DE2851645A1 - Immunochemical reagents with latex carrier - based on copolymer of styrene! with di:vinyl!-benzene or methacrylate! ( NL 31.5.79) - Google Patents

Abstract

The immunochemical reagents have a carrier comprising a styrene/divinylbenzene (S/DVB) copolymer latex, a hydroxylated or carboxylated S/DVB copolymer latex, or a styrene/methacrylate copolymer latex. The particle size of the carrier is pref. 0.1-1.0 (esp. 0.3) mu. Suitable latices are commercially available (e.g. "Estapor" products). The reagents, comprising an antigen or antibody adsorbed on the carrier, retain a constant sensitivity and specificity for long periods (e.g. 12-18 months).

Description

description

For measuring physiologically active substances in body fluids, such as blood, urine and the like., An immunochemical method is known which is the makes use of immunochemical activity of these substances.

When measuring an immunochemically reactive element, i. H. antigen or antibody, becomes an element (antigen or antibody) alone or an element in combination with the other (antibody or antigen) with one to be tested Fluid implemented. Due to the agglutination reaction or agglutination that occurs inhibition reaction, the immunochemically reactive element is measured. To the visual To facilitate confirmation of the reaction, small particles are usually considered Carrier used for antigen or antibody.

In the case of the agglutination reaction, the antibody (or antigen) adsorbed on the carrier composed of fine particles (this method is referred to as the awareness-raising process). Then the sensitized, out fine Particulate carrier mixed with a fluid to be tested. It reacts the antigen (or antibody) in the fluid with the antibody (or antigen), which is adsorbed on the finely divided carrier, resulting in a visible agglutination reaction conditional. There is no antigen (or antibody) to be measured in the tested fluid before, then there is no agglutination reaction. Is the amount of antigen (or Antibodies) extremely low, then the reaction does not appear visually as agglutination to recognize.

In the case of the agglutination inhibition reaction, the finely divided one becomes the first Carrier sensitized to the antigen (or antibody). Then the sensitized finely divided carriers together with the antibody (or antigen) with implemented in the fluid to be tested.

Although the method of carrying out the agglutination inhibition reaction is the same as that for carrying out the agglutination reaction the reaction result is the opposite. For example, there is no agglutination reaction, when the antigen (or antibody) to be measured is present in the fluid being tested.

The finely divided carriers are bentonite, kaolin, activated carbon, polystyrene latex as well as red blood cells. In addition to other materials were widely used Polystyrene latex is used as well as red blood cells.

A reagent for immunochemical measurement using such finely divided carrier is required to have one for the least amount of one tested Substance (antigen or antibody) to achieve sufficient sensitivity.

The reagent must be specific enough to deal exclusively with to react to the tested substance, it must also be able to demonstrate this sensitivity and maintain specificity during prolonged storage.

However, the conventional immunochemical reagents have Disadvantage that when the sensitivity of the measurement is increased, the reagent often shows a false positive reaction (e.g. when diagnosed with a Pregnancy the sample received from a non-pregnant woman a reaction, which indicates pregnancy, d. that is, no specific response present).

In addition, the sensitivity of conventional reagents can not be during can be retained for longer periods of storage.

The object of the invention is to provide a reagent for an immunochemical Measurement, as it is characterized by a measurement sensitivity that lasts even after prolonged storage and specificity. Furthermore, the invention is intended to provide an improved carrier for this reagent are available.

The invention relates to a reagent for an immunochemical measurement, which is characterized in that a styrene / divinylbenzene-co? olumerlatex (S. D. latex), a S. D. latex with hydroxyl or carboxyl groups introduced, or a styrene / methacrylate copolymer latex (S.M. latex) with a particle size of 0.1 - 1.0 F is used as a finely divided carrier.

The finely divided carrier used according to the invention is preferred a S.D. latex, S.M. latex, or a S.D. latex with hydroxyl or introduced Carboxyl groups, very particularly preferably a S. D. latex with introduced Hydroxyl groups.

The particle size of the finely divided carrier is preferably 0.8 - 1.0 p and especially about 0.3 p. If it is below 0.05, then it is The mass obtained in the reaction is too small to agglutinate with the bare Eye to recognize. If it is above 1.1, the reaction tends to be non-specific so that the carrier becomes unsuitable for use.

The above-described effect is demonstrated using a few test examples explained in more detail.

Experimental Example 1 A comparison is made between that according to the invention finely divided carrier and a conventional carrier carried out.

Using a S. D. latex, a S. D. latex with hydroxyl or carboxyl groups as well as an S.M. latex according to the present invention as well using a conventional polystyrene latex as a carrier become immunochemical Reagents according to that in the later following embodiment 1 described method produced.

These reagents are used at 2 to 80C during the period shown in Table I. specified period of time stored. After these periods of time have expired them for a change in their sensitivity as well as for the appearance of non-specific ones Responses tested by measuring human chorium gonadotrophormone (hCG). The sensitivity of the measurement is expressed as the degree of agglutination when measuring hCG of 1.0 iu / ml and 0.5 iu / ml were expressed while the non-specific Reaction as degree of agglutination when measuring the urine of a non-pregnant woman Woman who does not contain hCG is detected.

The ratings for the degree of agglutination are as follows: (++) visible strong agglutination (+) clearly visible agglutination (+) hardly visible agglutination (-) no visible agglutination as styrene / divinylbenzene copolymer latex (S. D. latex) a latex is used that is manufactured by Rhone Poulenc S.A. under the trademark 'Stapor PSl "lin is brought to trade.

Also introduced as styrene / divinylbenzene copolymer latex (S.D. latex) Hydroxyl groups is used a latex made by the same company under the trademark "Estapor PSI 156" is put on the market.

Also introduced as styrene / divinylbenzene copolymer latex (S.D. latex) Carboxyl groups, a latex is used which is sold by the company mentioned under the Under the trademark "Estapor PSI 162".

The styrene / methacrylate copolymer latex used is a latex which is commercially available from the company mentioned under the trademark "Estapor PSI 153". Table I. W + 1month 5nate6nate 9 D- + 12th months 18 lt I ------- - 1.0 .iu / \. ++. ++. . + i '.. .. f: $ SD atex .0.5 / mZ. . +. +. . +. . +. X SfrSt- 4 X - r; l ++. . ++, II latex .0.5 i4m. . + +. +. +. +. iazi- - - Invention groups 0 .iu / m% - - - - ~ ~ 0 + t + ~ + ~. + +. + ~ +, l SD latex 0.5: Lu / + + + + t 1-l: Stl $ - - I 0. iu /. -. . -,. -. . -. {: ~. ~ ~~. ~, So t latex -f- +, + + + + + .0. .i4. - - - 1.0 t * ++ * * * 4I ~ Polystyrene latex ffttt fleagens l 3 from left:: 1: + 4 E oo, oo õ oooooo 'õ o õ o ['lili ta 2 ffi Q | i a WX 'M' The SD latex, SD latex with carboxyl groups as well as the SM latex show a slight change in the measurement sensitivity and specificity after long storage, while the SD latex with hydroxyl groups undergoes no change and proves to be the best material. The conventional polystyrene latex suffers an early change in measurement sensitivity and specificity.

Experimental Example 2 Human placental lactogen (hPL) is called to testing substance used. According to the manner described in Example 1 is a Comparison carried out between the carrier according to the invention and the conventional one. The non-specific reaction is determined by the degree of agglutination in the Measurement of serum from a non-pregnant woman that does not contain hPL, expressed using the same ratings as in Example 1.

The results are shown in Table II.

Table II 1 + 't ~. + - 41 + 'months 3rd months 6 months 9 months 12 - -] I Trer. ! <.; ng / tn .4+. ---- .4+ I. SD Latex iOng / n +. +. + +. . +. i 0 ng / tn -. .-. .- .-. - S '4+. 4+ .4+ .4+ 4+ 4+ cu. i 'F ~ ~ t + l.0.ng/n + ore groups 0 ng / tn that it If: 20 ng / m * 4+ .4+ 4+. .4+ 4+ 8. D. latex t + ~~ ~~ ~ ~ l. +. +, + l: cv Q n'nZ - - - - - - 2.0 ng / m + 1. t +, l 4+ 7- ~ ~. l 0 ng / tn 1. + + l + 2.0 tt 4 * .4+ 4+ 4+ 4+. 4+ ffi polystyrene- .l, 0., ng / tn 6 + + + 4+ 4+ Reagent latex 4 / a, F aoar: t F aaoa F- SlS NH «H ~ cs H ~« H cs H $ XS o ', W z 3 The SD latex and the SM latex suffer a slight change in the measurement sensitivity and specificity after prolonged storage, while the SD latex with hydroxyl or carboxyl groups does not experience any change and has proven to be excellent.

The conventional polystyrene latex undergoes an early change in these Properties.

Experimental Example 3 A comparison of particle size is used of the S. D. latex with hydroxyl groups. Reagents are as in the case of the embodiment 3 produced.

During the periods of time given in Table III, the Reagents stored at 37 "C. After each period of time they will respond to a change the sensitivity and the occurrence of a non-specific reaction Measurement of hPL tested in a manner similar to Example 2.

The ratings of the degree of agglutination are the same as in FIG Example 1. The results are shown in Table III.

Table III I \ raqernn4st 1 1'nat 2 months 3 months 4 6 iate Partial- hPLN 2 llvknat] ubnate 2 size 1 I. 2 = + + + 0.01-0.05'11 1 + + + + . 2 OX e H 4+ 4+ 0.27-D.33, 1 is 4+ ++ 4+ 4+ 1 ° - - - - - i I. 2 ++ -it 4+ 4 + 1 0.4 1 0.6p 1 ++ 4+ 4+ i 4+ 1 . I. o - - - - + 2 ++ + F ++ ++ 4+ 1.1 - 2.0y 1 4+ ++ + t ++ 4+ O - -, + + 4+ With a particle size of 0.01-0.05 p, the agglutination is too small to be seen with the naked eye. At 1.1 - 2.0 ß the non-specific reaction is determined. At 0.27-0.6 µ none of the above occurs. In particular, good results are obtained at 0.27-0.33 p.

As set out in Examples 1 to 3, those are according to the invention immunochemical reagents using S. D. latex, S. D. latex with hydroxyl or carboxyl groups as well as S.M. latex with a particle size of 0.1-1.0 p as Carriers decidedly better than traditional reagents in which polystyrene latex is used as a carrier.

The antigen is used to prepare the reagent according to the invention or the antibody dissolved in a buffer solution; like a glycine-saline buffer, a phosphate-saline buffer, a borate-saline buffer or the like. With the above mentioned copolymer latex mixed and optionally heated. After this awareness process the latex is separated by centrifugation and again in the same buffer suspended.

To further improve the reagent according to the invention with respect to A protein that does not take part in this immunoreaction may affect storage stability (for example bovine serum albumin, egg albumin or cytochrome C) as a stabilizer be used.

The following examples illustrate embodiments of the invention.

Example 1 From the anti-hCG serum obtained from a rabbit that has been immunized against hCG becomes a d-globulin fraction Purified by salting out with sodium sulfate and then in 0.75% glycine salt buffer (pH 8.2) dissolved at a concentration of 0.06%.

20 ml of the solution obtained are mixed with 20 ml of a 10% suspension from S. D. latex with introduced hydroxyl groups (Estapor PSI 156, trademark der Rhone Poulenc S.A., France) with a particle size of approximately 0.3 ji mixed and heated to 370C over a period of 10 minutes.

The anti-hCG-Y globulin is adsorbed on the carrier. To centrifugal separation, the precipitate is again in 70 ml of the glycine salt buffer suspended to give a reagent. The measurement of the fluid to be tested using this reagent is carried out through an agglutination reaction so that the reagent can be brought into direct reaction with the fluid under test got to.

Example 2 hCG is in 1/15 M phosphate salt buffer (pH 6.4) in a Concentration of 0.03% dissolved. 50 ml of the resulting solution with 50 ml a 10% suspension of S. D. latex with introduced hydroxyl groups (Estapor PSI 156, trademark of Rhone Poulenc S.A., France) and set to 370C heated for a period of 30 minutes. In doing so, the hCG is attached to the wearer adsorbed. After a centrifuge separation, the precipitate is again in 175 ml of the phosphate salt buffer is suspended to obtain a reagent. The agglutination inhibition reaction if the liquid to be tested is measured using this reagent, it is so that it is necessary to combine the reagent with the liquid to be tested to react with the anti-hCG antibody.

Example 3 From the anti-hPL antiserum obtained from a rabbit which immunized against hPL becomes a t-globulin fraction by salting out with sodium sulfate and dissolving in 1/15 m borate salt buffer (pH 7.0) Purified at a concentration of 0.06%.

20 ml of the solution obtained are mixed with 20 ml of a 10% suspension from S. D. latex with introduced carboxyl groups (Estapor PSI 162, trademark der Rhone Poulenc S.A., France) with a particle size of about 0.3 p mixed and heated to 560C over a period of 20 minutes. It will the anti-hPL-t globulin adsorbs to the carrier. After a centrifuge separation the precipitate is resuspended in 70 ml of the borate salt buffer solution, wherein a reagent is obtained. An agglutination reaction is used to measure the too testing liquid using this reagent applied so that it is necessary is, the reagent to react directly with that to be tested “Bring liquid.

Example 4 hPL is in a glycine salt buffer solution (pH 8.2) in a Concentration of 0.05% dissolved. 50 ml of the resulting solution with 50 ml a 10% suspension of S. D.

Latex (particle size approximately 0.3 µm) with introduced carboxyl groups (Estapor PSI 162, trademark of Rhone Poulenc S.A., France) mixed and heated to 560C over a period of 30 minutes. The hPL will be an adsorbed on the carrier. After centrifugal separation, the precipitate becomes again suspended in 175 ml of the glycine salt buffer solution to obtain a reagent. An agglutination inhibition reaction is used to measure the liquid to be tested using this reagent is applied so that it is necessary to use the reagent and the anti-hPL antibody with the liquid to be tested for conversion bring.

Example 5 From the antiestriol serum obtained from a Xaninchen, the immunized against estriol-16.17 <lisuccinyl-BSA (abbreviated as estriol) is obtained, an r-globulin fraction is obtained by salting out with sodium sulfate purified and in 1/15 M phosphate salt buffer (pH 6.4) at a concentration of 0.08 % resolved. 5 ml of the solution obtained are with a 10% suspension of S. D. latex (particle size approximately 0.3 ii) (Estapor PSI 152, trademark of Rhone Poulenc S.A., France) and heated to-450C for a period of 60 Minutes heated. The anti-estriol-globulin is adsorbed on the carrier. After a centrifuge separation, the precipitate is again in 175 ml of phosphate salt buffer suspended to obtain a reagent. The agglutination inhibition reaction will to measure the liquid to be tested using this Reagenses are used, so it is necessary to mix the reagent with the liquid to be tested to mix and then add an estriol solution to the mixture to react.

Claims (2)

Reagent for an immunochemical measurement. Claims reagent for zinc immunochemical measurement, characterized in that its carrier consists of a Styrene / divinylbenzene copolymer latex, a styrene / divinylbenzene copolymer latex with introduced hydroxyl and carboxyl groups or from a styrene methacrylate copolymer latex consists. 2. Reagent according to claim 1, characterized in that the particle size of the carrier is 0.1 - 1.0 µ.