DE1227195B - Immunological reagents - Google Patents

Description

Immunological Reagents This invention relates to a method for Production of an improved antiserum which contains antibodies to human chorionic gonadotropin (hereinafter referred to as HCG) for use in immunological pregnancy examinations contains.

Pregnancy can be determined by detecting HCG in urine samples or serum; HCG is present during pregnancy but is usually absent otherwise. Since HCG is antigenic, the assay can be carried out by treating the sample with a reagent containing HCG antibodies and observing the immunological reaction according to any of the conventional methods. For example, pregnancy examinations have been described using the precipitin method (French patent 973 915), the complement determination method (Belgian patent 605517) and the agglutination method using either red blood cells (Belgian patent 605 826 and 610 085) or polystyrene particles (Belgian patent 613 680) .

An antiserum that contains HCG antibodies is produced by immunizing of an animal with HCG, together with an aid (preferably an oily Aids), collecting the animal's blood and separating the serum. However, the commercially available HCG is not pure, but contains antigenic impurities; and therefore the antiserum can contain not only HCG antibodies but also secondary antibodies produced due to the antigenic contamination. The secondary antibodies can be with the antigenic components of the urine or Serums respond whether or not HCG is present, and this non-specific response can give a false positive result in cases where HCG is absent is.

Research has been carried out to examine this difficulty Avoid using a highly purified HCG to immunize the animals, but even in this case it was often required special procedural steps turn on secondary antibodies to the impairment of the pregnancy examination to prevent. In Belgian patent specification 605 517, for example, the antiserum treated before use with human serum from non-pregnant women to the secondary Saturate antibodies.

It has now been found that the secondary antibodies in the titer with respect to the HCG antibody increase as the immunization is prolonged or when the immunization dose of HCG is large. It is therefore preferable to have one very short sequence of injections of moderate doses of HCG to use and to obtain a highly specific antiserum.

So far, an antiserum, which contained HCG antibodies, was under Using a sequence of many injections of HCG, often very large ones Cans, made. For example, in Belgian patent specification 605 826 Rabbit antiserum by injection of 12,000 international units of HCG produced in weekly intervals over 5 to 6 weeks. In the Belgian U.S. Patent 605,517 became antiserum by immunizing rabbits with 12,500 I. E. HCG three times at 3 week intervals, these being three treatments A fourth injection of 3000 I.E. HCG followed 4 to 6 weeks later. According to Belgian patent 613 680, rabbit antiserum was produced by intramuscular injection of 50,000 I. E. HCG, which 3 weeks later 3 intravenous Injections of 10,000 I.U. on consecutive days followed.

According to Belgian patent specification 610 085, a rabbit became nine Injections totaling 2250 I.U. administered.

However, when using the above methods, it could not the formation of secondary antibodies in the serum can be avoided.

According to the present invention there is provided an improved antiserum which HCG antibodies for use in immunological pregnancy examinations contains, produced by repeated intramuscular injection of HCG in animals and Obtaining the formed anti-HCG serum 1 to 3 weeks after the last injection, with two injections with a single dose of less than 1000 1.U / kg body weight be made.

Animals suitable for this purpose are mammals such as rabbits and Horse. It is advantageous to have two injections of 1500 I each in a rabbit. E. and two injections of 7500 I.E. each made in a horse.

- -To the. The antiserum can be used in pregnancy examinations of this invention, free of microbial contamination, recovered and packaged in ampoules are presented. The antiserum is expediently stored frozen, for example at -20 ° C. Optionally, the antiserum can be freeze-dried in ampoules and be made usable for carrying out the investigation. For use at a Haemab glutination pregnancy examination it can be combined with an equivalent amount of HCG-sensitive red blood cells freeze-dried as described in Belgian patent specification 605,826.

The following examples illustrate the invention: Rabbit serum. Rabbits weighing approximately 2 kg are injected intramuscularly into the soft with -1500 units of HCG (Leo) in 0.5 ml of saline, mixed and homogenized with an equal volume of incomplete Freund's aid. One such adjuvant comprises a water-in-oil emulsified system in which the aqueous component contains antigen and the oil is a mineral oil such as paraffin. The injection was repeated 28 days later. 10 to 14 days later, blood was taken from the animals, the serum was separated off and stored at -20.degree. The titers of the serum obtained by this method were between 1/2000 and 1/10000 in the case of a haemagglutination reaction with HCG-sensitized cell suspensions.

Horse antiserum A horse weighing approximately 600 kg became intramuscular in the throat with 7500 units of HCG (Leo) in 2.5 ml of saline, mixed and homogenized with an equal volume of incomplete Freund's device. The injection was repeated 28 days later. Blood was drawn from the horse 2 weeks later, the serum separated and stored at 4 ° C. The serum was made with 6,9-diamino-2-ethoxyacridine lactate ("Rivanol") treats the precipitation of non-specific haemagglutinins and other unwanted proteins are removed, creating the desired HCG antibody remained in solution. The treated antiserum was stored at 4 ° C. The titers of the serum obtained by this procedure ranged between 1 / 10,000 and 1 / 2o,000 in a hemagglutination reaction with HCG-sensitized cell suspensions.

When different antisera with HCG sensitized blood cells for specific antibodies and with NHS-sensitized blood corpuscles for ünspe = specific antibodies are examined, the comparison of the results shows the relative Amount of antibodies present.

The results of the investigations are listed below.

An antiserum which was produced using rabbits, these rabbits having been given two doses of 1500 units of HCG in Freund's solution 28 days apart, had the following relative titers: Titer HCG cells ..................... 1 / sooo NHS cells ..................... 1 / 20o After another dose of antigen as above, the following picture emerges: Titer HCG cells ...................... 1 / sooo NHS cells ..................... 1 / soo After another dose of antigen the following picture emerges: Titer HCG cells ..................... 1 / 500o NHS cells ..................... 1 / looo Since the amount of antibody is inversely proportional to the titer values, it follows that further injections only increased the concentration of the specific antibodies.

In the case of equine antisera, two doses of 7,500 units of HCG given 28 days apart would be found to give antisera with the following characteristics: Titer HCG cells ..................... 1 / 2o ooo NHS cells ..................... 1 / 5o It was found that this antiserum gave the lowest known ratio of specific to unspecific antibodies.

Claims (3)

Claims: 1. A method for producing an improved antiserum which contains HCG antibodies for use in immunological pregnancy examinations by repeated intramuscular injection of HCG in animals and obtaining the anti-HCG serum formed 1 to 3 weeks after the last injection, d a -characterized in that two injections are made with a single dose below 1000 IU / kg body weight. 2. The method according to claim 1, characterized in that in a rabbit two injections of 1500 I. E. are made. 3. The method according to claim 1, characterized in that that two injections of 7500 I.U. each are made in a horse. Into consideration Drawn pamphlets: French patent specification No. 973 915.