DE2657292A1 - METHOD OF PREGNANCY DETERMINATION AND MULTI-COMPONENT PACKAGING - Google Patents

Description

Methods for determining pregnancy and which can be used therefor Multi-component pack

The invention relates to an enzymatic color test for Determination of pregnancy and a multi-component pack that can be used for this purpose.

There are already various methods for determining human chorionic gonadotropin (hereinafter abbreviated as HCG-) known. In some processes (biological processes), certain animals, such as rabbits, mice, frogs and the like can be used. These biological tests have serious disadvantages: there must be many animals available stand and are maintained that meet the specific requirements, making the process time-consuming, becomes complicated and costly.

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Some other tests (immunological Tests) in which certain cerum-

diagnostic compositions can be used (see e.g. Israeli patents 19 301 and 34-960).

Furthermore, a color test is also known in which antibody-enzyme conjugates used (see PEBS Letters, Volume No. 2, July 1974 pages 215-217).

Both for the implementation of the immunological tests and for the implementation of the color tests have to be complicated Apparatus are used and they can only be carried out in an appropriately equipped laboratory and with qualified Personnel are carried out

! .lan therefore endeavored to develop a test that also luhr · can be done so that any woman can easily determine whether or not she is pregnant. It is clear that for :? such a test should not require any complicated apparatus and that it should be carried out on the basis of simple instructions should·

The invention thus relates to a method for determining pregnancy, in which an enzyme-labeled anti-human chorjongonadotropin immunoglobulin G (hereinafter abbreviated to Anti-HCG-IgG) is mixed with any body fluid of the woman; this mixture is then mixed with a insolubilized anti-HCG-IgG (gel) mixed and after a predetermined period of time the whole thing is mixed with a developer dye mixed solution.

The abbreviations HCG and IgG used here stand for Human chorionic gonadotropin or immunoglobulin G.

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Various known enzymes can be used as enzymes for carrying out the method according to the invention: however, preferred enzymes are horseradish peroxidase, alkaline phosphatase and glucose oxidase.

The dye solutions to be used are a function of the enzyme used. For example, for the Horseradish peroxidase preferably o-D: "_ anisidine in an ELjCp solution. Another suitable compound mentioned should be is benzidine.

Any suitable source can be used to obtain the anti-HCG IgG as long as the material obtained is sufficient is specific. Suitable sources for obtaining this material include rabbits, Goats and other animals or birds may be considered.

In a preferred embodiment of the invention, there is one one or two of the substances listed below to make the test more sensitive:

a) normal ^ -globulin obtained from any suitable source, e.g. from rabbits, sheep or goats;

b) some free anti-HCG IgG obtained from the same sources as the gel and labeled with an enzyme is. These compounds, if present, are first mixed with the anti-HCG IgG labeled with the enzyme

Any fluid of the Woman, preferably the urine, used, verden. The fiction o Proper procedure for determining pregnancy can be in anyone commercial laboratory, in every pharmacy, but also by every woman can be carried out at home.

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In a "preferred embodiment of the invention" the entire mixture before developing with the dye solution washed several times with a suitable washing solution, for example a borate-sodium chloride solution.

The test can be translucent in any suitable Vessel, e.g. in a test tube, in a bag., On a Paper and the like.

When the woman is pregnant, i.e. when there is HGG in the body fluid is contained, a color appears after the addition of the developer solution, which is appropriate for the enzyme and the used corresponding dye is specific. If no color appears about 1/2 hour after adding the developer solution, this is it an indication that the woman is not pregnant.

The invention also relates to a multi-component pack (Test pack), which consists of the components for carrying out the test according to the invention, namely from

a) an anti-HCG IgG labeled with an enzyme,

b) an insolubilized anti-HCG IgG and

c) a suitable dye solution.

In a preferred embodiment, the anti-HCG IgG labeled with an enzyme also contains a normal γ- globulin and / or free anti-HCG IgG.

Component a) is preferably lyophilized in a Form stored and put on the market.

Of course, not all of these components need to be contained in a single package (set). If one or

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two of the above components packed separately this is also still within the scope of the present invention

The invention is illustrated in more detail by the following examples, without, however, being restricted thereto.

example 1 Manufacture of the Anti-HCG IftG

Normal rabbits were intramuscularly 3 times (once a week) in the soccer balls each 0.6 ml of a suspension of HCG, homogenized with an equal volume of the complete Freund * already Adj'uvan, is injected. The final concentration of HCG was 0.5 mg / ml. The activity (titer) of the HCG can vary from 2800 to 3600 units (g) / mg and is in no way critical to the success of the immunization. Pas HCG was dissolved in a 0.85% sodium chloride solution. It can also be in any other suitable buffer solution with a pH range of 6.8 to 8.5. One week after the last injection, the rabbits would bleed out left and the serum was separated by centrifugation. The unspecific antibodies were by 1/2 hour Incubate with an equal volume of insolubilized normal human serum separated from the serum and then the solution was centrifuged.

The specific anti-HCG-IgG was determined by adding a 60 % v / v saturated ammonium sulfate solution (75% w / v) precipitated from the serum to the anti-HCG serum. The resulting precipitate was buffered in a Dissolved saline and dialyzed against the same buffered saline for 2 to 3 days.

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The borate salt solution used in this example and in the following examples was prepared as follows:

I.) KaCl 0.85 %

II.) H ₃ BO ₃ 12.37 S

KaOH 0.52 g

ITaCl 8.00 g

H ₂ O to 1000 ml

In each case 97 ml of I.) were mixed with 3 ml of II.).

The concentration of the anti-HCG IgG was spectrophotometric determined at a wavelength of 280 mn. An optical density (CD) of 1.4 corresponds to 1 mg IgG / ml.

It is important to check that the preparation obtained is free from peroxidase activity.

Example 2 Manufacture of insolubilized anti-HCG IgG

The reaction was carried out under the white light of a fluorescent lamp. After mixing 2.5 ml of a bisacrylamide solution with 2.5 ml of the borate saline solution, 1 ml of proteins (containing 30 to 50 mg of anti-HCG IgG and another suitable protein source, such as normal rabbit IgG, bovine serum albumin (ESA) and the like at a concentration of 100 mg / ml) and 0.1 to 0.3 ml of riboflavin (from an aqueous solution of 0.2 mg / ml), a photoelectric reaction occurred. After incubating the above mixture for 1/2 hour at 25 to 36 ° C, an insoluble gel appeared. The gel was homogenized and washed 3 times in a borate saline solution. The amount used for testing was about 0.1 to about 1.0 is filled gel · The Bisacrylamidlosung was prepared as follows:

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1 s of bis- (N, N'-methylenebisacrylamide) (G ₇ E ₁₀ Ii ₂ O ₂ ) and 4 g of acrylamide (GH ₂ GHGOIiH ₂ ) were dissolved in 19 ml of distilled water.

Example 3 Production of the anti-HCG IgG labeled with an enzyme

5 mg of horseradish peroxidase was dissolved in 1.0 ml of a 0.3 molar sodium bicarbonate solution. 0.1 ml of o-fluorodinitrobenzene in absolute alcohol (1%) was added to this solution and the mixture was mixed slowly for 1 hour. 1.0 ml of an O, C6niolarene atrium peroodate solution was added and mixing was continued for 30 minutes. Then 1.0 ml of 0.16 molar ethylene glycol was added and mixing continued. The mixture was then dialyzed against a carbonate buffer (0.01 M, pH 9.5) for one day at 4 ° G. 5 mg was added to the anti-HCG IgG and the mixture was mixed for 2 to 3 hours at ambient temperature. The mixture was then dialyzed against a 0.01 M buffer, pH 7 »1 * at ^L. The Pufia? \ 7 was prepared by mixing: 17.5 ml Na ₂ IIPO ₄ 0.15 +

32.5 ml of KH ₂ PO ₄ .0.15 +

50 ml saline solution (0.85, #iges HaCl)

In this way about 90% immunoglobulin was labeled and 70-90 % peroxidase was coupled with the IgG. The labeled anti-HCG IgG was produced by passing the dialysate through a column with Sephadex G200 (PHAHIiACIA AE). The anti-HCG labeled with the enzyme can be stored in freeze-dried form in a concentration suitable for one or more tests. The enzymatic activity was measured in a spectrophotometer under visible light with a wavelength of 460 nm (0.1 ml test mixture, 2.9 ml dye, incubation for 3 minutes at room temperature). It became 1 dropper

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6 η HGl added to stop the reaction, and that Read the result in the spectrophotometer. The enzyme activity, expressed by Hillieunheiton (mU) was like is calculated as follows:

A = OD absorbance.
Example 4 - Execution of the test

The reagents given below are put into a tube mixed together and incubated for 1/4 to 1/2 hour:

a) C, 1 ml of an anti-HCG labeled with an enzyme with a Enzyme activity of 65

b) 0.1 ml of normal rabbit γ- globulin (or from a sheep or goat) at a concentration of 10 to 100 Hg / ml and

c) 0.1 ml of the body fluid to be examined, preferably urine.

By adding 0.1 to 0.5 mg of anti-HGG IgG, the Reaktior be made more sensitive.

After the yor incubation period, the mixture is transferred to another Tubes transferred, which the filled, insoluble made anti-HCG IgG gel in a volume of about 0.3 ml contains. This composition lasts for 15 to 30 minutes incubated. Thereafter, the composition is 4 times with the

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Borate saline solution washed (by centrifugation or by passing the solution through a "| pi§ ^ pf or through Immersing a nylon bag in the solution). After the last wash, 2.5 ml of the developer dye solution are added (prepared as indicated above) is added, the composition is shaken well and left to stand for about 5 minutes calmly. If HCG is present in the body fluid being examined, a reddish-brown color will appear.

Preparation of the dye solution

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0.5 ffil of a 0.3% HoO ^ solution are added to a 0.01 M Pbüsphatbuffer with a pH value of 6 "up to a volume of ^r 0 KiI. Then 0.41 ml of a 1% aqueous o-dianisidine solution was added to the above buffered solution.

Patent claims:

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Claims (1)

Claims Procedure for determining blackness, thereby ge '· -: · Braise rejects that an enzyme-labeled anti-ECG - I3G mixed with a woman's body fluid, de .; "; then the mixture with an anti-HCG IgG that has been made insoluble (Gel) mixed and the whole thing close to one before a specified period of time with a developer dye solution is mixed. 2. The method according to claim 1, characterized in that £ as SnzT'Tn horseradish peroxidase is used. 3. The method according to claim 1 or 2, characterized in that the dye solution is o-dianisidine in a HpO ^ solution is used. 4-, method according to one of claims 1 to 3 »characterized in that that the enzyme-labeled anti-ECG IgG is mixed with normal V - globulin. 5. The method according to any one of claims 1 to 4, characterized in that that the anti-HCG-IgG labeled with an enzyme is mixed with free anti-HCG-IgG. G. Method according to one of claims 1 to 5? characterized, that urine is used as the body fluid. 7- method according to one of claims 1 to 6, characterized in that that the mixture is washed before the addition of the dye solution. 709828/0600 BADORtGINAL δ. Method according to claim 7 »characterized in that a borate-common salt solution is used as the wash solution Y / ird. 9 · Multi-component pack (reagent set) for implementation of the method according to any one of claims 1 to 8, characterized marked that she (he) consists of a) an anti-HCG IgG labeled with an enzyme, b) an insolubilized anti-HCG IgG and c) a suitable dye solution. 10. Pack according to claim 9, characterized in that the labeled with an enzyme anti-HCG IgG with normal γ-globulin is mixed. 11. Pack according to claim 9 or 10, characterized in that the rait an enzyme-labeled anti-HCG IgG with free Anti-HCG-IgG is mixed. 12. Pack according to one of Claims 9 to 11, characterized in that that it contains component (a) in a lyophilized form. 709828/0600 ORIGINAL BATHROOM