Method to determine a specific substance capable of binding.

The invention relates to a method for the determination of a specifically bindable substance by incubation of the sample solution with at least three receptors R₁, R₂ and R₃, of which R₁ and R₂ are each capable of binding to and R₃ is specifically bindable with the substance to be determined and measurement of the occurring during the reaction agglutination and a suitable reagent therefor.

In body fluids and tissue, the substances are found, which are capable of binding to a specific binding partner and serve as parameters for specific diseases or the health of the human body. These include haptens such as. For example, hormones, proteins such as tumor markers, protein hormones and viral proteins and antibodies. The determination of drugs in the blood is to monitor drug treatment often required. Since these substances often only occur in very small amounts, are used for their detection method according to the immunoassay principle. There are many variants. The various immunological assay methods can be divided into homogeneous and heterogeneous methods. In the heterogeneous method, a solid phase reaction is always involved to separate the bound portion of the labeled component from the unbound. In this mode of the marker can be determined well, disadvantageous, however, that the heterogeneous reaction takes a long time.

In the homogeneous method variant there is no separation of bound label and unbound label so that a differentiation between bound and unbound label must be carried out by other methods.

There are various possibilities. So conjugated enzymes can for example be used as a marker, only receive their enzyme activity when bound to the antigen to be determined or hapten or be activated by the substance to be determined. A further possibility is to use as a label a fluorescent substance whose fluorescence is shifted by binding to the substance to be determined either in a different wavelength range or whose polarization is changed.

The disadvantages of these known processes are, in particular, that the sample often contains the test interfering components, which makes a sample pretreatment for eliminating these substances required. In addition, a complicated optimization for each parameter is required for. B. have the enzymes are modified depending on the parameter.

Furthermore, a method is known from EP-A-0079962, in which the composition containing the hapten to be determined with solution haptenbeschichteten latex particles or haptenbeschichtetem albumin is brought into contact. By adding bindable with the hapten antibodies are an agglutination. Since the bound to the latex particles or to the albumin hapten competes with the hapten contained in the sample, the agglutination reaction is the lower, the more hapten contained in the sample. The disadvantage of this method is that for each substance to be determined special particles must be provided and each parameter must be optimized individually.

Another method for detecting proteins, which is based on the analysis of an agglutination reaction, is known from DE-A-27 49 956th Antibodies against the subject substance to be bonded directly to the agglutinierbaren particles. This binding, however, the reactivity of the antibodies may be impaired. Moreover, such a determination method susceptible to interference from rheumatoid factors.

Disadvantage of all known competitive homogeneous agglutination immunoassays is that the raw materials have to be very complicated parameter specifically optimized. In all of these tests are made opposing requirements for optimal differentiation and optimal sensitivity, on the one hand, the concentration of the particulate reagent should be limited, so that the competing reaction with the sample is also very useful and should be on the other hand highly concentrated and highly marked the particulate reagent to a sufficient to achieve signal change per unit of time. Reconciling these requirements leads to limited sensitivity and susceptibility, which can often be eliminated by specific sample pretreatment.

Object of the present invention was therefore to provide a homogeneous assay method is available that allows the detection of substances with high sensitivity and accuracy and the disadvantages described above has not.

This object is achieved by a method for determination of a specifically bindable substance by incubation with three receptors R₁, R₂ and R₃, of which R₁ and R₂ are each capable of binding to and R₃ is specifically bindable with the substance to be determined and measurement of the agglutination occurring in the reaction , which is characterized in that, as receptor R₁ which comprises a conjugate of a partner of a specifically binding pair P and of a substance S which corresponds to that substance to be determined or is a derivative thereof and at least one epitope substance to be determined, whereby R₁ only a binding site for R₂ has, as R₂, a receptor which has at least two binding sites for P but is not an erythrocyte and as R₃ a receptor which has at least two binding sites, of which at least one binds specifically with an epitope of the substance to be determined or of S , is used, and R₂ is added simultaneously with the receptors R₁ and R₃ or after a certain period of time or R₂ and R₃ are premixed and added after addition of the sample R₁.

The inventive method is suitable for the determination of practically all detected in body fluids or tissue extracts empower a specific binding substances, low concentrated substances are also well documented as highly concentrated. The sensitivity and accuracy of the method is improved over the previously known methods. The invention provides the ability to quickly and reliably perform with simple reagents provisions.

The method is particularly suitable for the determination of monovalent specifically bindable substances. while a substance is referred to as monovalent, having only one binding site for a specifically bindable partner. As examples may be mentioned here haptens, z. B. drugs. Likewise, substances can be determined, the multiple binding sites for specifically bindable partners possess such as protein hormones such as HCG or TSH, antigens and proteins, tumor markers like CEA, viral proteins and antibodies.

Under epitope is understood in the description of the present invention a binding site that can specifically bind with another substance. Examples of epitopes are antigenic determinants on antigens and haptens or even specific binding sites on proteins.

The sample solution is incubated to three receptors R₁, R₂ and R₃. The R₁ and R₂ receptors are capable of binding to each other, and R₃ is specifically bindable with the substance to be determined. Different reaction principles which can be carried out with the method according to the invention are shown in FIG. 1. A preferred process variant is designated as 1a implementing form. Here is a sample solution receptor R₁ which is a conjugate of a partner of a specific binding with each pair P and the substance to be determined S and has only one binding site for R₂, and R₃, which is the substance to be determined specifically bindable receptor added , The solution then compete the part south of R₁ and the substance to be assayed for binding to R₃. If R₃ example, a bivalent antibody, then form the following complexes:<dl id="dl0001"><dt>R₃,</dt><dd>at both paratopes each R₁ is contraindicated connected via S;</dd><dt>R₃,</dt><dd>at both paratopes the substance to be determined is bound, and</dd><dt>R₃,</dt><dd>is bound at one paratope the substance to be determined and at the other paratope R₁ through S.</dd></dl>

Simultaneously with the two other receptors or after a certain period, the receptor R₂ is added, at least two, and preferably having a plurality of binding sites for P but is not an erythrocyte. Thus it comes to binding of the receptor R₁ through P to receptor R₂. Of the present in the solution complexes can only complexes which have at least one receptor R₁ bound, bind to R₂, and only those in which R₁ two receptors are bound to R₂ can, produce agglutination, the back or a photometrically detectable turbidity caused. turbidity change. The more contained by the substance to be determined in the solution, the less R₁ is bound, the less agglutination occurs and the smaller the increase in haze. Thus, the scope of agglutination is an indirect measure of the substance to be determined. This can be evaluated via a calibration curve.

The implementing variant b) is designed to detect bivalent substances, for example antibodies. The principle is the same as in variant a). Here again, only the complexes in which the two binding sites of R₃ are each an R₁ is bound, cause agglutination.

In variant c) a conjugate of one used with the substance to be determined specifically bindable receptor and a partner of a specific binding pair as R₃. In incubating the sample solution with R₁ and R₃ in turn compete to be determined substantia R₁ and for binding to R₃. Here, either the binding substance to be determined at a receptor R₃ R₁ or a molecule. After incubation with R₂ the proportion P of the receptor R₃ binds to R₂. If the substance to be assayed bound to the other binding site of R₃, so no cross-linking via R₂ is possible; However, the conjugate R₁ bonded to the other binding site of R₃, the receptor R₁ provides a second binding site for R₂, and it comes to networking. In this form of implementation less R₁ is more bound, the more there is of the substance to be determined in the solution, and accordingly the Agglutinationsbildung is then lower.

The detailed shape d) is a variant of the form of implementation a), but wherein a receptor is used as receptor R₃, which still has another binding site in the form of P besides the two binding sites for the substance to be determined or S. On incubation of the test solution with R₁ and R₃ again the same form complexes as in variant a). In contrast to variant a), however, both the receptors R₃ is bonded at both paratopes R₁, R₃ and the receptors for which at a paratope R₁ and on the other the substance to be determined is bound to cause agglutination. This embodiment is therefore particularly suitable for the detection of substances which are highly concentrated.

For the invention process defined principle there are so many implementation variants. In any case, at least three receptors are required. The substance to be determined can be any compatible to a specific binding substance, and in particular as defined above, a hapten, monovalent, bivalent or polyvalent antigen, antibody or protein to be.

The first receptor R₁, a conjugate is used which consists of a partner of a specifically binding pair P and of a substance S which corresponds to that substance to be determined or is a derivative thereof and at least one epitope comprising substance to be determined and only one binding site for R₂ has. Specifically, each binding pairs are known per se. Suitable binding pairs (P-R₂) are in particular biotin-streptavidin or avidin; Hapten-antibody; Antigen-antibody; Konkavalin antibody; Sugar-lectin; Hapten-binding protein, eg, thyroxine binding globulin and thyroxine or oligopeptide-antibody.

Particular preference is streptavidin or avidin-biotin is used as the binding pair. Particularly preferably, the receptor R₁ contains biotin.

The percentage S of the receptor R₁ may preferably correspond to the unchanged substance to be determined. It may also be a derivative of the substance to be determined or a portion of the substance to be determined such as a protein epitope can be used. It is only essential that the proportion of S with R₃ is capable of binding, wherein it is not absolutely necessary that S and the substance to be determined bind with the same binding strength to R₃.

The preparation of the conjugates are prepared in a manner known per se (eg. B. analog Eur. J. Biochem. 131 (1980) 333-338).

The second required for the inventive method receptor R₂ has at least two binding sites for P and preferably has a plurality of said complementary to P partner of a specifically binding pair on. The receptor R₂ mediated agglutination of the forming in the reaction complex. Since a variety of other partners of the specific binding pair in the reaction system are present, leads a course in the test solution of existing low level of bindable with this partner substance not interfere. The receptor R₂ may be a substance that is already naturally possesses several binding sites for P, for example, streptavidin or antibodies. Receptor R₂ may be polyvalent and having a plurality of binding sites for P or be a polymer complementary to the P partner of the specific binding pair, such as polystreptavidin. The individual partners are then bonded either directly connected or over bridges linked. Processes for preparing such polymers are known to the expert and require no further explanation.

In a further embodiment, the receptor R₂ consists of a carrier material to which a plurality of the complementary specific binding partner to P are bound. As support materials, particles can be used, which usually have a size of 50 to 1000 nm. Suitable materials include polystyrene, finely divided silica, or cross-linked albumin. The coating of these particles with the specific binding partner is subject to the skilled worker methods. Method thereof are described for example in EP-B-0073611 and US Patent No. 4,703,018.

The so coated or polymerized receptors R₂ are universal and therefore not parameter specific for the novel process.

Essential for carrying out the method according to the invention that the receptor R₁ possesses only one binding site for R₂, that is to say that each receptor R₁ can only react with one R₂ is. This is essential, because otherwise R₂ could be linked solely by the receptor R₁ and thus agglutination would cause that is not attributable to the substance to be detected S.

The third invention is an essential component of the receptor R₃ which has at least two binding sites, of which at least one binds specifically with an epitope of the substance to be determined or of S. This receptor is selected after each substance to be determined. Here a variety of receptors are suitable. For the determination of haptens, proteins, DNA or sugar, it is particularly advantageous to employ antibodies or other receptors such as, for. Example, naturally occurring binding proteins, such as thyroxine binding globulin against these substances or their fragments. For the determination of DNA, it is also advantageous to use a complementary DNA as receptor R₃.

If the substance to be determined has only a single binding site for R₃, so, in a preferred embodiment, the receptor R₃ addition of a binding site for the epitope of the substance to be determined or S A different binding site for a member of a specific binding pair in the form of also for R₁ used P Partners on. R₃ is then preferably either a conjugate of a partner P of a specifically binding pair and of a substance having a single binding site which binds specifically with an epitope of the substance to be determined or of S, more preferably a conjugate of an with an epitope of substance to be determined or of S specific binding Fab fragment and P, or a conjugate of a partner P of a specifically binding pair and a receptor which has two binding sites for the epitope of the substance to be determined or S.

Receptor R₃ may also include two identical binding sites that bind to an epitope of the substance to be determined or S. Suitable as receptor R₃ are, for example, monoclonal or polyclonal antibodies, Fab₂ fragments or conjugates of Fab fragments and partners of a specific binding pair. If the receptor R₃ is intended to have more than two binding sites can conjugates of antibodies, F (ab) ₂- or F (ab ') ₂ fragments used with a partner P. Here are especially biotinylated antibodies and antibody fragments which are specifically bindable with the substance to be determined, suitable.

In another embodiment, the receptor R₃ is only formed during the determination, for. Example, of an antibody against TBG and two molecules of an antibody against TBG or Fab fragments, and two Fab fragments.

The process may be carried out in one or more stages. The evaluation is performed by measuring the extent of agglutination. Methods for this are known. Good example is the photometric turbidity measurement, the measurement of scattered light by nephelometry, particle counting or photon correlation spectroscopy (PCS).

Since each of the receptors and the substance to be determined can react only with the specifically intended for it reactants, it is possible to incubate all of the receptors and the sample together and carry out the process in one stage. This is particularly advantageous in carrying out the process in an automated analyzer.

multistage process variants are preferred for the detection of very low concentrations or very highly concentrated substances. In the detection of highly concentrated in the sample solution substances the embodiment described below is preferred. This receptor R₁ is pre-reacted with the receptor R₃ first. It react the proportion S of the receptor R₁ which corresponds to the substance to be determined, and R₃ together. Subsequently, the sample solution is added. The devices in this sample solution substance to be determined now displaced already bound receptor R₁ from the binding. As in the test solution, the proportion of substance to be determined is high, this displacement reaction takes place sufficiently quickly. Subsequently receptor R₂ is added and then measured the increase in haze. This type of test execution, although less sensitive, but very suitable for highly concentrated samples.

A further preferred embodiment of the inventive method is suitable for detecting very low concentrations of substances. Examples hormones and drugs are mentioned. This receptor R₂ is first premixed with receptor R₃, and then the sample was added. Thereby responding present in the sample substance to be determined with the receptor R₃. An agglutination can not take place because of the missing is the binding to the receptor mediating receptor R₂ R₁. Subsequently receptor R₁ is added, which is capable of binding with the receptor R₃ and with the receptor R₂. Again, the increase in haze is measured after a certain period. Those implementing variant is very sensitive.

as a fully competitive test procedure is suitable for medium concentration parameters. In this case, the same receptor R₁, R₃ receptor and sample solution are incubated. Here, the substance to be determined and the proportion contained in the conjugate of the receptor R₁ S compete for binding to receptor R₃. After the addition of receptor R₂ agglutination is then measured again.

Also suitable is, this variant of the inventive method for the detection of haptens which can be coupled with an antibody specific for it protein, wherein said binding protein has only one binding site for the hapten. An example is thyroxine which is specifically bound by thyroxine binding protein.

The implementation of all process variants is preferably carried out in a buffered solution. Buffer systems for this method are known per se. Particularly suitable for this purpose are GOOD-buffers and phosphate buffers.

According to the invention, a method is provided which is carried out quickly and easily and provides a concentration-dependent of the substance to be determined, a measurement signal. As the body responsible for immunological competition system and the signal-forming system are separate, the sensitivity of detection is increased according to the invention. This can be quickly and reliably determined quantitatively using simple reagents, even low concentrations of haptens.

Another object of the present invention is a reagent for the determination of specific bindable substances, which is characterized in that it receptor R₁ which is a a conjugate of a partner of a specifically binding pair P and of a substance S which corresponds to the substance to be determined or derivative thereof and at least one epitope comprising substance to be determined, whereby R₁ possesses only one binding site for R₂, receptor R₂ which has at least two binding sites for P but is not an erythrocyte and receptor R₃ which has at least two binding sites, of which at least one specifically binds to an epitope of the substance to be determined or of S, contains.

The reagent according to the invention may include physically separated from each other in a preferred embodiment, the individual receptors R₁, R₂ and R₃. In another embodiment, the receptors R₁ and R₃ may be premixed and then the reagent, the mixture of R₁ and R₃ and R₂ receptor physically separated therefrom. It is also possible to premix the receptors R₂ and R₃, in which case the reagent comprises a mixture of R₂ and R₃ as well as physically separated from R₁.

This reagent is suitable for determining a plurality of parameters in body fluids and tissue extracts.

In a preferred embodiment, the reagent in addition contains buffer substances. Particularly preferably, it contains phosphate buffer or Good buffer.

The invention is illustrated by the figure and the examples.<dl id="dl0002"><dt>Fig. 1</dt><dd>shows a scheme for the reaction principles of various preferred embodiments of the method according to the invention.</dd></dl>

In all variants shown receptor R₂ has two binding sites for P. As receptor R₁, a conjugate of the substance to be determined and a partner P is used in each case.

Fig. Figure 1a shows a variant that is suitable for the detection of substances that have only one binding site for receptor R₃.

As receptor R₃ is here with respect to the used substance to be determined bivalent receptor.

Variant 1b) serves for the determination of bivalent substances (such as antibodies). As receptor R₃ is with the substance to be determined specifically bindable bivalent receptor is used.

Description 1c) is suitable for the detection of monovalent substances. Here, a conjugate of a receptor having a binding site for the substance to be determined and a partner P used as receptor R₃.

Description 1d) is suitable for the detection of monovalent substances. As receptor R₃, a conjugate is used which has two binding sites for the substance to be determined and a partner P.

example 1

<ul><li>a) Preparation of Streptavidin-Latex Streptavidin at a concentration of 2 mg / ml in 15 mmol / l imidazole buffer, pH 7.5, 100 mmol / l NaCl with chloromethylstyrene particles (latex, d = 70 nm, corresponding to US-A-4,703,018) at a concentration of 2 incubated wt .-% for 24 hours at 55 ° C and stirred. After the reaction mixture was centrifuged for 60 minutes at 20,000 rpm, the supernatant is decanted and the precipitate was collected in 200 mmol / l glycine buffer, pH 7.5, containing 0.5% bovine serum albumin. By appropriate dilution, a 1 wt .-% sodium streptavidin-latex reagent is prepared.</li><li>b) Preparation of the hapten-biotin conjugates For this purpose, J. Biochem -xanthine (DE-A 28 05 961) is n-butyloxycarbonyl-tetrajodothyronin and 1-methyl-3- (3'-carboxypropyl) coupled via pentamethylenediamine with biotin as described in Eur.. 131 (1980) 333-338 described. This gives T₄-biotin conjugate. Analogously, Theophylline is coupled to biotin.</li></ul>

example 2

Determination of T₄

A reagent composed of:    900 »l 0.1 mol / l phosphate buffer, pH 7.5, with 2%    Polyethylene glycol 40000    20 »l sample (aqueous T₄ standards)    20 »l of a 0.1 mg / ml solution of a polyclonal    Antibody to T₄    20 »l of 1% latex reagent (example 1) is incubated at 37 ° C and then added 20 »l of a conjugate of biotin and T₄ concentration 10⁻⁶ mol / l 5 minutes.

There the kinetics of the increase in haze was measured and the change in absorbance at 405 nm / unit time (5 minutes) determined. This gave the following values ​​were obtained:<tables id="tabl0001" num="0001"><table frame="all"><title>Table I</title><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Sample / ng / ml T₄ /</entry><entry namest="col2" nameend="col2" align="center">mE / 5 min</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">0</entry><entry namest="col2" nameend="col2" align="right">620</entry></row><row><entry namest="col1" nameend="col1" align="right">50</entry><entry namest="col2" nameend="col2" align="right">500</entry></row><row><entry namest="col1" nameend="col1" align="right">100</entry><entry namest="col2" nameend="col2" align="right">410</entry></row><row><entry namest="col1" nameend="col1" align="right">200</entry><entry namest="col2" nameend="col2" align="right">230</entry></row><row><entry namest="col1" nameend="col1" align="right">500</entry><entry namest="col2" nameend="col2" align="right">60</entry></row></tbody></tgroup></table></tables>

example 3

Determination of theophylline

It is prepared by mixing a reagent:    300 »l 100 mmol / l Tris buffer, pH 7.5    7 »l of 1% latex reagent (example 1),    7 »l polyklonalar antibodies to theophylline (Fab₂" concentration 5 mg / ml ") and incubated for 5 minutes at 37 ° C. Then 5 x 10⁻⁶ mol / l of a conjugate of theophylline and biotin added and determines the change in absorbance at 450 nm with a time difference of 4.2 minutes. The results are shown in Table II.<tables id="tabl0002" num="0002"><table frame="all"><title>Table II</title><tgroup cols="2" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center">Sample uM / L theophylline</entry><entry namest="col2" nameend="col2" align="center">mE / 4.2 min</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="char" char=",">0</entry><entry namest="col2" nameend="col2" align="right">1050</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">0.5</entry><entry namest="col2" nameend="col2" align="right">880</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">2.5</entry><entry namest="col2" nameend="col2" align="right">800</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">5</entry><entry namest="col2" nameend="col2" align="right">550</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">12.5</entry><entry namest="col2" nameend="col2" align="right">320</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">25</entry><entry namest="col2" nameend="col2" align="right">230</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">50</entry><entry namest="col2" nameend="col2" align="right">70</entry></row><row><entry namest="col1" nameend="col1" align="char" char=",">125</entry><entry namest="col2" nameend="col2" align="right">40</entry></row></tbody></tgroup></table></tables>

example 4

Comparison with the prior art

The inventive method was compared to a procedure analogous to EP-A-0073611. As coated "carrier" streptavidin was used which 4 T₄ molecules per "carrier" includes.

The preparation of this "carrier" was made by mixing 20 »l of a conjugate of biotin and T₄ (concentration 10⁻⁴ mol / l) with 20» l of streptavidin (concentration 2.5 x 10⁻⁵ mol / l) were combined, and 30 minutes was incubated. After completion of the reaction contained this reagent T₄, which was coupled via biotin to streptavidin.

The comparative experiment:

820 »l of 50 mM phosphate buffer, pH 7.5, 4% polyethylene glycol 40,000 were added together with 40» l of Streptavidinreagenzes prepared as described above and a sample (0 mol / l T₄) was added. Simultaneously 100 »l of a solution of 3 mg / l antibody was added against polyclonal antibodies to T₄ and followed the extinction per 3 minutes at 340 nm.

Inventive method variant A:

820 »l of 50 mmol / l phosphate buffer, pH 7.5, 4% polyethylene glycol 40,000 were mixed with 20» l T₄ conjugate and biotin (concentration 10⁻⁴ mol / l in methanol) and 100 »l T₄ polyclonal antibody against ( pooled 3 mg / ml) and incubated for 5 minutes.

After addition of 20 »l of streptavidin (concentration 2.5 x 10⁻⁵ mol / l) the change in absorbance per 3 min monitored at 340 nm.

Inventive method variant B:

820 »l of 50 mmol / l phosphate buffer, pH 7.5, 4% polyethylene glycol 40,000 are mixed with 20» l of streptavidin (2.5 x 10⁻⁵ mol / l) in 200 mM phosphate buffer, pH 7.5, and 100 »l polyclonal antibody to T₄ (3 mg / ml ₎ together and incubated at 37 ° C for 5 minutes. After addition of 20 »l T₄ conjugate and biotin (concentration 10⁻⁴ mol / l) the change in absorbance per 3 min monitored at 340 nm.

The results of the comparative experiment are:

Rates: 35 mE Inventive method variant A: 646 mE Inventive method variant B: 414 mE It can be seen that under analogous conditions with the inventive method, a higher sensitivity can be obtained over the method according to the prior art.