CS276912B6 - Stabilized reagent for determining proteins in biological fluids - Google Patents

Abstract

The reagent contains a dye composition 3- [N-ethyl-N- / 3-methyl-4-4- / 4-fluoroaniline / [phenyl] -2-methyl-4 (N-ethyl-N- [3- (soda sulfonate / phenyl)] methyl] aminophenyl}> / methylene-2,5 cyclohexadiene-1-ylidene /] ammonobenzene sulfonate. Contains 1 liter of aqueous solution 250 to 300 mg of dye, 0.85 to 0.95 mole hydrochloric acid, 0.15-0.20 mol sodium chloride and 0.15 to 0.20 mol polyhydric an alcohol such as monoethylene glycol or glycerin.

Description

The invention relates to a stabilized reagent for determining the concentration of proteins in biological fluids

Laboratory practice in protein purification and isolation as a diagnostic need of clinical practice often requires rapid and accurate determination of protein concentration in aqueous solutions with relatively high sensitivity and, if possible, using a minimum volume of sample to be analyzed. These direct criteria have been met in recent years by a method based on the formation of a colored complex of a protein with a suitable dye. Such a dye is e.g. 3 - [[N-ethyl-N- [3-methyl-4- [4- (4-ethoxyanilino) phenyl] - <2-methyl-4- {N-ethyl-N- [3- (sodium sulfonato) phenyl] methyl]} aminobenzene sulfonate Coomasie * brilliant blue G-250 / Color Index no. 42655 /, in which A.H. Reisner et al. (Reisner A.H., Nemes P. and Bucholz G .: were the first to observe A. Anal. Biochoem. 64/1975/509 / that it exists in two color forms. Oxide leucoform in an acidic environment red, which changes to blue by adsorption of the protein in the same environment. This knowledge was used by M.M. Bradford and developed a method for the determination of proteins in aqueous solutions of phosphoric acid with the addition of ethanol. / Bradford M.M .: Anal. Biochem. 72, / 1976/248 /. However, it was found / Spector Z .: Anal. Biochem. 86/1978 142 / that the dye reaction solution is unstable during storage and therefore needs to be corrected at two-week intervals, which is not welcome for routine operation. It has also been found that the dependence of absorbance on protein concentration is not linear with the method used [Sedmak H.J. and Grossbery S.E .: Anal. Biochem. 79/1977/544 /, which is an unwelcome moment in the analytical method.

These shortcomings are addressed by the stabilized regency of the present invention, which has substantially higher storage stability, and the dependence of the adsrobance on the measured protein concentration is strictly linear in the range of up to 2.0 g. ¹ .

SUMMARY OF THE INVENTION The reagent for determining the concentration of proteins in biological fluids contains 0.000285 to 0.000342 moles of 3 - [[N-ethyl-N- [3-methyl-4 - / - 4- [4-ethoxyanilino] dye. [phenyl] - <2-methyl-4- {N-ethyl-N- [3- (sodium sulfonato) phenyl methyl]} aminophenyl> methylene-2,5-cyclohexydien-1-ylidene]] imoniobenzene sulfonate, 0.85 to 0.95 moles of hydrochloric acid, 0.15 to 0.2 moles of sodium chloride and 0.15 to 0.2 moles of ethylene glycol or glycerol per liter of aqueous solution.

It has been found that the most suitable acid in which said dye dissolves most rapidly and forms an oxidized red leucoform is hydrochloric acid. It has also been found that the highest sensitivity of blue staining by the addition of protein is achieved in a very small range of hydrochloric acid concentrations at the optimum dye concentration. With increasing concentration of acid at a constant concentration of dye, the sensitivity of the color reaction to proteins decreases and the maximum sensitivity is reached in the acid concentration range of 1.175 to 0.195 mol.l ^-1 , which is lower than in previously published works. In the indicated concentration range, according to the assay procedure, the presence of 10 mg of protein causes a change in absorbance of up to 0.65 to 0.75.

The addition of polyhydric alcohols and sodium chloride to the reaction solution accelerates the equilibrium of the formation of the color complex of the dye with the protein. The equilibrium is stabilized within 5 minutes according to the proposed procedure. In addition, the alcohols and sodium chloride present increase the stability of the reaction stock solution, so that it is not necessary to check the sensitivity of the solution as often. It was found that the stock solution according to the invention, even after 3 months of storage at 15 to 30 ° C, had the same sensitivity as the freshly prepared solution. It has also been found that a method of dissolving the dye is important in the preparation of the reaction solution. The rate of dissolution depends on the rate of dissolution and equilibrium of the leucoform of the dye.

Example 1

250 mg of dye composition 3 - [[N-ethyl-N- [3-methyl-4-4- (4-ethoxyanilino) phenyl] -2'-methyl-4- {N-ethyl-N- [3- / sodium sulfonato (phenylmethyl)} aminophenyl> (methylene-2,5-cyclohexadiene-1-ylidene)] ammoniobenzene sulfonate Coomasie briliant blue G-250 was dissolved in a solution consisting of 80 ml of concentrated hydrochloric acid (36-37%), 160 ml of distilled water, 10 g of sodium chloride and 10 ml of ethylene glycol. After dissolving the dye, the reaction solution was made up to a total volume of 1 liter with distilled water and filtered through filter paper to remove insoluble dye residues, respectively. insoluble impurities. The solution thus prepared, which is a stock solution which is diluted with distilled water in a ratio of 1 volume of stock solution and 4 parts of distilled water before use for the determination of proteins. After mixing, the solution is working, it can be used immediately to measure the concentration of proteins in the solutions. The absorbance of the working solution against water at a wavelength of 595 nm was 0.370. When measuring the protein concentration in control samples e.g. of human blood protein at a declared concentration of 66.7 g (1/100 nl of serum) is diluted with 3.2 ml of distilled water. Then, 2x1 ml of the protein determination working solution is metered into the dry colorimetric cuvette with a micropipette, whereby the reaction solution is sufficiently mixed at the same time.

The reaction solution immediately turns blue and the absorbance is measured at 2 to 5 minutes when the absorbance is maximum. In this case, the absorbance relative to water was 1.115, i. after subtracting background 0.745. The calibration curve of the absorbance as a function of the protein concentration is linear over the entire range from 0.2 to 2.0 g / protein.

Similar results were obtained using bovine albumin serum, the measured values were within the reproducibility of the method.

Example 2

The procedure is as in Example 1, except that 300 mg of dye are dissolved in a solution consisting of 85 ml of concentrated hydrochloric acid and 170 ml of distilled water, 15 g of sodium chloride and 15 ml of glycerol. The finished stock solution after dilution to working solution had an absorbance of 0.420. Control sera gave comparable absorbances as in Example 1 at the same protein concentrations.

The invention has application wherever protein solutions are used, both in research and industrial application in various branches of biotechnology and in clinical diagnostics.

Claims (1)

3 CS 276912 B6 PATENT REQUIREMENTS Stabilized regimen for protein concentration determination in biological fluids containing 3 - [[N-ethyl-N- (3-methyl-4-4- / 4-ethoxyanilino / phenyl] -2-methyl- 4 - {- ethyl-N- [3- (soda sulphonato / phenylmethyl)} aminophenyl} methylene-2,5-cyclohexadiene-1-ylidene]] - ammonium benzene sulphonate, characterized in that it contains from 0.000285 to 1.000 mg / l in 1 liter solution. 0.000342 mol of dye, 0.85 to 0.95 mol of hydrochloric acid 0.15 to 0.20 mol of hydrochloride and 0.15 to 0.20 mol of polyhydric alcohol, for example monoethylene glycol or glycerin. End of document