The method of obtaining the latex diagnosticum


(57) Abstract:

The invention relates to medicine, and more specifically to Microbiology. The aim is to increase the information content of diagnosticum and sorption capacity of the latex. This objective is achieved in that get polygroove serum by simultaneous immunization of rabbits with a mixture of antigens of the causative agents of plague, glanders, melioidosis, tularemia and brucellosis in the ratio 0,1 : 1 : 1 : 6 : 4, as sensitin used immunoglobulins from polygrapholog serum. The latex sensitization was carried out at the ratio of 10 mg of carrier 0.1 mg globulins, at a PH of 7.0 to 7.4, for 4 h at room temperature, then for 12 h at 4C with constant uniform stirring. The method provides an increase in globulin load per unit of media and allows you to cut at least 2 times the time of analysis, 4 times to reduce the cost of drugs is relatively prototype using the received diagnosticum. table 1.

The invention relates to medicine, and more specifically to Microbiology, and for laboratory diagnosis of especially dangerous infections.

There are various ways to obtain diagnostics using artificial HV effect. cholerae serogroup 01 with sensitivity 5106-1107CFU/ml complicated and requires expensive equipment and chemicals.

Known also diagnosticum polymer microspheres with a diameter of 4-5 microns as carriers, covalently bound to immunoglobulins from plague commercial agglutinating sera. The activity of diagnosticum with typical strains of the plague pathogen grown at 37aboutWith a made 3 to 105MT /ml, but the sensitivity with respect to the microorganism cultivated in the 28aboutWith much lower - 5 107MT /ml.

The closest analogue (prototype) is a method of obtaining or antibody-based test diagnosticum, in which as the carrier used suspension polyacrolein latex with a diameter of spheres of 1.8 μm, activated casein and tannin. Latex particles were senzibilizirani immunoglobulins plague serum in a ratio of 0.15 mg immunoglobulin 50 mg media exposure 3 h at room temperature and constant stirring. Then sensitized suspension was besieged and twice washed with 0.15 M NaCl. The washed suspension sensitised polyacrolein media di. liofilizirovannoe the contents of capsules suspended in 4 ml of 0.05 M FSB pH 7.2 and used for setting the reaction of agglutination of latex.

Was marked by a high specificity and sensitivity obtained diagnosticum. However, if you need sharp increase in the volume of discovering the unknown pathogen dangerous infections of bacterial origin, for example in terms of epidural, commercial immunoglobulin nanodiagnostics not suitable. Each received sample should be investigated at least four different drugs, which is very cumbersome and unacceptable, especially in the field.

Offer polygrapholog latex diagnosticum four times reduces the number of analyses and saves backpropagate.

The aim of the invention is a method for polygrapholog immunoglobulin diagnosticum to five species of bacterial pathogens of especially dangerous infections that can increase the number of adsorbed immunoglobulins on the unit carrier and to reduce the time of analysis using the obtained diagnosticum.

This objective is achieved in that get polygroove savoryi and brucellosis in the ratio 0,1: 1: 1: 6: 4. Immunoglobulins from sera allocate Caprylic acid by well-known methods. Polyacrolein particles with microspheres of 1.8 μm sensibiliser immunoglobulins polygrapholog serum according to the following procedure. Poured in the tube is equal to the volume of 1.25% latex suspension and the suspension of immunoglobulins to protein concentration of 50-100 μg/ml and stirred on a magnetic stirrer at a temperature of 20-23aboutC for 4 h Then the process continues at a temperature of 4-5aboutWith 12 hours the Mixture is washed 0.15 M solution of the FSB and the precipitate resuspended in 1 ml of medium drying, consisting of a mixture of 2% solution of bovine serum albumin and 5% sucrose solution. Diagnosticum poured into ampoules of 0.5 ml and freeze-dried. Before setting reaction of agglutination of the latex content of the ampoule is dissolved in 5 ml of 0.05 M FSB pH of 7.2.

Thus, the application of the proposed combination of techniques (getting polygrapholog serum, sensitization of latex polypropolyne immunoglobulins) allowed to give way to a new quality to get polygrapholog diagnosticum, which allows you to detect any of the five bacterial pathogens of especially dangerous infections in contaminated samples, which significantly tx2">

P R I m e R 1. Prepare buffered 0.15 M NaCl pH 7.2 and use it as a planting liquid. 1 ml of 1.25% suspension polyacrolein latex poured to 1 ml suspension poligraphic immunoglobulins with a protein concentration of 100 μg/ml and stirred on a magnetic stirrer at 20aboutC for 4 h and Then stirring is continued at 4aboutFrom 12 o'clock Sensitized latex precipitated by centrifugation at about 3 thousand. /min, washed 0.05 M solution of the FSB and the precipitate resuspended in 10 ml of the same solution. Received diagnosticum put the reaction of agglutination of latex. Controls: 1) diagnosticum with distributing fluid; 2) diagnosticum with suspension heterologous microorganisms. See a positive reaction (umbrellas) in the wells containing the plague, glanders, melioidosis, tularemia brucellosis in concentration 6105MT /ml In controls-compact discs on the bottom of the hole.

P R I m m e R 2. Sensitization as in example 1, but as the distribution of the liquid used buffered 0.15 M NaCl pH of 7.0. When setting the reaction of agglutination of latex see "buttons" on the bottom of the hole in the controls, i.e., the result is doubtful.

P R I m e R 3. Sensitization of latex as worthwhile in the background, i.e. the result is doubtful.

P R I m e R 4. Serves 4 diagnosticum with protein concentrations of 50 and 100 µg/ml (1 pair) and 50 and 100 µg/ml (2 pair), as in example 1, with the first 2 sensibiliser only 4 h, and the other two additional 12 h at 4aboutC.

Received diagnosticums put agglutination reaction latex, using suspensions of killed microorganisms.

The results RAHL shown in the table.

Thus, as can be seen from the table, increasing the load globulin per unit of media leads to spontaneous agglutination in the control, hindering polygrapholog of diagnosticum known method, and the proposed method allows to achieve a specified target.

P R I m e R 5. For the study enrolled 20 field samples suspected to be infected with bacterial pathogens of especially dangerous infections. The analysis was carried out using a commercial nanodiagnostics plague, Sanogo, melioidosis, tularemia, brucellosis. Just put 80 reactions. Simultaneously examined the samples by diagnosticum Polygraphy latex. Conducted 20 tests. The results for all the samples obtained in the first case, after 6 h (3 min n 24 ml (6 ml x 4) nanodiagnostics and 6 ml polygrapholog of diagnosticum, respectively, in the 1-m and 2-m cases.

As you can see by the results, the time saved, diagnostic preparations, tablets, reduced labor costs for the study samples. Moreover, the effect of the application polygrapholog latex diagnosticum will increase with increasing amounts received for analysis of samples.

Thus, the proposed method managed to get polygrapholog diagnosticum, allowing to reduce in 2 times the time of analysis, 4 times to reduce the amount of consumption of drugs relative to the prototype and to increase globulin load per unit of media.

(56) USSR Author's certificate N 1596926, CL G 01 N 33/53, 1989.

The METHOD of OBTAINING the LATEX DIAGNOSTICUM by sensitization polyacrolein latex immunoglobulins plague serum, characterized in that, to increase the information content of diagnosticum and enhance the sorption capacity of the latex particles, sensitization is carried immunoglobulin antisera containing additional immunoglobulins to Sapa, melioidosis, tularemia and brucellosis and obtained by immunization of rabbits with the corresponding antibodies in the ratio 0,1 : 1 : 1 : 6 : 4, moreover, sensitization take 0.1 mg globulin 10 mg of latex particles, ingibuvannya.


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The invention relates to biotechnology and can be used to obtain stable forms of immunobiological preparations

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EFFECT: immune serum with increased specific activity.

2 tbl, 2 ex

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1 tbl, 3 ex

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6 tbl, 4 ex

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7 ex, 7 tbl

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2 tbl, 2 ex

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2 tbl, 2 ex, 4 cl

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2 tbl