CS242609B1 - Production method of an agent for determination of an early pragnancy and pathological statuses in gynaecology - Google Patents

Abstract

The way the preparation is made is that that at least two species are immunized pure human choriogonadotropic preparation hormone, the antibodies obtained are tested during serum and urinary immunization non-pregnant women, then they are selected animals that produce the lowest antibody the degree of cross reaction from which the obtained antibodies are reacted with urine and serum proteins of non-pregnant women and purified antibodies obtained at least from two animal species, they are tested in sandwich layout with zero content sample choriogonadotropic hormone and se sample containing at least 1 IU choriogonadotropic hormone per ml sample.

Description

Method for production of a preparation for the detection of early pregnancy and pathological conditions in gynecology

The method of manufacture of the composition comprises immunizing at least two animal species with a pure human preparation of choriogonadotropic hormone, obtaining antibodies obtained during immunization with serum and urine proteins of non-pregnant women, then selecting animals that produce the lowest degree of cross-reaction from which they are obtained. the antibodies are reacted with the urinary and serum proteins of non-pregnant women and the purified antibodies obtained from at least two animal species are tested in a sandwich arrangement with a sample containing zero choriogonadotropic hormone content and a sample containing at least 1 IU choriogonadotropic hormone per ml sample.

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The invention relates to a method for the production of a specific composition for the detection of early pregnancy and for the diagnosis and monitoring of certain pathological conditions associated with the presence of human chorionic gonadotropin in body fluid, for its detection and quantification in urine, serum or plasma by the Enzyme Linked Immunosorbent Aásay antigen, labeled antibody.

Human chorionic gonadotropin is a glycoprotein hormone physiologically produced by the placenta. The hormone appears in the peripheral circulation (serum, plasma and then urine) about 9 days after conception and its level gradually rises. Thus, the detection of chorionic gonadotropin means the detection of pregnancy, and the more sensitive the method of detection, the sooner pregnancy can be detected. Early detection of pregnancy is of considerable medical importance. In addition to own information for women and doctors is necessary in various pregnancy pathologies such as ectopic pregnancy and others. Quantitative determination of chorionic gonadotropin is very important in monitoring endangered pregnancy and in monitoring therapy for hCG-producing tumors. Early and reliable recognition of pregnancy is also necessary for the widespread implementation of the so-called mini-abortion method, ie induced abortion at the earliest stage.

Currently, tests based on the method of preventing hemagglutination are used to determine pregnancy. The disadvantage of these tests is their low sensitivity, which allows to detect pregnancy at the earliest in the period of about 40 '. their applicability only for urine tests and the impossibility of direct quantification of chorionic gonadotropin. In addition to these tests, latex-based tests are known which suffer from the same disadvantages and are particularly suitable for rapid orientation examination. Furthermore, there are highly sensitive tests for radioimmunoassay baxi. These kits are

242 609 in themselves very expensive, but they also require an expensive evaluation device, i.e. a pulse counter. The kits have a very short shelf life and work with radioactive material poses a health risk.

The possibility of using an immunoenzymatic method to detect chorionic gonadotropin has been known for some time. The principle of the sandwich method is that the first antibody binds to an immunosorbent, e.g., ice on the wall of a polystyrene tube. The tube is then washed, removing any unbound antibody molecules. Add a sample containing choriogonadotropic hormone - an antigen that binds to the immobilized antibody. Wash the tube again. Add a second enzyme-labeled antibody that binds to the antigen from the other side of the molecule and washes the tube. A substrate with a colorless chromogen is added - the substrate is degraded by the enzyme, causing the chromogen to stain. The reaction stops after a period of time A colored reaction in the tube therefore indicates that the antigen under test has been present. Unstained contents indicate the absence of antigen. The color intensity corresponds to the antigen concentration. The dependence of absorbance on antigen concentration can be expressed by a calibration curve, from which the desired amount of antigen can be read back from the measured absorbance. The basic principles were elaborated in a rough form by B.K.Van Weemen and A.H.W.M. Schuurs / Van Weemen, B.K. Schuurs,

A. H. W. M., FEBS Letters 15 (3), 1971, pp. 252-256; Van Weemen,

B. K., Schuurs, A. H. W. M., FEBS Letters 45 (2), 1974, pp. 215-218. Also, many recent, high quality experimental works are not suitable for practical routine use / Hamada, K. et al., Endocrinol. Japanese. 25 (5), 1978, pp. 515-517; Iwasa, S. et al., J. Biochem. 89 (4), 1981, pp. 1091-1099; Tomoda, S. et al., Endocrinol. Japanese. 27 (6), 1980, pp. 785-788; T. et al., Acta Endocrinol. (KbH), 97, 1981, pp. 562-568. Competitive ELISA assay reported by U.M. Joshi / Joshi, U.M. et al., Obstet. Gynec., 57 (2), 1981, pp. 252-254, is suitable for routine use, but has a relatively low sensitivity and the specificity of the assay is not further specified. Quality immunoenzymatic test / Mohta, H.C., MacDonald, D.J., Glin, Chim. Acta 121, 1982, pp. 245-250), which uses a commercial antibody against the beta subunit of chorionic gonadotropin, is highly specific, but the preparation of the anti-beta subunit antibody A-242 609 is relatively complex, which would be manifested in commercial production in increased the cost of one examination.

Human chorionic gonadotropin is composed of two non-identical subunits, called alpha and beta, the alpha subunit being almost identical to the alpha subunits of other hormones, especially the luteinising hormone, while the beta subunit is specific for the choriogonadotropic hormone. Immunization with the whole molecule of choriogonadotropin is very simple and inexpensive as the preparation does not need to be specially treated, but it leads to an almost complete cross-reaction of the antibodies with the luteinizing hormone. While choriogonadotropic hormone is specifically pregnancy, luteinizing hormone occurs at relatively high levels in non-pregnant women. A test using such an antibody is then not suitable for proving pregnancy - at least not at an early stage when choriogonadotropin levels do not exceed luteinizing hormone levels. Pregnancy can only be determined at a later stage when choriogonadotropin levels are already high enough.

This disadvantage can be overcome by immunization with only the specific beta subunit of the hormone. The resulting antisera are then capable of partially specific reactions with choriogonadotropin in the presence of luteinizing hormone - i.e. they preferentially react. The degree of specific reaction varies between antisera individually. The problem with this procedure lies in the very difficult isolation of the chorionic gonadotropin beta subunit, which is very costly, and that immunization of animals rarely leads to the production of a specific antibody.

These disadvantages are overcome by a method of making the composition comprising immunizing at least two animal species with a pure human chorionic gonadotropic hormone preparation, obtaining antibodies obtained during immunization with serum and urine proteins of non-pregnant women, and selecting animals that produce the lowest grade antibodies. cross-reactions from which the obtained antibodies are reacted with the urine and serum proteins of non-pregnant women and purified antibodies reacting primarily with choriogonadotropic hormone are obtained from at least two species of animals in a sandwich arrangement with a zero chorio content

242 809 gonadotropic hormone and containing a sample containing at least 1HJ of choriogonadotropic hormone per ml of sample.

The effect of the invention lies in a simpler method of preparing specific antibodies and then optimizing them for specific detection of low levels of choriogonadotropic hormone.

The advantage of the immunoenzymatic test for the determination of choriogonadotropic hormone is its high sensitivity and specificity, comparable to imported radioimmunoassay test kits, with the cost of one test being considerably lower, the test also having a longer shelf-life, of months compared to weeks, no costly evaluation required. a conventional spectrophotometer is sufficient and there is no risk of working with the radioisotope. Said immunoenzymatic test can be performed in urine, qualitative determination and indicative quantification, serum and plasma, qualitative determination and quantification, and can be performed in healthcare facilities of all stages and is feasible in a single work shift. The assay can be performed not only in tubes but also on microtiter plates.

The following example illustrates the invention in more detail.

Example 1

Antibodies were prepared by immunizing pigs and sheep with purified chorionic gonadotropin with an activity of 10,000 IU choriogonadotropic hormone / mg. A suspension of aluminum hydroxide with paraffin oil and sorbitan trioleate was used as an adjuvant in animal immunization. The antibodies obtained were tested by immunodiffusion for the degree of cross-reaction with cross-reacting hormones, in particular with luteinating hormone, and for the degree of reaction with urinary and serum proteins, and the antisera with the lowest degree of cross-reaction were selected. These tests were already performed during immunization and non-compliant animals were discarded. The obtained high-quality antisera were loaded on columns containing preferably urine-coupled agarose gel, which were isolated from a large number of male urine - 500 donors and female urine in postmenopause. In this way, the antiserum of antibody molecules cross-reacting with urine pro- ducts, which arose from an immune reaction against a small amount of contaminating material present in a highly pure preparation, was freed. Since cross-reacting luteinizing hormones are present in the urine, the procedure used not only suppresses non-specific interactions, but also cross-reactions against these hormones. The antisera were further grown on a column containing solid-bound CNBr Sepharose 4B bound proteins obtained from the serum and urine of non-pregnant women.

The obtained purified sera containing mainly antibodies reacting with choriogonadotropin hormone were combined with samples containing no choriogonadotropin hormone and with samples containing 1 U of choriogonadotropin / ml. Antibody combinations were selected that had an absorbance with a sample having a choriogonadotropin zero content of less than 0.2 units and a sample having a choriogonadotropin content of 1 IU greater than 1.0 absorbance unit. Pig antibody was selected as antibody I and showed a sensitivity of 6m IU choriogonadotropin / ml. The sheep antibody was tested with a sample containing 20 mlU choriogonadotropin / ml and 20 mlU luteinizing hormone / ml and showed a 4-fold higher sensitivity to choriogonadotropin than luteinizing hormone.

On this II. the peroxidase was bound to the antibody.

The constructed calibration curve expressed a linear dependence of absorbance on a concentration ranging from 10 to 1000 mlU of choriogonadotropin / ml. Similarly, a combination of antibodies was prepared by immunizing rabbits and sheep.

Antibodies were included in the hCG detection and quantification kit. The kit contained 5x20 ml lyophilized antibody I and 1 dose of peroxidase labeled specific specific antibody II, 5x1 ml lyophilized comparator choriogonadotropin as standard, 15 ml of 100x binding buffer solution, alkaline pH bicarbonate buffer, 100 ml 20x concentrated washing solution containing detergent 1x 50 ml of diluent I for dilution of antibody II and 250 ml of diluent II for dilution of samples, 60 ml of substrate solution and 6x10 mg of O-phenylenediamine in homogeneous mixture with 90 mg of potassium sulphate.

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Five 96-well polystyrene plates per plate were delivered as carrier. Polystyrene tubes can also be used.

The kit is intended for the detection and semi-quantitative determination of human chorionic gonadotropin in urine and quantitative determination in serum. The examination is based on the principle of Enzyme Linked Immunosorbent Assay. Pig antibody I is bound to the walls of the wells and, after rinsing, the standard and test samples are applied to the wells. Choriogonadotropin, if present, binds to bound antibody during incubation. After washing, peroxidase-labeled sheep antibody II is applied. The labeled antibody binds to the antigen - choriogonadotropic hormone. After washing, peroxidase activity is detected, which is proportional to the antigen concentration in the sample. The test results are known in about 6 hours.

One hundred morning urine samples from healthy menopause women aged 45 to 60 years were used to determine the degree of cross-reaction.

The urine of these women contains an increased amount of cross-reacting hormones, which in an unknown sample could misinterpret the test result in terms of false positivity. After statistical. the evaluation of the whole immunoenzymatic assay is determined by a level that cross-reacting hormones are no longer likely to exceed, and the detection of higher levels in an unknown sample can already be considered a specific ”finding of choriogonadotropin. This level corresponds in serum, plasma and urine to 200 mlU of choriogonadotropin / ml sample, and levels between 50 and 200 mlU of chriogonadotropin / ml can be considered as a possible pregnancy and are therefore considered transient and the test is repeated the following day. This specific level also covers the preovulatory increase in cross-reacting hormones in the menstrual cycle.

Conventional water or motor vacuum pump equipment and conventional spectrophotometers capable of measuring 1 ml of the sample at 492 nm to two decimal places, such as Spekol 10, are used to perform the test.

AND

- | δ Test example; ^{242 609}

Morning urine samples from a healthy 281 year old woman who planned to become pregnant were tested during the menstrual cycle. The menstrual cycle was regular - 29 days, the woman had already delivered one healthy baby. Urine samples were tested as follows:

the lyophilized anti-hCG antibody was dissolved in the binding solution, which is an alkaline pH bicarbonate buffer. 0.3 ml of dissolved antibody was pipetted into one tube into 3 cm5 polystyrene tubes. After 20 hours at +4 ° C, incubation until the next day in the refrigerator, the binding of the antibodies to the tube walls was terminated.

The solution was aspirated from the tubes and each tube washed 3 times with binding buffer. Standard and test urine samples, 0.2 ml each, were then applied to the tubes and stored in a thermostat at 37 ° C for 2.5 hours.

The contents of the tubes were aspirated, the tubes washed twice with a detergent-containing solution to block the remaining free binding groups.

0.2 ml of enzyme labeled second antibody solution was applied to each tube. The tubes were incubated in a thermostat at 37 ° C for 1.5 hours.

The contents of the tubes were aspirated and the tubes were washed 4 times with detergent-containing solution. 0.2 ml of a 0.1% solution of o-phenylenediamine in a buffer containing hydrogen peroxide was applied to the tubes and incubated for 40 min. at 37 ° C. Positive samples decompose hydrogen peroxide by the enzyme, which results in a color reaction. The reaction was stopped by adding 1 mL of 0.5 N sulfuric acid to each tube. The result is determined by photometry by determining the absorbance of standard and unknown samples and evaluated as above.

In the follow-up cycle, pregnancy was detected on day 29 of the cycle, when the level of chooriogonadotropin was in the area of transient results and the pregnancy was subsequently verified on day 30 of the cycle, when the result was already positive.

Claims (1)

SUBJECT OF THE INVENTION Method for the manufacture of a composition for the detection of early pregnancy and pathological conditions in gynecology, obstetrics and oncology associated with the production of human chorionic gonadotropin by an immunoenzymatic sandwich method, characterized in that at least two animal species are immunized with pure human choriogonadotropic hormone preparation. serum and urinary proteins of non-pregnant women, then selecting animals producing the lowest degree of cross-reaction, from which the antibodies obtained react with urinary and serum proteins of non-pregnant women, and purified antibodies obtained from at least two animal species are tested in sandwich; arrangement with a sample containing zero choriogonadotropic hormone content and a sample containing at least 1 IU choriogonadotropic hormone per ml of sample.