FI58566C - FOERBAETTRAT FOERFARANDE ATT FRAMSTAELLA INDIKATORSYSTEM FOER BESTAEMNING AV ANTIGEN-ANTIKROPPSREAKTIONER MED HJAELP AV HEMOLYS I GEL - Google Patents

Description

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RoS cw: o JC 'n ^ (51) Kf.rn.Vci.3 G 01 H 33/5 * FINLAND — FINLAND (21) 790598 (22) HakemltpClvI — An * 6kitlnp4 «g 22.02.79 (23) Afloipllvt— GIMcMtadag 22.02.79 (41) Tullut ivIklMkii - BlMt offamtfg 23 08 80 ^ »tantti. ) ft r.kift «rlh» llltu * (44) Date of NiMMUulpanon Jt kuLfulkalMn - rtmtt- oeh raflftarttyralaan AmMcm iittagd och utUkrMtm pubiiearatf 31.10.80 (32) (33) (31) Ρ) Τ »4 · **⁇ u Helsinki 51, Suami-Finland (FI) (72) Pertti Ilkka Väänänen, Helsinki, Finland-Finland (Fl) (7 ^) Fermion Oy, Patent Department (5 ^ 4) with the aid of an antigenic-antichromatic reaction system with the presence of hemolysis in the gel

It is an object of the invention to provide an improved method of preparing an indicator system for determining antigen-antibody reactions by gel hemolysis.

Gel hemolysis, or the HIG method, is used in hospitals and laboratories to show that a person is or has been suffering from a specific disease. The HIG method then shows that the person's blood or serum contains an antibody to the pathogen in question. This HIG method can also be used to identify a particular antigen with a known antibody.

HIG methods use an agarose plate or gel prepared in a specific buffer containing erythrocytes to which antigens or antibodies have been bound. The bond between erythrocytes and antigens or antibodies can be further strengthened with chemical agents such as chromium trichloride. The gel also contains complement.

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The test sample, for example serum, is added to a well in an agarose gel and incubated. Hemolysis of the plate around the well indicates that the test sample contains the antigen or antibody to be determined. Hemolysis is based on the following mechanism. The antibody or antigen in the test sample reacts with the antigen or antibody on the agar plate and sensitizes the complement, causing the erythrocytes to break down, i.e. hemolysis.

Indicator systems based on the HIG method are described, for example, in the previous applicant's Swedish patent application SE 7608297, in the Wellcome Foundation's patent publication DE 2532283 and in Schildt et al. Intern. Symp. on Immunity to Infections of Respiratory Systems in Man and Animals. Develop. Biol. Standard, vol. 28 pp. 253-272. In the indicator systems described in the above publications, complement is present in the gel. In this case, the shelf life of the agar plate is limited, ie no more than 3-4 weeks. If complement is omitted and added only as in the present invention when using a plate, the shelf life of the indicator system can be increased to 3-4 months.

Skaug et al. Acta Path. Microbiol. Scand. Sect. B vol. 83, ss. 36.7-372 and Strannegärd et al. J. Clin. Microbiol, vol. 1_ (6), ss. 491-494 have disclosed a method of pouring a complement solution onto a plate. Using the Skaug method, the test sample is first added and incubated for a few hours. The complement solution is then poured onto a plate and incubated again for 20 hours. In the Strannegärd method, incubation times are 7-24 hours with serum and about two hours with complement, respectively. When complement is added as a solution, its even application to the gel, which is often elevated at the edges, is cumbersome and the hemolysis rings may be uneven as a result, which in turn makes it difficult to measure the diameter of the hemolysis rings. In addition, two different incubations prolong the results.

In order to overcome the above-mentioned disadvantages, a fast, reliable and longer-lasting indicator system according to the invention and a method for manufacturing this indicator system have been developed.

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In the system according to the invention, an agarose plate containing only erythrocytes and antigens or antibodies attached thereto is prepared in a certain buffer environment, which bond can be further strengthened by a chemical substance such as chromium tri k Lori di 1la. The complement of the indicator system is impregnated by dipping a film having a suitable pore size of 0.5-5 μm, most preferably 1.2 μm, into the complement solution and drying the film. Various cellulose ester mixtures or cellulose triacetate can be used as the film material. The absorbed film is lyophilized and stored at + 4 ° -6 ° C and used as needed.

Alternatively, the complement can be absorbed onto the blotting paper sheet. However, the film material is easier to standardize and the complement spreads more evenly from it than from the absorbent paper.

Using the system of the invention, the test sample is added to the plate and incubated for one hour. The membrane containing complement is then placed on a plate and incubated. The hemolysis rings are smooth and easy to measure against the white membrane.

The system according to the invention is easy to use. No specially trained staff or specialized laboratories are required. The system can be used where it is needed, such as in hospitals and health centers. The test is fast to complete and the results are simple and straightforward to read.

Improved shelf life of the indicator system means that plates can be manufactured in larger batches and stored.

This facilitates the industrial manufacture and distribution of the system. If the shelf life is poor, the boards must be prepared just before use and special transports must also be arranged. Better shelf life also ensures that users who need the system less frequently can take advantage of it. The above points show that such a system of indicators with better durability is of great technical and economic importance.

The invention is illustrated by the following examples of influenza, mumps and rubella: 58566

Example 1 Preparation of HIG plate with influenza virus antigens a) Preparation of plate

Mix in a test tube 0.5 ml of rooster red cells, 0.5 ml of influenza virus-containing allantoic fluid with a haemagglutination titre of 640 HAU / ml and 0.5 ml of 0.1% CrCl3'6H2O in 0.15 M NaCl.

After 5 minutes, 30 ml of 0.05 ΙΊ phosphate buffer pH 7.2 with 0.15 M NaCl, 0.003 M CaCl 2 and 0.001 H MgCl 2 and 0.2% bovine albumin are added.

Centrifuge and wash the cells once more with the same buffer. The cell pack is then centrifuged and resuspended to 10% in the above buffer without bovine albumin.

Mix 0.6 g of agarose in 39 ml of 0.05 M phosphate buffer pH 7.2 with 0.15 M NaCl and heat in a water bath until the agarose has melted. Cool the agarose to 43 ° C and add the 10% aliquot (5 mL) prepared above. Immediately pour the agarose red cell mixture, for example, into a 7.5 ml plastic plate with a 1.5 mm deep and 6 x Θ cm well. Once the agarose has solidified, 0 2 mm holes are punched in it for serum samples.

b) Preparation of complementary film

Complement (guinea pig serum) is diluted 1: 3 with 0.05 M phosphate buffer pH 7.2 with 0.15 M NaCl.

A 6 x 6 cm membrane filter membrane made of a cellulose ester mixture with a pore size of 1.2 [mu] m is cut and placed in turn to float on top of the complement solution. After aspiration of complement (approx. 0.7 ml / piece), the membrane is placed on a steel tray, where it is transferred to a freeze dryer. The lyophilized films can be stored, for example, in a heat-sealed plastic bag in the refrigerator.

5 58566 c) Use 5 ul of serum is added to the well of the HIG plate and allowed to diffuse for 1 hour. The complement membrane is then placed on the surface of the agarose and the plate lid is closed again. Incubate for 1B hours at 37 ° C in an oven, then measure the haemolysis ring formed around the well, which easily separates from behind the plate against the white membrane. The diameter of the hemolysis ring is proportional to the antibody concentration in the sample.

Example 2 HIG plate rubella for antibody assay

The plates are prepared as in Example 1 a), but the influenza antigen is replaced by a rubella virus culture medium with a haemagglutination titre of 512 HAU / ml.

A cellulose triacetate film having a pore size of 1.2 μm can also be used to prepare the complement film, which is treated as in Example 1. This film absorbs about 0 to 5 ml of complement.

Used as described in Example 1 c).

Example 3 HIG plate for mumps antibody assay The HIG plate is prepared according to Example 1a], but the influenza antigen is replaced by an allantoic fluid containing mumps virus. with a haemagglutination titre of 640 HAU / ml.

A membrane filtration membrane made of a cellulose ester mixture with a pore size of 0.8 .mu.m can also be used as a complementary membrane.

This membrane absorbs about 0.65 ml of complement.

Used as described in Example 1 c).