DD249768A5 - METHOD FOR DETERMINING A PARTNER OF AN IMMUNE REACTION - Google Patents

Abstract

A method and a reagent for the determination of a partner in an immunological reaction by the immunoassay principle at a temperature between 15 and 40 DEG C is described, where one of the reactants is present in solid phase, and a surface-active agent with an HLB above 20 is added. <IMAGE>

Description

I ^3/3

H (OCH ₀ CH ₉₎ (OCH _{CH-0), (0} CH -CHO) (CH ₀ CH ₀ O) "~ H

N-CH-CH-N

H (CCH ₀ CH ₀₎ (OCH-CH ₂₎ (CH ₀ -CHO) (CH ₀ CH ₀ O) H

"YLL ^Y '*"

in which χ has a value between 10 and 50, preferably between 15 and 30 and y has a value between 30 and 150, preferably between 40 and 120.

19. A reagent according to claim 18, characterized in that the compound has a proportion of Poiyoxyethylengruppen from 50 to 90%.

20. A reagent according to any one of the preceding claims, characterized in that it also contains a system for detecting an enzyme activity used as a label.

See 1 page table

Field of application of the invention

The invention relates to a method for determining a partner of an immune reaction according to the immunoassay principle at a temperature between 15 and 40 ⁰ C, wherein one of the reactants is in the solid phase.

Characteristic of the known state of the art

Methods for immunological determination are widespread. There are both homogeneous and heterogeneous phase processes. In the heterogeneous phase embodiment, one of the reactants is bound to a support. To carry out the immuno determination in heterogeneous phase, various process variants are now known, e.g. the sandwich method, the indirect method and the competition method. In the sandwich method, an antibody is bound to the carrier, the test solution is added, whereby the specific antigen contained in the test solution is bound to the antibody. Then, a labeled specific antibody for the antigen-antibody complex, or for parts of the complex, is added, which then binds to the complex. The labeled antibody can then be used to calculate the amount of antigen. In the indirect method, an antigen is bound to the carrier material. Test solution is added thereto, the antibody specific for the adsorbed antigen contained in the test solution reacting with the antigen. Upon addition of a labeled antiglobulin, the antiglobulin binds to the antigen-antibody complex and the amount of unknown antibody in the test serum can again be determined via the labeled antiglobulin. In the competition method, one of the bound partners of the immune reaction is bound to the support material. You then give a solution; which contains both the other part of the immune reaction present in an unknown amount, as well as a known amount of labeled other partner of the immune reaction. Both partners, the labeled and the unlabeled, compete for the binding sites of the carrier-bound partner of the immune response. For a second sample add a standard solution containing only marked partners. Mankannnun by determining the labeled partners in standard and sample from the difference calculate the amount of unknown reactant.

Further possibilities for the determination arise in the case of an at least bivalent antigen with the aid of three receptors, which may be antibodies or antibody fragments. When performing an immunoassay with three heterogeneous phase receptors, one of the three receptors is always insoluble, while the other two are soluble, with one of the two soluble receptors labeled while the other is not labeled. The insoluble receptor is then directed against the unlabeled soluble receptor. To carry out the determination, various process variants are possible, both single-stage and multi-stage, in which the addition of the individual receptors can be varied.

Another variant of the method consists in adding the antigen to be determined in the sample in the first step simultaneously with a soluble, unlabelled receptor and a soluble labeled receptor in solution and then the resulting soluble sandwich complex in a second step by binding to an insoluble receptor insoluble. To mark the reactants, various possibilities are known. Thus, one of the reactants can be radioactively labeled, in which case the measured radioactivity allows a calculation of the reactant to be determined. Furthermore, it can be labeled with an enzyme. It is then after carrying out the usual steps of immunoassay, a substrate for the enzyme, which is used as a label added and then calculated by its reaction, the concentration of the reactant to be determined. Another possibility is the labeling with a fluorescent substance, wherein the fluorescence can then be determined directly or with a dye, the evaluation is carried out spectro-or photometrically.

Immunoassays are growing in importance because they have a very high sensitivity and with the help of this method substances can be detected up to the Picog ramm area. Immune determinations in a heterogeneous phase, however, are falsified to varying degrees by non-analyte-specific interferences, so-called non-specific disorders, which have already been referred to as "matrix effect", "background" and "unspecific binding" These unspecific disorders may be due in part to unknown components of the sample, and the susceptibility of the immunoassay may be impaired by non-specific interactions of the interfering factors with the immunoreagents or analyte used. That non-specific interference in immunological determinations in plasma is more pronounced than in immunological determinations in serum Therefore, the recovery in determinations in the plasma is much lower than in the serum, while in competitive tests the Wieder is too high in the plasma. The cause of this disorder was because of the clotting proteins, which are too high in the plasma. The cause of this disorder was therefore attributed to the coagulation proteins, which are still present in the plasma, but not in the serum. The clotting proteins attach themselves to the surface of the carrier material and thus hinder the antigen-antibody reaction or the antigen-conjugate reaction.

It has hitherto been known to add certain detergents to the incubation environment to reduce nonspecific interactions in immunological determinations. However, a disadvantage of this method is that also the desired, i. the analyte-specific antigen-antibody reaction is affected in its extent, which in turn results in a decrease in sensitivity. In addition, in the known application of detergents there has been the problem that in solid phase immunoassays, the surfactant could displace the adsorptively bound molecule from the solid surface. The function of Immunoassays.wird thereby impaired.

Thus, it has been known that detergents such as Tween 20 produce plasma binding in the incubation buffer. Detergents of this type, however, have the disadvantage that, in concentrations higher than 0.01%, they detach the reactant bound to the support material, which in turn leads to a negative influence on the immunoassay.

Object of the invention

The object of the invention is to provide an improved method for the determination of a partner of an immune reaction in which non-analyte-specific and therefore undesirable non-specific disorders are suppressed in heterogeneous phase immunoassays. Further, it is an object to find such a method in which neither the reaction between antibody and antigen / hapten is impaired, nor adsorptively bound reactants are desorbed from the carrier, nor when enzyme-labeled immunoreagents are used as the label that reduces enzymatic activity becomes. Another object of the invention is also an acceleration of the immune response and thus a shortening of the analysis time.

Explanation of the essence of the invention

The invention has for its object to provide reaction conditions with which the objectives of the invention are achieved.

According to the invention, a method for determining a partner of an immune reaction according to the immunoassay principle is provided at a temperature between 15 and 40 ° C, wherein one of the reactants is in the solid phase. The method according to the invention is characterized in that a surface-active agent with an HLB value of more than 20 is added to the incubation medium of the immune reaction.

It has surprisingly been found that detergents with an HLB value greater than 20 can suppress undesired reactions in immunological determinations and at the same time neither impair the reaction between antibody and antigen or hapten nor cause desorption of the adsorptively bound reactants In addition, the addition of the agent according to the invention leads to an acceleration of the immune reaction.

Surfactants based on alkylene oxides having 2 to 4 carbon atoms, which may optionally contain as central molecule an alkylene diamine having 2 to 4 carbon atoms, are preferably used as surfactants with an HLB value above 20 nonionic block copolymers.

Preferred block copolymers are compounds of the general formula I ₍

HO (CH ₂ CH ₂ O) _a (CCHCH ₂ O) _b (CH ₂ CH ₂ O) _a H, OH ₃

in which a can assume a value between 30 and 150 and particularly preferred between 60 and 100 and b a value between 15 and 60 particularly preferentially between 25 and 40. These compounds are also called poloxamers. A trade name is Pluronic ^R , for example.

In the group of poloxamers, compounds which have a proportion of hydrophilic polyoxyethylene in the total molecule of 60 to 90% have proven particularly suitable.

Preference is furthermore given to block copolymers of the general formula II ₍

CH ₃ CH ₃

H (OCH CH _{0 0) (0} OC) (CH ₀ CHO) (CH CH O) H

ζ ζ γ ζ x-. / ^ * ** ^J

N-CH ₀ -CH ₀ -N

H (OCH CH _{0 0)!} (OC ₀₎ ^ ₍₀ CH CHO) (CH CH ₀ O) H

2 2 y 2 χ X Z \ ZZ γ

I CH ₃ CH ₃

which are referred to as poloxamines, in which χ the values of 10 to 50, more preferably 15 to 30 and y can take the values 30 to 150, particularly preferably 40 to 120 used. A trade name for such poloxamines is z. B. Tetronic ^R.

Among the above-mentioned surfactants, those whose HLB value is between 24 and 30 are most preferred.

The surfactant is used in an amount of 0.1 to 5%, based on the weight of the entire reaction mixture.

More preferably, in the process of the present invention, poloxamines having a proportion of hydrophilic polyoxyethylene groups of 50 to 90% are used.

The addition of the surfactant according to the invention brings both advantages in addition to plasma and in addition to serum. In both cases, there is an acceleration of the reaction as well as a prevention of nonspecific disturbances, in particular in the case of the particularly sensitive determinations in the plasma.

The inventive method is applicable to all types of plasma. Thus, it can be used both for plasma, which has been added for stabilization with EDTA, as well as for plasma, which has been treated with heparin or citrate.

The addition of the surfactant may be made to any solution used for the determination. In a two-stage process, particularly good results are obtained when the detergent is added to both buffers, ie for each of the two stages. For this purpose, the solid phase carrying the one reactant, first treated with a first incubation solution, the buffer and additives, such as. B. IgG contains. It is then washed and then added in a second stage, the conjugate solution and incubated again. The conjugate solution contains conjugate and buffer as well as optionally other customary additives. The conjugate itself consists of an immunological reaction partner for the substance to be determined or for the solid phase partner, eg. Example, an antibody against the antigen to be determined in the labeled state. The sample solution is expediently added to the incubation buffer. However, good results are also achieved if the addition of the surface-active substance to the incubation solution or to the conjugate solution takes place.

The addition of the surfactant to the sample solution itself is less favorable. In the finished incubation solution must indeed be present a sufficiently high concentration. Now, if the agent is added via the sample solution, which is used in only a very small amount, the concentration of surfactant therein must be very high. At very high concentrations, however, undesirable effects could occur.

To carry out the method according to the invention, a reagent is used which contains mobilized and immobilized partners of the immune reaction and which is characterized in that it contains a surface-active agent with an HLB value of more than 20. This reagent may also contain conventional ingredients. Suitably it also contains buffer substances, for example phosphate buffer, citrate buffer, borate buffer and the like and / or bovine serum albumin and / or preservatives. If an enzyme is used as a label in the immune reaction, the reagent also contains a system for detecting the enzyme activity.

Preferably, the surfactant reagent comprises a nonionic block copolymer based on alkylene oxides having 2 to 4 carbon atoms, optionally containing an alkylene diamine having 2 to 4 carbon atoms as the central molecule.

With particular preference it contains as surface-active agent a compound of general formula I or II having the ranges indicated for the indices a, b, χ and y. Particularly preferably, surfactants having an HLB value between 26 and 30 are used. The surfactant is contained in the reagent in a concentration of 0.1 to 5%.

With the method and reagent according to the invention it is possible to improve the recovery in immunoassays in a heterogeneous phase. The addition of the agent used according to the invention further accelerates the immune reaction.

In addition, undesirable side reactions are suppressed, in particular the non-specific addition of conjugate to the solid phase, without there being any increased detachment of the bound reactants.

embodiment

The invention is explained in more detail below with reference to some examples.

example 1

Plastic tubes with antibody to TSH labeled with 125 I by the chloramine T method were loaded into phosphate buffer at 40 mmole / L at pH 7.4. The concentration was 2 / itg / ml. The loading was with a solution of 0.9% sodium chloride and 0.3% bovine serum albumin, type II. The loading volume was 1.5 ml. The incubation time for the loading was 22 hours. After that, with 1.8m! reloading a post-load solution for V2 hours. The tubes were dried for 24 hours with the tube up. The slides were then treated with detergent. In these tubes, the desorption of the antigen or the antigen-antibody complex was then measured. The results are shown in the following Table I.

Table 1

detergent concentration desorption antigen Wall AK 1.2% buffer solution - 3.2% 74% Tween 20 (V) 1% 58% 65% Tween 20 (V) 0.1% 50% 45% Tween 20 (V) 0.01% 30% 85% Triton X100 (V) 1% 61% 89% Triton X100 (V) 0.1% 62% 78% Triton X100 (V) 0.01% 53% 85% PluronicL64 (V) 1% 64% 39% PluronicL64 (V) 0.1% 21% 10% PluronicL64 (V> 0.01% 6% 7% PluronicF68 (E) 1% 9% 6% PluronicF68 (E) 0.1% 5% 5% PluronicF68 (E) 0.01% 5% - Tetronic707 (E) 1% 5% - Tetronic707 (E) 0.1% 4% - Tetronic707 (E) 0.01% 4%

V = comparison, surfactant with HLB <20

E = surface-active agent according to the invention with HLB>

Example 2

The recovery in the TSH test using different detergents was checked. Plasma was used with EDTA, with heparin or with citrate. At the same time, the TSH test was carried out with serum. In this test the recovery was 100%. The values obtained for various detergents are shown in the following table:

Table 2

detergent

Detergent concentration Incubation buffer Conjugate buffer

recovery

EDTA

plasma

Heparin plasma

Citrate plasma

without- - TritoxinX405 0.1% Tween 20 0.005% PluronicF68 1% PluronicF68 1% PluronicF87 1% PluronicF87 1% PluronicF88 1% PluronicF88 1% PluronicF88 1% PluronicF98 1 % PluronicF98 - PluronicF127 1% PluronicF127 1% PluronicF38 1% PluronicF108 1% Tetronic 707 1% Tetronic 707 1% Tetronic 707 - Tetronic 908 1% Tetronic 908 1% Tetronic 909 1% Tetronic 909 1% Tetronic 1107 1% Tetronic 1107 1% Tetronic 1307 1% Tetronic 1307 1%

1% 1% 1%

1% 2%

1% 1%

1% 2%

1% 1% 1% 1%

40-70 40-70 - 66% 81% - 88% 89% 89% 98% 97% 93% 105% 98% 99% 99% 105% 100% 106% 100% 112% 106% 93% - 103% 92% - 105% 104% 105% 99% 98% 102% 98% 98% 99% 103% 98% - 101% 95% - 102% 86% 94% 105% 92% 101% 100% 97% 105% 98% 94% 102% 96% 83% 96% 98% 84% 100% 97% 94% 99% 90% 84% 95% 98% 96% 101% 110% 103% - 105% 99% - 105% 96% - 107% 103% ,

Serum value = 100%

Analogous experiments were also carried out in the CEA test. The results were the same as in the TSH test. It has been found that as the surfactant level increases, not only does recovery in the plasma increase, but also the absorbance of the zero standard is lowered. This means that the non-specific conjugate binding is reduced by the addition of the surfactant.

Example 3

To demonstrate the effect of various detergents on the non-specific binding of conjugate, various surfactants were tested. The results are shown in Table 3 below.

Table 3

detergent concentration Ext. O standard without - 68 mE Tetronic 707 1% 43 mE Tetronic 707 2% 26mE Tetronic 908 1% 1OmE PluronicF68 2% 22 mE PluronicF88 , 2% 18mE

Example 4

It was tested to what extent the surface active agent used according to the invention can accelerate the TSH test without the extinction of the zero standard increasing. For this purpose various surfactants were tested. The results are shown in the attached figure.

Example 5

Several TSH tests were performed. The acceleration, based on a standard, was determined which can be achieved with the agents used according to the invention. The values listed in Table 4 below were obtained with the various surfactants.

Table 4

surfactant Conc. default % % ΔΕ 100.5 PluronicF68 1% 0.847 110 PhjronicF68 2% 0.926 147 PluronicF68 5% 1,238 114 PluronicF88 1% 0.775 112 PluronicF88 2% 0,762 147 PluronicF88 5% 1,002 118 PluronicF98 1% 0.831 112 PluronicF98 2% 0.789 172 PluronicF98 5% 1,213 102 PiuronicF38 1% 0.697 102 PluronicF38 2% 0,695 92 PluronicF38 5% 0.627

Claims (18)

1. A method for the determination of a partner of an immune reaction according to the immunoassay principle at a temperature between 15 and 40 ⁰ C, wherein one of the reactants in the solid phase, characterized in that one comprises a surface-active agent having an HLB value of more than 20 added. 2. The method according to claim 1, characterized in that the determination is carried out in a plasma sample. 3. The method according to claim 1 or 2, characterized in that nonionic block copolymers based on alkylene oxides having 2 to 4 carbon atoms, which optionally contain an alkylenediamine having 2 to 4 carbon atoms as the central molecule, are used as surfactants. 4. Process according to one of Claims 1 to 3, characterized in that surfactants with an HLB value between 24 and 30 are used. 5. The method according to any one of claims 1 to 4, characterized in that as surface-active compounds of the formula I, CH ₃
(CH ₂ CH ₂ O) ₈ (CH-CH ₂ O) ₅ (Ch ₂ CH ₂ O) ₈ where a can assume a value between 30 to 150 and b a value between 15 and 60. 6. The method according to claim 5, characterized in that one uses block copolymers in which a has a value between and 100 and b has a value between 25 and 40. A process according to any one of claims 4 to 6, characterized in that block copolymers having a proportion of hydrophilic polyoxyethylene groups of 60 to 90% are used. 8. The method according to any one of claims 1 to 4, characterized in that one uses as surfactants compounds having the following formula II: ν 3, 3 H (OCH ₂ CH ₂ ) (OCH-CH ₂ ) _{x V} ^ _χ (CH ₂ -CHO),. (CH ₂ Clf ₂ O) H N-CH "-CH" -N ^
^ ^{2 2} \ H (CCH ₀ CH ₀₎ (CCH-CH ₀₎ "^ ₍₀ CH -CHO) (CH ₀ CH ₀ O) H z t. y \ Zx x2) y Z 2. CH- CH- where χ can have a value between 10 and 50 and y a value between 30 and 150. 9. The method according to claim 8, characterized in that one uses block copolymers in which χ has a value between 15 and 30 and y has a value between 40 and 120. 10. The method according to any one of claims 8 or 9, characterized in that the proportion of polyoxyethylene groups is 50 to 90%. 11. The method according to any one of the preceding claims, characterized in that the surface-active agent is introduced together with the incubation solution. 12. The method according to any one of the preceding claims, characterized in that the surface-active agent in a concentration of 0.1 to 5%, based on the total solution, is added. 13. A reagent for carrying out a method according to any one of claims 1 to 12, comprising mobilized and immobilized partners of an immune reaction, characterized in that it contains a surfactant having an HLB value of more than 20. 14. A reagent according to claim 13, characterized in that it contains as surface-active agent nonionic block copolymers based on alkylene oxides having 2 to 4 carbon atoms, optionally containing an alkylenediamine having 2 to 4 carbon atoms as the central molecule contains. 15. A reagent according to claim 13 or 14, characterized in that the surface-active agent has an HLB value between 20 and 30. 16. A reagent according to any one of claims 13 to 15, characterized in that the surface-active agent is a compound of the formula I. CH ₃ (CH ₂ CH ₂ O) _a (CH-CH ₂ O) _b (CH ₂ CH ₂ O) _a " in which a has a value between 30 and 150, preferably between 60 and 100 and b has a value between 15 and 60, preferably between 25 and 40. 17. A reagent according to claim 16, characterized in that the compound has a proportion of Poiyoxyethylengruppen of 60 to 90%. 18. A reagent according to any one of claims 13 to 15, characterized in that the surface-active agent is a compound having the general formula II