SU1529124A1 - Method of diagnosis of mastitis - Google Patents

Abstract

The invention relates to medicine, in particular to surgery, and can be used to diagnose mastitis. The aim of the invention is to increase accuracy and early diagnosis. For this, a sample of breast milk surveyed in a volume of 0.20-0.04 ml with a drop of a 0.01% aqueous solution of methylene green is applied to the upper surface of a column of 15% polyacrylamide gel and subjected to electrophoretic separation in 0.9 n . Acetic acid for 3 hours. After staining the gel with the amideciferous dye 10B, the spectrum of acid-soluble milk proteins was visually evaluated and the path length of individual protein francs was measured. When a cathode strip with R _F = 0.47-0.49 appears in relation to methyl green, an early stage of mastitis is diagnosed with respect to methyl green. The method is relatively uncomplicated, allowing reliably distinguishing between mastitis and lactostasis at the preclinical stage. The accuracy of the method is 97%.

Description

The invention relates to medicine, in particular to surgery, and can be used to diagnose mastitis.

The aim of the invention is to increase the accuracy of the method and early diagnosis.

The method is carried out as follows.

A sample of milk in a volume of 0.02-0.04 ml with a drop of a 0.01% aqueous solution of methyl green is applied on the upper surface of a pre-prepared column of polyacrylamide gel and for 3 hours at a current of 2.0 mA electrophoretic separation of proteins is performed on the tube

in an acidic solution until the methyl green reaches the finish zone of the gel. After removing the gel from the tubes, they are stained with a solution of amid black dye 10 V for 30-40 minutes. and then washed unbound dye, visually assess the spectrum of acid-soluble proteins and measure the length of the run of individual protein fractions. When a cathode band with E 0.47–0.49 appears on electrophores in relation to methyl 1-lenoma, the early stage of mastitis is diagnosed.

Example 1. Sample milk of a healthy puerperal in a volume of 0.02 ml

„Ii.

together with a drop of 0.01% aqueous dye of methyl green is applied on the upper surface of a previously prepared column of polyacrylamide gel, which was placed in a 0.5 x 10 cm glass tube. The preparation of polyacrylamide gel and the electrophoresis procedure are carried out according to the recipe of Pani m and Chalkly. Polyacryl amide gels are prepared by mixing 3 solutions: solution A: 60% acrylamide (W / V); 0.4% methylene bisacrylamide (W / V) in H-jO; solution B: 43.2% acetic acid (V / V); 4% tetramethylethylenediamine (V / V) in HjO; solution C: IOM solution of urea.

All solutions can be prepared for storage and stored in the cold (+ 4 ° C) in a temny glass dish with a ground-in lid for 2-4 weeks. Immediately before the analysis, the solutions are drained in the following ratio A: B. 2: 1: 5. Next, a polymerization catalyst — ammonium persulphate (40 mg per 32 ml of the mixture; 50 mg per 40 ml of the mixture) is added to the mixture. The catalyst mix is thoroughly mixed and poured with a pipette or syringe into glass tubes to a height of 9 cm, and then distilled water is layered onto the mixture. Within 60-90 minutes, the gel is polymerized in the tubes. After completion of the polymerization, established to form a clear boundary between the gel and water and the appearance of the syneresis of the gel, wait 1 hour and transfer the tubes with the gel to an apparatus for electrophoretic separation (you can use a domestic apparatus of the PEF-1 brand or a Hungarian apparatus of the Reanal company). 69). In the upper (anodic) and lower (cathodic) chambers pour 0.5 l of 0.9 n. a solution of acetic acid in water (buffer), with a current of 2-3 mA per tube, a pre-electrophoresis procedure is carried out, during which components that interfere with the analytical electrophoresis are removed from the gel. After finishing the NIN electrophoresis, the buffer in the upper and lower chambers is replaced with fresh. Trans electrophoresis is carried out for 5-16 hours to constant values of current and voltage. Polyacrylamide gel gels can be prepared 12 hours before analytical electrophoresis. Samples of the analyzed milk with a drop of 0.01% aqueous solution

methyl green is applied to the top surface of the gel. They are quite dense, so fast diffusion of proteins into the acidic solution for electrophoresis is not observed. The electrophoresis is carried out at a constant current of 2.0 mA per tube for 3 hours. At the end of the electrophoresis, fixed upon reaching the indicator dye methyl green - the lower finishing part of the gel column. The gels are removed from the tubes in the traditional way and stained for 30 min with a 0.1% solution of amide-black 10 V dissolved in 20% ethanol, 7% acetic acid, the rest. After staining, the gels are washed away from the unsupported dye, either electrophoretically or by changing the solution of 0.9 n acetic acid. The spectrum of acid-soluble milk proteins can be divided into two zones. In the first upper zone, 5 basic protein fractions are represented. The second zone in the middle part of the gel is represented by a single cathode strip with C 0.37-0.38. Rr is defined as the ratio of the mileage of a given protein band to the mileage of methyl green, i.e. to the total length of the gel. The range of Rr values is due to the fact that the protein strip after staining has a certain thickness, and therefore the determination of its run length in the gel can be carried out both along the front and

on the falling edge of the strip.

I. Example 2. Milk sample in

a volume of 0.02 ml taken from the puerperal patient with lactostasis clinically diagnosed undergoes electrophoretic analysis in the same way as in example 1. In the spectrum of the acid-soluble proteins of the milk of this patient, an additional cathode band with Rf 0.47-0 was detected, 49. Consequently, the patient has an initial stage of inflammation in the mammary gland in the absence of its clinical manifestation. The diagnosis was confirmed by clinical observations over the next seven days.

Example 3. A sample of milk in a volume of 0.02 ml taken from a puerperal who has been clinically diagnosed with lactostasis is subjected to electrophoretic analysis in the same way as in example 1. In the spectrum of the acid-soluble milk proteins of this patient

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compared with the norm is not detected. The diagnosis is lactostasis (milk stagnation), which is confirmed by subsequent clinical observations.

Using the proposed method, it is possible to unambiguously diagnose the initial stage of developing mastitis in cases where known biochemical and physical methods do not allow this, as well as to differentiate inflammation from lactostasis. The accuracy of the method is 97%, which is 16% higher than the prototype method.

Claims (1)

Invention Formula A method for diagnosing mastitis by means of a biochemical study of milk, characterized in that, in order to increase the accuracy of the method and early diagnosis, an electrophoretic separation of milk proteins is made of 0.9 n. Acetic acid in a 15X polyacrylamide gel and when an electrophoregram appears in the cathode strip with Ilr 0.47-0.49 compared to methyl green, mastitis is diagnosed.