IMMUNOENZYMETIC VETERINARY TEST - SYSTEM FOR
PROGESTERONE DETERMINATION
Garavshina G.R., Chistyakov P.G.
«Radiopreparat" enterprise, INP, Academy o f Sciences, Uzbekistan Republic
«Radiopreparat" enterprise specializes in manufacturing of radioisotope labeled compounds including radioimmunological test - systems for qualitative and quantitative determination of components of the blood and markers of a viruses. By the reason that the sensitivity of RIA is in 2 - 4 times less then sensitivity of immunoenzymetic test systems we have started the development this type of immunodiagnostic kits where as the detector acts the Peroxidase from horse redish instead of Iodine-125 as in RIA. One from the latest our developments is immunoenzymetic test system as the alternative variant of RIA for determination progesterone in blood of animals.
Steroids represent the class of biologically important hormones with the similar carbon skeleton. These molecules are the extremely high-powered regulators of velocities of biochemical reactions. Progesteron is most known of steroid hormones:
ch
3c = o
In women it is secreted by the cells of the corpus luteum of the ovary in luteinum phase and placenta. It effects on endometrium together with the estradiol changing the proliferative phase of the menstrual cycle to the secretory phase. Progesteron level reaches its maximum on 5 - 7 day following ovulation. Unless insemination occurs the level of progesterone decreases. Conversely, if the impregnated ovule was implanted, the corpus luteum continues to secrete
greater amounts of progesterone up to 12 weeks of pregnancy. A placenta joins then in the action and it becomes a main area of hormone production.
In blood progesterone is both free and in bound condition (with the proteins albumine and transcortine). The half-life of the hormone is several minutes; two thirds of the progesterone is inactivated in a liver and is secreted as free pregnandiol, glucuronide of pregnandiol and sulphate pregnandiol. In veterinary practice the determination of the progesterone level nporecTepoHa in blood serum or plasma by means of immunological methods of analysis is used for confirmation of the ovulation and to provide the indicator of the luteinization. The kit of reagents ensures high sensitive direct determination of progesterone in blood serum or plasma of animals.
The solution of total antibodies to the Progesterone-BSA (Bovine Serum Albumin) complex is prepared for sensibilisation of the polystirol plate wells. 0,2 ml of the solution of total antibodies to progesterone is added into the every well. The plate is covered with the film and kept for night in a household refrigerator at 4°C. After the termination of the incubation the contents of the plate is removed by shaking. Then 0,25 ml of the buffer «for supressing sorbence activity of the plate wells»containing (0,1 M of Diethylamine HCl, pH 9,5, 1 % -BSA) is added into the every well and keep 1 hour at 4°C. . After the termination of the incubation the contents of the plate is removed by shaking. The calibration tests were prepared then in the concentration range 0 - 60 ng/ml.. Marking of the plate wells and adding of the samples was carried out according to the following scheme which is indicated below (where 1
12 are the numbers of the wells, A-H - numbers of the plate rows). The scheme of the analyzed samples to be added into the wells is the next:
1 2 3 4 5 6 7 8 9 10 11 12 A 0 10 X1 X5 X9 X13 X17 X21 X25 X29 X33 X37 B 0 10 X1 X5 X9 X13 X17 X21 X25 X29 X33 X37 C 0,5 20 X2 X6 X10 X14 X18 X22 X26 X30 X34 X38 D 0,5 20 X2 X6 X10 X14 X18 X22 X26 X30 X34 X38 E 1 40 X3 X7 X11 X15 X19 X23 X27 X31 X35 X39 F 1 40 X3 X7 X11 X15 X19 X23 X27 X31 X35 X39 G 5 60 X4 X8 X12 X16 X20 X24 X28 X32 X36 X40 H 5 60 X4 X8 X12 X16 X20 X24 X28 X32 X36 X40
The period of ime of the analysed samples adding should not exceed 15 minutes. Into the wells 1 is added 0,1 ml of calibration tests containing 0 ng/ml progesterone (rows A and B), 0,5 ng/ml progesterone (rows C and D), 5 ng/ml progesterone (rows E and F), 10 ng/ml progesterone (rows G and H), into the wells 2 - 20 ng/ml progesterone (rows A and B), 40
ng/ml progesterone (rows C and D), 60 ng/ml progesterone (rows E and F). Into the rest of wells are added in the duplicates by 0,1 ml of the analyzed blood serum or plasma of animals
(X).
Then the solution of the conjugate is prepared. 0,1 ml of the conjugate solution is added to all wells of a plate. The plate is covered by the tape and incubated during one and half hour mines at the temperature 37°C. The substrate mixture is prepared ten minutes before the termination of incubation. After the termination of incubation the wells of the plate are washed out by 0,2 M Phosphate buffer containing 0,05 % Twin - 20. 0,1 ml of substrate mixture is added to all wells of the plate. The plate is covered by the tape and incubated at room temperature during 10-30 minutes in a dark place. The stop - reagent (7% Sulfuric acid) is added into the wells after the termination of the incubation. The optical density of the wells is detected be the photometer of vertical scanning at the wavelength 492 nm. The typical calibrating curve for quantitative determination of the progesteron is shown at the Figure 1.
O.D
Figure 1. The typical calibrating curve for quantitative determination of the progesteron.
The indicated data are testifying about the high sensitivity of the developed immunodiagnostic test-system. The offered method ensures high specificity of the analysis.The sensitivity of the offered test-system is about 0,1 ng/ml at range of determination from 0 to 60 ng/ml. For determination it is necessary 0,1 ml of blood plasma or serum of animal for the double determination. Duration of the analysis is 2 hours.
