing reagent is epsilon P / (epsilon P - epsilon R), where epsilon R and epsilon P are the molar absorptivities of the reagent and the product, respectively. We derived a factor for the case when more than one reagent absorbs light at the same wavelength as the measured product. This factor is independent of the concentration of reagent(s) and can correct the determined result or absorbance for the consumption of light-absorbing reagent(s) during the reaction. (+info)
A candidate reference method for determination of total protein in serum. I. Development and validation.
(7/13)
We developed a candidate Reference Method for measuring total serum protein by use of the biuret reaction. The method involves a previously described biuret reagent (Clin. Chem. 21: 1159, 1975) and Standard Reference Material (SRM) 927 bovine albumin (National Bureau of Standards) as the standard. At 25 degrees C, color development for 30 or 60 min provides identical serum protein values. Glucose (up to 10 g/L) and bilirubin (up to 300 mg/L) do not interfere. Hemoglobin, at 3 g/L, increases apparent serum protein by 0.4 g/L. The presence of dextran in serum causes easily detected turbidity, but this interference can be eliminated by centrifuging the reaction mixture. Therapeutic concentrations of ampicillin, carbenicillin, penicillin, oxacillin, nafcillin, chloramphenicol, cephalothin, and methicillin in blood do not interfere, nor do triglycerides up to 10 g/L. Within-run and day-to-day standard deviations of the method are 0.1 and 0.4 g/L, respectively. (+info)
Prevention of interference by dextran with biuret-type assay of serum proteins.
(8/13)
In assay of serum proteins by use of the biuret reaction, dextran can cause turbidity by formation of an insoluble complex of dextran with copper and tartrate (or EDTA) in strongly alkaline solution. Whether or not the turbidity occurs depends on the tartrate concentration: turbidity is maximal at about 10 g/L, absent at 20 g/L or more, and only slight and delayed at 4 g/L. Two biuret reagents, containing respectively 5.6 and 22.5 g of tartrate per liter, obviate the interference, but the former is suitable only when a short (5 min) incubation is used. Both reagents show linear calibration curves and yield virtually identical results. (+info)