CS242621B1 - A method for determining human chorionic gonadotropin in immunoglobulin preparations prepared from substitute raw materials - Google Patents

Abstract

The determination consists in diluting the immunoglobulin preparation with a buffer of pH 6.5 to 8.0 with the addition of serum albumin, for example bovine albumin in an amount of 0.005 to 1%, after which the content of human chorionic gonadotropin is determined by the immunoenzymatic method. The immunoglobulin preparation can be diluted with a buffer of pH 6.5 to 8.0 with the addition of serum albumin and sodium chloride to a maximum concentration of 0.3 M. It is preferable to use a phosphate buffer. It is also preferable to use immunoenzymatic methods, in which one of the immunoreagents is adsorbed onto a solid carrier.

Description

Method for determination of human chorionic gonadotropin in preparations of immunoglobulins prepared from surrogate raw materials

The assay consists of diluting the immunoglobulin preparation with a pH of 6.5 to 8.0 with the addition of serum albumin, for example bovine albumin, in an amount of 0.005 to 1%, followed by human chorionic gonadotropin content by immunoenzymatic method. The immunoglobulin preparation can be diluted with a pH 6.5 to 8.0 buffer with the addition of serum albumin and sodium chloride to a maximum concentration of 0.3 M. It is preferred to use a phosphate buffer. It is further preferred to use an immunoenzymatic method in which adsorption of one of the immunoreactors to a solid support is carried out.

242 621

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The invention relates to the determination of human chorionic gonadotropin in purified and unpurified preparations of immunoglobulins prepared from surrogate raw materials - eg erymasa, postpartum and abortion sera using the enzyme immunoassay method. Said starting materials comprise human chorionic gonadotropin (hCG), which during manufacture is transferred from the raw material to the final preparation. The presence of hGG in the final preparation is undesirable because it is a biologically active substance - a hormone - produced by the human placenta under physiological circumstances. This hormone in the final formulation is introduced into the recipient-patient organism, which is very undesirable, although these are relatively small doses. The child may also be a patient and any external hormonal effects are undesirable. In order to develop an effective method for removing hCG from immunoglobulin preparations in the manufacture thereof, a reliable method must be available to quantitatively determine the hormone in the preparations.

Currently, only hemagglutination tests (Wide) are available. L .: Lancet, ii, 1969, p. 863, or latex agglutination (01son, Adduci, Amer. J. Clin.Pathol., 39, 1963), s.589 / ·

These assays are poorly sensitive to the determination of hCG in protein media, such as immunoglobulin preparations. They are greatly influenced by the composition of the preparation being determined and the results are biased.

Determination of hCG in immunoglobulin preparations by radioimmunoassay (RIA, Talow, Berson, Nature, Lond., 184, 1959, p. 1648) has been shown to be satisfactory. However, the kits for this determination are only available from imports from the foreign exchange area. My method and a number of disadvantages, necessnihst building special isotope laboratories, for the evaluation of results it is necessary to cost

- 2 242 621 commercial kits have a short shelf-life of several weeks, work must be carried out according to strict safety regulations and used radioactive reagents must be stored inactivated at designated locations. Moreover, working with radioisotopes presents a health risk. The cost of one examination is relatively high.

The above-mentioned drawbacks are eliminated by the process according to the invention, which comprises diluting the immunoglobulin preparation with a buffer of pH 6.5 to 8.0 with the addition of serum albumin, for example bovine albumin, in an amount of 0.005 to 1%. gonadotropin.

The immunoglobulin preparation can be diluted with a pH 6.5 to 8.0 buffer with the addition of serum albumin and sodium chloride to a maximum concentration of 0.3M. Phosphate buffer is preferred.

It is further preferred to use an immunoenzymatic method in which adsorption of one of the immunoreactors to a solid support is carried out.

Assay Example: Human chorionic gonadotropin in immunoglobulin preparations manufactured by ÚSOL o.p. Prague - Lot No. 2/83, 4/83, 5/83, 8/83, 12/83 in the form of a 16% solution. The content of hCG in lyophilized preparations of immunoglobulins manufactured by Imuna Šarišská Michelana, batches 70/72, 26/77 was also determined, from which also 16% solutions were prepared. The determination was performed on microtiter plates of type P manufactured by KOH-I-NOOR Dalečín.

Determination procedure:

1 / Lyophilized anti-hCG antibody was dissolved in buffer and 0.2 ml antibody solution was applied to each well of the plate. After 20 hours at +4 ° C (incubation until the next day in the refrigerator), the binding of the antibody to the wall of the wells is complete. The solution from all wells was sprayed into the sink and each well of the plate was washed 3 times with buffer.

2 / hCG standards were applied to wells of 0.1 ml each in pairs of 2 wells (always in pairs), diluted immunoglobulin preparations were diluted into the remaining wells (dilution is performed by geo 1 242 621 metric series 2 ⁿ 0.01 M phosphate buffer) 0.14 M NaCl 0.01% merthiolet 0.1% bovine serum albumin pH 7.5 (other suitable buffers may also be used) in a volume of 0.1 ml per well. Dilution of samples is performed to allow the resulting absorbance values to be recalculated against the calibration curve (from multiple points). The standards and samples plate was then incubated for 2.5 hours in a thermostat at 37 ° C.

3 / The contents of the wells were sprayed into the sink and each well washed twice with buffer. 0.1 ml of enzyme-labeled second anti-hCG antibody (horseradish peroxidase is used as enzyme) was applied to each well. The plate was incubated

1.5 hours in a thermostat at 37 ° C.

4 / The contents of the wells were sprayed into the sink and the plate washed 4 times in succession with buffer. 0.1 ml of chromogen substrate solution was applied to each well. The plate was incubated for 40 minutes in a thermostat at 37 ° C. Decomposition of the substrate occurs by the enzyme, which is manifested by a change in color of the chromogen.

5 / The reaction in all wells was stopped by the addition of 0.05 ml of 4 N h ₂ sd ₄ .

The resulting staining of the well contents is read on a photometer. The higher the concentration of hCG in the sample, the more intense the reaction and the more intense the chromogenureaction color is quantitative. Color can be read photometrically - as absorbance. If the sample to be examined did not contain hCG there was no binding to the first layer, the subsequent layer also had no possibility of binding, so there was no enzyme present in the test tube after flushing, no substrate degradation and chromogen color change.

If several unknown samples of known hCG / so-called hCG concentration are assigned to unknown samples. For example, a calibration curve can be constructed by plotting known standard concentrations and their respective absorbances on two perpendicular axes. A calibration curve is plotted from the absorbance of the samples to which hCG standards have been applied, and hCG concentrations are subtracted from the detected absorbance of the diluted immunoglobulin samples. To plot the curve and calculate the concentration

- 4 242 β? 1 hCG computers can also be used. Test tubes can be used instead of microtiter plates, where each well of the plate is a small tube.

Results / by recalculation of microcomputer Tesla PMD-85 /:

I. preparations of immunoglobulins ÚSOL o.p. Prague

batch no. hCG unit content (IU / ml) in a 16% solution determination error in% / variation coefficient / 2/83 20,557 18.59 4/83 27,711 25.49 5/83 24,982 9.37 8/83 40,899 20.43 12/83 36,853 9,857 II. immunoglobulin preparations Šarišské Michalany

Human chorionic gonadotropin was not detected in the lots 70/72 and 26/77. It was then found by the manufacturer that these lots were not prepared from substitute raw materials, but from blood of healthy non-pregnant donors (hCG free of raw materials).

Repeated experiments have shown that the immunoenzymatic method can be used to determine human chorionic gonadotropin in immunoglobulin preparations prepared from secondary raw materials. The assay method of the invention is 200 times more sensitive than hemagglutination or latex-agglutination tests, and does not suffer from the disadvantages of radioimmunoassays.

The test can be carried out on tubes in any laboratory equipped with a conventional spectrophotometer, using only pipettes of suitable volumes, a standard thermostat and household raw materials. It is not limited by safety regulations and the cost of a single assay is low. The use of the immunoenzymatic method for the determination of hCG brings the possibility of sensitive and accurate quantification of hCG in immunoglobulin preparations and can contribute significantly to the health care of the population.

Claims (4)

OBJECT OF THE INVENTION 242 621 A method for the determination of human chorionic gonadotropin in immunoglobulin preparations prepared from surrogate raw materials, characterized in that the immunoglobulin preparation is diluted with a pH buffer 6.5 to 8.0 with the addition of serum albumin, for example bovine albumin, in an amount of 0.005 to 1.00% by weight, after which the human chorionic gonadotropin content is determined by immunoenzymatic method. 2. The method according to claim 1, wherein the immunoglobulin preparation is diluted with a buffer of pH 6.5 to 8.0 with the addition of serum albumin and sodium chloride to a maximum concentration of 0.3 M. 3. The method of claims 1 and 2, wherein the immunoglobulin preparation is diluted with phosphate buffer. 4. The method of claims 1 to 3, wherein the human chorionic gonadotropin is determined by an immunoenzymatic method, wherein adsorption of one of the immunoreactors to a solid support is performed.