IL34544A - Serological diagnostic preparations consisting of antigen-antibody system and a method for producing them - Google Patents

Description

Serological diagnostic preparations consisting of antigen-antibody systems and a method for producing them LIOENCIA TALALMAOTOKAT ERTEKESITO VALLALAT C: 32820 The invention relates to a method for producing serological diagnostic preparations consisting of antigen-antibody systems, apt to be stored and ready for use in slide test reactions.

Serological methods based on antigen-antibody reactions permitting to indicate the presence of various proteins aa well as normal or pathological antibodies have already been known. Some examples of such methods are the following: Indication by means of serological diagnostic preparation of the growth hormone (P. Weieer: Eine Methodik der Be-stimmu g hypophysarer Gonado ropine mittels Hamagglutinationa Hemmungsreaktion, Zbl. Gyn¾k., 87 /1965/ » H. 46 . S. I569-157 ) ; Of the Chorlonogonadotropic Hormone appearing in the Urine of Pregnant Women (Joyce L. Bell: Comparative Study of Immunological Tests for Pregnancy Diagnosis, Journal of Clinical Pathology, Vol . 22 , p . 79 /1969/ ) ; of Pathological Antibodies in Certain Thyroid . Diseases (Martti Oka, Pentti SeppSla and Leena Mikkonen: Latex Slide Test in Thyroid Diseases. Acta Medica Scandinavica, Vol. 179 » fasc. 2 A966/ ) ; Of the Pathological Protein in the Serum of Bheumati Patients (Singer, J. ., Plotz , Ch. M.: Am. J. Med. 21 , 888 /1956/ , Tonder, 0. : Studies on the Mechanism of the Waaler-Rose Test, Norwegian University Press, Bergen, Oslo, 1962 ) ; Of the Pathological Antibodies in Infectious Mono-nucleosis (Beatrice Saemann-Naville: Mononucleose-Schnell- test, Objekttrager-Agglutination formolisierter P erdeerythro c ten. - Schweiz. med. Wsch. 6 , Nr. 45(· 1649-1651 /1960/ , Hoff. G. and Bauer S.: A New Rapid Slide Test for Infectious Mononucleosis J. Amer. med. Ass. JAMA 194, 351 /1965/ ) etc.

In some of the above mentioned test methods the antigen or antibody required for the test is bound to lyophilized sheep er hrocytes, in others to synthetic materials, e.g. particles of polystyrene-latex. Most of these serological methods require voluminous laboratory facilities, but even the simplest ones are rendered more complicated by the fact that, in addition to the serum or urine to be tested, lyophilized or fluid, preserved reagents are also needed. The dissolution of the reagents, as well as their administration from the appropriate storage vessels renders otherwise simple methods complicated, so that skilled laboratory personnel is required for carrying them out.

It is the object of the invention to eliminate the described deficiencies of the immunological methods based on erythrocyte or latex agglutination, and to provide a method permitting to apply the necessar reagents ready for use onto the test slide by evaporation. Thereby one can achieve the major advantage that merely the serum or urine to be tested is required for various serological diagnostical tests; moreover, these serological methods can be considerably simplified with the aid of the method according to the invention.

The method according to the invention consists in first mixing the complexes of pre-treated sheep erythrocytes or latex-antigen and antibody-complexes with the aqueous solution of natural, semisynthetic or synthetic polymers or synthetic resins, which are compatible with the biological substance, apt to form a film after drying, and apt to be dissolved upon the addition of water, which may contain a bactericide agent, if desired, applying said solution, onto the test slide, drying it on the test alide, whereby a reagent ready for use, apt to be stored Without decrease of activity is obtained. After the drying •r evaporation the reagents are covered with a protective coating preventing the scaling off, dissolution or decomposition, whereby the reagents are preserved.

As film-forming synthetic or semi-synthetic, water-Boluble polymers one can use polyvinyl alcohol, polyvinyl pyrrolidon^ polyvinyl methylether, carboxy methyl cellulose, or methyl cellulose and other water-30luble linear polymers. Aa film-forming aynthetic resin one can use products on a urea-formaldehyde, melamine-formadehyde, casein- ormaldehyde, or alk d resin basis, which are suitable for satisfying the requirements of water solubility, and compatibility with the biological substances. As film-forming natural polymers the use of gelatine or glue i3 suggested, for example. As bactericide agent added to the aqueous solution of the film forming substance one can use e.g. merthiolate (sodium ethyl-mercury-(ll)-thiosalycilate) or 30dium thiazide.

The test slide may consist of metal, glass, mirror or a plastic material, which may be coated, if desired, with varnish. The varnish-coated slides are preferably allowed to dr during 24 hours, and the aqueous solutioi of the antigen-antibody complexes with film-formin eubatanoes are applied only thereafter onto the surface of the slide which has been dried after the application of the varnia .

The preparation produced according to the invention and dried on the slide is excellently suited for carrying out diagnostioal procedures based on direct and passive haeaagglutina ion, wherein micro-amounts dried on the. alide and apt to be atored for long periods of time are used as reagent. A considerable advantage of the process according to the invention is, in addition to the substantial economies to be realized, that the reagents may be used in an extremel rapid and simple manner directly in the consultation room or at the bedside of the patient, after drying them to ready-for-uae state in a much, simpler manner than hitherto without the need for laboratory equipment, as a means for medical diagnosis. The test method according to the invention has the further advantage that when the reaction between diagnostic preparation plus serum or urine has been completed, the protective coat ia re-formed on the test substance in a reversible manner, so that is is possible to store the teat result fixed on the alide.

All one has to do for using the reagent obtained according to the invention for a diagnostic test is to apply on it a drop of the test material, whereupon the water content of the test material will dissolve the protective coating and the desired reaction takes place, The following Examples 3erve to illustrate the method according to the invention more particularly, without, however, limiting the scope of the invention to what has been described in the Examples.

Example 1 Preparation of a th reoidea-anti en serological diagnostic preparation,, To a suspension of th reoidea-antigen bound to poly-stirene-Latex particles prepared by a known- method. (TA-test = Hyland Laboratories, Los Angeles, Calif. USA) a solution of polyvinyl alcohol in a volume equivalent to 50 # of the suspension is added. It is advisable to use one of the commercial polyvinyl alcohols, such as Yinavilol 4-88 (Mon-teca ini), or Rhodovilel 4/125/P (Rhqne-Poulenc ) . The con- ■ centration of the polyvinyl alcohols in water or in a solution of physiological sodium chloride is abt. 18 to which merthiolate or sodium thiazide is added as bactericide agent diluted in a ratio of 1:5000.

One drop (0.05 ml) of the suspension so prepared is applied onto a metal 3lide coated with varnish, and the water content of the drop is evaporated* If required, one can use a fan to promote evaporation. The main requirement for the r quality of the metal alide is to permit a ver clear indication and evaluation of the result o/f the diagnostical / test. Empirical results have shown that it is preferable to use a metal slide prepared by vacuum casting from a annealed, nickel-plated and magnetized metal of high purity and low gas content, having the following composition: G 0.06 # Si 0.04 $ P .0.22 % S 0.02 $ Mn 0.2 ' Cu 0.03 % Al 0.03 # Fe 99.35 # After evaporation a protective film is formed which will preserve the reagent for a considerable period of time in a state ready for use. Instead of a polyvinyl alcohol of the above described quality one can also use methyl cellulose for preparing the diagnostic composition.

The teat for thyreoid antibodies may be carried out in the followin .manner uaing the reagent according to the invention: 0.05 m of serum to be tested are applied onto the antigen dried on the varnished metal slide, whereupon the polyvinyl alcohol coating on the reagent ia dissolved.

After waiting for 30 seconds the substance ia stirred, then the alide is gently rocked, whereafter the results of the agglutination test can be evaluated in the usual manner after the lapae of minutes.

Example 2 Gamma globuline- antigen serological diagnostic preparation.

To a suspension of gamma globuline-antigen bound to polystyrene-latex prepared in a known manner (manufacturera : Human Oltoanyagtermelo es Kutato Intezet, Budapest, HUMAN Institute for the Research and Production of Immunological Substances) an aqueous aolution of methyl cellulose ' is added in an amount corresponding to 50 % of the auapenaidn. The methyl cellulose is employed in a # solution. If desired, a bactericide agent diluted with water in a 1: 5000 ratio ia added to the suspension. The suspension is applied onto a varniBh-coated metal alide as deacribed in Example 1 and dried.

The testin of the rheumatic factor is effected by meana of the gamma globuline antigen preparation as described in Example 1, with the difference, however, that the serum to be tested is previously diluted to a 1: 10 ratio by means of a boric acid buffer having a pH 8 , 2 .

Example 3 Gamm globuline-antigen serological diagnostic pre- paration.

To a suspension of gamma globuline-antigen bound to sheep erythrocyte preserved with a 3 % formaldehyde solution, prepared in a known manner, a solution of polyvinyl alcohol corresponding in volume to 50 a of the suspension is added. The concentration of the polyvinylalcohol is identical with that used in Example 1.

The suspension so prepared is applied onto a varnish coated metal slide according to Example 1 and dried. Instead of a metal slide, the suspension may als^o be applied on a mirror surface.

The test is carried out in respect of the rheumatic factor using the gamma globuline antigen preparation in the manner described in Example 2.

Example 4 Chorion gonadotropic hormone antigen and antibody serological diagnostic preparation.

To a suspension of chorion gonadotropic hormone antigen bound to polystyrene-latex particles ( Gravindex-Ortho Diagnostics, Raritan, New Jersey, USA; Pregnosticon- Planotest = Organon, Oss, Netherlands; Prepurex = Burroughs Wellcome and Co., London, England; DAP-Test = Wampole Lab. Stamford, Conn., USA) a polyvinyl alcohol solution, of a concentration as described in Example 1, is added in a volume corresponding to 0 % of the suspension. Simultaneously with the aaid antigen a solution is prepared from the immunserum containing the anti-chorion gonadotropic hormone antibodies diluted by means of the above said polyvinylalcohol solution, to such an extent that its amount employed in the reaction be neutralized by 1.5 - 2 I.U.HGG per ml. urine. 0.05 ml. portions of the antigen suspension prepared as described and of the solution of immuneserum each are separately applied onto the varnished metal slide and dried as described in Example 1.

Pregnancy tests may be carried out by means of the preparation described according to the invention and suitable, among other, as pregnancy indicator in the following manner: Onto the varnished metal slide carrying dried antigen and immuneserum 0.2 ml portions each of urine are applied, whereupon the polyvinyl alcohol film coating the reagents is dissolved. After waiting for 30 seconds the drops are stirred (blended): after having waited for 4 more minutes the metal slide is gently rocked, whereafter the results of the agglutination test can be evaluated within one minute's time.

Example 5 Chorion gonadotropic hormone antigen and antibody sero-logical diagnostic preparation.

To a chorion gonadotropic antigen, conserved with a 3 % formaline solution prepared in a known manner and, if desireds bound to sheep erythrocytes, one adds a solution of polyvinyl alcohol concentrated as described in Example 1» in an amount corresponding to 5 # of the volume of the suspension* One proceeds further as described in Example 4.

Pregnancy tests may be carried o'ut by means of the test substance prepared according to the invention and suitable, among others, as pregnancy indicator, in the following manner: The procedure described in any of the Examples may be performed, using as film forming substance gelatine or glue, also polyvinylpyrrolidone, or carboxy methyl cellulose, as well as precondensates based on synthetic resins such as urea-formaldehyde, melamiiie-fo maldehyde etc.

Claims (15)

'ν - 11 - WHAT WE CLAIM IS:

1.. A method for preparing serological diagnostic preparations consisting of antigen-antibody systems, apt to be stored and ready for use in slide test reactions, in which the complexes containing pre-treated 3.heep erythrocytes or latex-antigen and antibody complexes, or the suspensions of said complexes are mixed with the aqueous solution of natural, semi-synthetic or synthetic polymers or synthetic resins, which is compatible with the biological substance, apt to form a film after drying and apt to be dissolved upon the addition of water, containing a bactericide agent, if desired, applied onto the slide and dried.

2. A method according to claim 1, in which polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methyl ether, carboxy methyl cellulose, methyl cellulose or some other linear polymer is employed as film-forming synthetic or semi-synthetic , water-soluble polymer.

3. A method according to claim Ί, in which the film-forming synthetic resin is an aqueous solution of a composition on an urea formaldehyde, melamine formaldehyde, casein formaldehyde or alkyd resin basis,

4. A method according to claim 1, in which the film-forming natural polymer employed as film-forming natural polymer is gelatine or glue .

5. A method according to claim 1, in which merthiolat (sodium ethyl mercury-ll~thi03alycilate) or sodium thiazide is employed as bactericide agent.

6. A method accordin to claim 1, in which a metallic, glass, mirror or plastic plate is used as test slide.

7. A method according to claim 1» in which- a metallic, glass, mirror or plastic plate coated with varnish ia employed as teat slide.

8. A method for producing serological diagnostic preparations consisting of antigen antibody systems* substantially as hereinbefore described.

9. A serological diagnostic preparation containing as biological substance antigen-antibody systems ready for use in slide reactions comprising complexes of pretreated sheep erythrocytes or latex-antigen and latex-antibody systems in combination with aqueous solutions of natural, semisynthetic or synthetic polymers or synthetic resins, which is compatible with the biological substance, apt to form a film after drying -and to be dissolved after the . addition of water, comprising further a bactericide agent if desired, applied onto a slide and dried,

10. A serological diagnojtic preparation in accordance with claim 9» comprising as film-forming synthetic or semi-synthetic polymer polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl methyl ether, carboxy methyl cellulose, methyl cellulose or some other polymer of linear structure.

11. A serological diagnostic preparation in accordance with claim 9» comprising as film-forming synthetic resin an urea formaldehyde, melamine formaldehyde, casein formaldehyde or an alkyd resin.

12. A serological diagnostic preparation in accordance with claim 9» comprising as film-forming natural polymer gelatine or glue.

13. A serological diagnostic preparation in accordance with claim 9 » comprising as bactericide agent merthiolate (sodium ethyl mercury-thlosaiicylate) or sodium thiazide,

14. A serological diagnostic preparation in accordance with claim 9 » wherein the test slide ia a metal, glass, mirror or .plastic plate.

15. . A serological diagnostic preparation in accordance with claim 9 , wherein the test slide ia a metal .plate coated with a synthetic varnish.