DE1253934B - Process for the production of a diagnostic agent for the determination of protein in biological fluids - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. CL:

GOIn

German KI .: 421-3 / 55

Number: 1 253 934

File number: B 70383IX b / 421

Filing date: January 18, 1963

Opened on: November 9, 1967

The qualitative and quantitative detection of protein in biological fluids, e.g. B. in the urine, is of great diagnostic importance, especially with kidney diseases, but also with poor circulation, Atherosclerosis, hypertension, diabetes. There are a number of methods that can be used to detect Protein became known, ζ. B. the sulfosalicylic acid sample, acetic acid cooking sample, nitric acid ring sample, Picric acid sample; all of these methods are based on precipitation reactions and do not require laboratory equipment not feasible. These reactions are therefore not useful for quick diagnostic purposes Consideration; this requires a simple color reaction, which can also be done by untrained staff can be performed routinely, e.g. B. with the help of test papers. ·

A color reaction suitable for the detection of protein was first used by F eig 1 et al. (Mikrochim. Acta, 2, 1937, p. 107). This reaction takes advantage of the so-called "protein error" known with various indicators: If the pH value of the reagent is kept below the indicator change range, the addition of protein solution causes a color change that is independent of the pH value and is caused by the protein content; the color intensity of the resulting color depends on the concentration of protein in the solution to be tested. F Eig 1 worked out this proof of protein as a spot reaction, using the potassium salt of tetrabromophenolphthalein ethyl ester and acetic acid as reagents. To produce protein test papers based on the Feigl reaction, the acetic acid must of course be replaced by a non-volatile acid; According to the method of British patents 814 223, 826 066 and 840 362, an acidic buffer substance, such as, for. B. Citrate Buffer. However, the protein test strips obtained in this manner does not completely satisfy, because the color contrasts of the negative and positive protein sample in concentration ranges differ very little below 0.1 ° / ₀ protein; the positive: the test is more or less yellow-green, the negative test is green-yellow and is darker than the dry test strip. The sensitivity of these test papers also leaves something to be desired (detection limit about 0.03 to 0.01% protein). However, it is necessary for the doctor to be able to detect protein in the urine in the same dilution as is possible with the acetic acid boiling test; accordingly, 0.005 per cent. protein in the urine must still be able to be correctly diagnosed.

In the case of the above-mentioned British

Method of making a
Diagnostic agent for the determination of protein in biological fluids

Applicant:

C. F. Boehringer & Soehne G. m. B. H.,

Mannheim-Waldhof, Sandhoferstr. 112-132

Named as inventor:

Dr. rer. nat. Hans-Georg Rey,

Dr. rer. nat. Peter Rieckmann,

Mannheim-Waldhof

2

Patent specifications, the absorbent carrier is impregnated with a solution which contains the buffer substance together with the dye indicator. It has now surprisingly been found that protein test papers which react much more sensitively are obtained if the buffer substance and the dye / indicator are applied to the carrier separately, ie one after the other. The test papers obtained in this way are colored pale yellow and give strong blue colorations with protein-containing urine, which are graded according to the protein concentration and clearly stand out from the yellow coloration of the negative test. It can be detected properly ° / ₀ protein to 0.005; even lower protein concentrations can still be determined. With normal urine as well as with alkaline urine, the color of the paper does not change.

The method according to the invention for producing a diagnostic agent for determining protein in biological fluids, consisting of a dye indicator, which has the so-called "protein error", an acidic buffer substance and a absorbent carrier, is accordingly characterized in that the absorbent carrier is first with impregnated with the buffer solution and then treated with the dye solution.

709 687/178

Claims (5)

3 4 Examples of such are used as dye indicators costume that has a major protein defect in an Indian "" ... Cator change in the acidic pH range, in particular Composition of the buffer solution: special tetrabromophenolphthalein ethyl ester and tert. Sodium citrate 9.8 g tylester and tetrabromobenzaurine. Of great weight 5 citric acid 3.5 g The choice is the selection of a suitable solution. Sodium Lauryl Sulphate 0.05 g means for the indicator. Of course, slightly distilled water must ad 100 ml Volatile solvent can be used so that the test strips dry quickly after impregnation. Composition of the indicator solution:
If the indicator is now in methanol or acetone io tetrabromophenolphthalein ethyl ester ... 40 mg dissolved and with this solution an already with buffer methylene chloride ad 100 ml substance-treated paper is impregnated a test paper that is used with a green base color of filter paper (Schleicher & Schüll No. 2316) is interspersed with several bruises. It was first treated with the buffer solution given above now found that one is uniformly pale yellow and then air-dried. Afterward Colored protein test papers are obtained when the paper is impregnated with the indicator solution Solvent for the dye indicator halo and dries again. The dried paper is gene hydrocarbon used. For this come rolled, and the rolls are transverse to where you want them z. B. chloroform and carbon tetrachloride in question; Cut width of 5 mm. The in this way Methylene chloride is particularly suitable. The 20 test strips held are slightly yellow in color As a buffer, every buffer system can be used and does not change its color if it can be used with protein, that are moistened to a pH value below 7.0 a free normal urine. Protein im can be asked; citrate buffer is preferred; urine is shown by blue discoloration, depending on the d. H. an aqueous solution of citric acid and protein content are graded. So can protein in the urine tert. Sodium citrate, is used. The optimal pH value 25 is still acceptable at a concentration of 0.005% at about 5.0. This finding can be proven to be very excessively free. surprisingly, because this pH value is too. The protein test strips described above are already on the "alkaline side" of the aforementioned Color indicators. In the above cited before the process of patent application B 66255 VIb / In the literature, it is expressly emphasized that the 30 55 f is between a white colored and a transparent optimal pH values for the polyterephthalic acid coated on the so-called rente polyethylene "Protein defects" sealed in by ester foil based on dye indicators. Proof of protein a little below the pH envelope - Here you lead the test paper strips from point of the dye, that is, in its acidic range, 5 mm width between the two above-mentioned rich «, lie (cf. for example British patent 35 plastic films (width approx. 30 cm) divided by two 840 362, pH 3.5 to 4). Even if the finished rollers, one or both of which are heated. Reagent paper according to the present invention in a thermal contact are the art solution is immersed without egg white, the fabric-foils are welded or the paper strips Indicator not alkaline from its acidic color range in one or both sides with the plastic around. An explanation for this pH-4 ° welded. Shallow grooves on the rollers which by the simple measure of afford the safe welding of the foils in addition separate application of buffer substance and coloring of the paper strips. The one with the paper strips Substance solution according to the present invention causes welded plastic strips to be transverse has not yet been found. cut to the test paper strip. Man The production of the diagnostics according to the invention by this method keeps narrow foil sealants takes place in the usual manner: the suction strip 60 mm long and 5 mm wide, the capable carriers, e.g. B. filter paper, is first with the at one end a reagent paper section of Impregnated buffer solution and then contained in the approximately 5 x 5 mm size. The paper is Air dried. The test solution is expediently added to the buffer, covered on both sides with foil one of the usual surfactant 50 solution will dissolve through the open cut edges, z. B. Sodium lauryl sulfate or polyoxyethylene sucked. sorbitan monolaurate; It has proven to be useful when using the of the test paper accelerated absorption of the test strips with a second, non-impregnated one Test liquid and thus a faster and equal paper strip (filter paper Schleicher & Schüll No. 1450) A moderate coloration of the reagent paper is achieved. Then 55 sealed together; the sealing is in this the filter paper treated with the indicator solution is carried out in such a way that the to be combined delt, dried again and cut to size. The strips on top of each other are the test strips Protein test strips obtained in this way can be considered through the transparent film and the non-impregnated one these are used or the paper strips are removed from the white-colored film using the process. Patent application B 66255 VIb / 55 f is covered with plastic 60.
Foils are sealed. In the latter case it has proved to be expedient if one of the test claims:
sealing paper with a second, non-impregnated paper strip; This ensures a first method for producing a more uniform coloring of the test strip for diagnosis with a diameter of 65 for the determination of protein in biological control of the detection reaction. Liquids, consisting of a dye The following example is the inventive indicator which explains the so-called “protein-compliant process” in more detail. error «, an acidic buffer substance and an absorbent carrier, characterized in that that the absorbent carrier is first impregnated with the buffer solution and then treated it with the dye solution. 2. The method according to claim 1, characterized in that the solvent for the Dye indicator a halogenated hydrocarbon is used. 3. The method according to claim 1 or 2, characterized in that buffer solution and dye solution be applied to filter paper and this is then dried. 4. The method according to claims 1 and 3, characterized in that with the buffer solution and the dye solution impregnated filter paper strips are dried and wrapped in plastic films be sealed. 5. The method according to claims 1, 3 and 4, characterized in that the test paper with a second, non-impregnated paper strip is sealed together.