SU1490650A1 - Method for quantitative analysis of steroid hormones - Google Patents

Abstract

The invention relates to biochemistry and may find application in medicine, agriculture, experimental biology. In order to increase the accuracy of the analysis, the test sample is treated with a specific receptor protein (CRP) for steroid hormones (SG) in the presence of steroid hormones labeled with a luminescent substance (SGL). Then, using hydroxylapatite, SG and SGL are separated from SRB. The amount of SG in the test sample is judged by the luminescence intensity in the phase containing SG and SGL associated with CRP. For the implementation of the method, CRP to SG isolated from the decidual tissue is used. 1 tab.

Description

The invention relates to biochemistry, in particular to methods for determining steroid hormones, and may find application in medicine, agriculture and experimental biology.

The aim of the invention is to improve the accuracy of the method.

This goal is achieved by using a specific receptor protein for steroid hormones, derived from decidual tissue, as a hormone-binding substance, and hydroxyapatite is used to separate bound and unrelated hormones.

This protein provides a link between the hormone and a high binding constant (dissociation constant cd), i.e. more specific binding than the binding of a hormone to antibodies (K 10 M) of this hormone.

The method is carried out as follows.

Example 1. Obtaining specific receptor protein for progesterone.

Specific progesterone receptor protein was obtained from human decidual tissue as follows.

To 1 g of tissue washed with blood saline (at 4 ° C) and homogenized in a flow homogenizer

 co o o: ate

APG-1, 5 ml of ice-cold buffer pH 7.4, containing 10 mM TRIS-EC1, 1.5 mM EDTA and 0.02% Na azide were added. The resulting mass was besieged at USOOD for 1 h, the pellet was discarded, and a supernatant containing 4.5 mg of protein / ml was subsequently used.

The resulting supernatant was treated for 1 hour at 0 ° C with freshly prepared trypsin solution (8 mg / ml 1 mM HCl) at a rate of 20 μm trypsin per 1 mg of protein and then a soy trypsin inhibitor at a rate of 2 μg inhibitor per 1 mg trypsin (to stop the proliferation) .

To the resulting hydrolyzate was added mercaptoethanol to a concentration of 1 mM, and then, at O C, a saturated solution of ammonium sulfate (in 1 mM ED and 1 mM mercapto ethanol) to 20% by volume. This mixture was stirred on a magnetic stirrer for 40 min to form a protein precipitate, and centrifuged for 15 min at lOOOg. The precipitate was discarded, and a saturated ammonium sulfate solution in EDTA and mercaptoethanol was added to the supernatant to 40% concentration by volume.

The protein precipitate from this mixture was ground on a magnetic stirrer and precipitated by centrifugation. The supernatant was discarded.

The precipitate was dissolved in a buffer containing 10 mM Tris-HCl (pH 7.4), 1.5 mM EDTA and 1 (mercaptoethanol (TEM). A specific prescription protein for progesterone was isolated

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about 80 mg of protein was applied, the current strength was 5 mA / cm gel, the elution rate was 15 ml / h. Separation time

(output of the fraction with the required receptor protein) did not exceed 22 h.

After isotachophoresis, preparations containing specific receptor protein to progesterone were concentrated through an Amikon membrane filter and applied to a column (16x31 mm with cooling) with DEAE cellulose (DE-52 Whatman), equilibrated with TEM buffer-. rum. Ion exchange chromatography was performed by stepwise elution with TEM buffer and TEM buffer containing 0.15 M KCl. Neutral formalin was added at 0 ° C to the protein solution eluted in the presence of KCl at a concentration of 0.5% and after 45 minutes the isolated protein was concentrated on an Amikon membrane filter (to remove Tris and HCl) and lyophilized. Lyophilized progesterone receptor protein preparations were stored in a refrigerator and used to determine progesterone in biological fluids and tissues.

Each batch of lyophilized progesterone specific receptor protein (SRV) preparation was tested for binding titer with progesterone labeled with luminol (PL). For this, a portion of dry CPB was dissolved in 0.1 M sodium phosphate buffer pH 7.5, containing 1 mM EDTA and 0.1% sodium azide (HEA buffer), and titrated with 100 µl of GSH. The concentration of CPB that binds 50% of the hormone in a 500 pg sample

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from this solution by gel-filter 40 GSH was used in further analysis on a column (1100 x 20 mm with cooling) with Sephadex G-150. Fractions containing a specific receptor protein for progesterone (present as a working concentration.

Pr and M e r 2. Determination of serum progesterone

For the study, samples were taken for the consistency of this protein was determined with the help of 5 blood coils of three women (16 weeks

pregnancy) with a volume of 0.5 ml, which were cooled to 4 ° C, and 100 μl were taken from each sample into incubation tubes for subsequent analysis.

The method of receptor-luminescence analysis was collected, concentrated through an Amikon membrane filter, and further purification was performed by isotachophoresis in a 200 × X 20 mm column with cooling in a polyacrylamide gel (, 2%) containing 8 M urea, 60 μl of ampholins ( pH 6-8) per 1 mg of protein. The leading buffer was tris-phosphate (pH 6.6), the trailing electrolyte — Tris-B - -amushokapronovy (pH 8.9), the elution buffer — the leading buffer containing 1 mM mercaptozthanol. On column

zah as working concentration.

Pr and M e r 2. Determination of the content of progesterone in the serum.

For the study, serum samples were taken from three women (16 weeks

pregnancy) with a volume of 0.5 ml, which were cooled to 4 ° C, and 100 μl were taken from each sample into incubation tubes for subsequent analysis.

To construct a calibration curve, control samples were prepared containing 100, 200, 500, 1000, 2000, 5000 and 10000 pg (picogram / ml) of progesterone, 100 µl each were also taken into incubation tubes.

In the selected test and control samples were added 100 µl of progesterone solution labeled hatchway.

NOLOM (PL) containing 500 pg / ml PL and 100 µg each of the solution containing the specific receptor protein for progesterone (isolated from the decidual tissue, as described in Example 1) in an amount of 1.1 mg / ml, which provides 50 % PL in a sample containing 500 pg PL (see example 1).

The resulting mixtures were incubated at A ° C and shaken for 60 minutes (the progesterone-receptor protein complex was formed during incubation).

To separate the progesterone-receptor complex from the free progesterone and the remaining components of the blood serum, hydroxylapite was added in an amount of 30 µg to each incubated sample and kept at 4 ° C for 10 minutes. The incubated samples — hydroxylapite with a progesterone-receptor complex — were applied to the filters (each for 1 filter) and washed with cold HEA buffer (0.1 M sodium phosphate buffer pH 7.5, containing 1 mM EDTA and 0.1% azide on three ). Then each sample was transferred (along with

Comparative characteristic of the specificity and sensitivity of the methods of RIA and RNA when determining progesterone

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The content of progesterone in the serum was determined by the methods of RIA and RLA in 8 women who have 9 weeks of gestation and 8 women who have, 16 weeks of gestation.

10 µl of 5 M NaOH was added to each vial and incubated at 60 ° C for 60 minutes. Samples were cooled to room temperature, 300 µl of liquid was taken from them for further investigation, added to each

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filter) in a separate vial and filled with NEA buffer (200 μl in each sample).

To isolate the isoluminol from the progeteroie-repeater complex, 100 µl 5 M NaOH was added to the samples and incubated at 60 ° C for 60 minutes.

Q Samples were cooled to room temperature, 300 µl each were taken (with a pipette with filter) and placed in luminometer cuvettes.

Then, up to 100 µl of horseradish peroxidase solution was added to the samples, and no later than 30 seconds, 100 µl 10 m each was added.

The chemoluminescence intensity was measured in a luminometer for 20 seconds and the values obtained were used to construct a calibration curve (the dependence of the luminescence intensity on the progesterone concentration in the control samples) and

5 determine the amount of progesterone in the serum samples of the examined women.

The minimum amount of progesterone, as determined by standard methods in various ways, is presented in the table.

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23, 96 39.62i1.79

The test sample of 100 µl horseradish peroxidase and 10 m were immediately measured in the luminometer for the luminescence intensity of each of the samples for a period of 20 s.

The table shows that in the quantitative determination of progesterone in standard solutions, the accuracy and, consequently, the specificity of RLA and RIA are almost the same. However, when determining the hormone in the blood serum of pregnant women (of different gestational age), the specificity of RLA is more than 2 times higher than the specificity of RIA.

Claims (1)

Invention Formula Method for quantitative determination of steroid hormones by treating blood serum with a hormone-binding substance in the presence of labeled steroid hormones, removal of free hormones, followed by determining the amount of label in the fraction of antibody-related hormones, characterized in that, in order to increase the accuracy of the method, a receptor protein from the decidual tissue is used as a hormone binding agent, and to remove free hormones roxylapatite.