DE3541611A1 - Method for the determination of a hapten on the competitive immunoassay principle - Google Patents

Abstract

To determine a hapten on the competitive heterogeneous immunoassay principle, the hapten-containing sample is incubated with defined amounts of a) labelled anti-hapten antibody (antibody conjugate) and b) a hapten conjugate which consists of one or more hapten molecules bound to a carrier which is capable of specific binding and c) with a binding partner, which is present in an insoluble phase, for the carrier present in the hapten conjugate, the liquid and the solid phases are separated, and the label in one of the phases is measured.

Description

The invention relates to a method for determination a hapten based on the principle of competitive hetero gene immunoassays using a labeled Anti-hapten antibody (antibody conjugate), as well as a reagent suitable for carrying out the method.

Many substances, particularly in clinical Chemistry are demonstrated due to their to call low molecular weight haptens, d. H. Compounds that are not inherently immunological Can trigger reaction in connection with a However, carriers induce the formation of antibodies can. Because on the one hand these connections are often very low concentrations, down to the picomole range, occur and on the other hand these connections a Can trigger an antibody reaction Detection method an immunological method.

From J. Clin. Invest., 39, 1157, 1960, it is already known to use haptens in a competitive radioimmunoassay to prove, in which the hapten of the sample with radioactive hapten labeled with hapten-specific antibodies competes, so the application of the well-known competi tive radioimmunoassays on Haptene. The use of radioactive markings means restricted Storability and is with a complex disposal connected.

Another procedure in this direction is one Bringing improvement is the process known as ELISA enzyme-labeled hapten and Hapten the sample for solid phase bound antibodies  against the hapten. The disadvantage of this method is in that for each connection to be verified, that is, for every special hapten, a special solid phase combination whose antibody must be used. That means, that for the detection or determination of each individual Connection special media on which the solid phase with the antibody is provided have to.

The object of the invention was therefore a method and to create a suitable reagent in which for many different haptens the same solid phase can be used so that a kind of with Binding partners coated carriers, as a called matrix for many different determinations can be used.

This object of the invention is achieved by a ver drive to determine a hapten according to the principle of competitive heterogeneous immunoassays using labeled anti-hapten antibody (antibody perconjugate), which is characterized in that the sample containing hapten with certain amounts

• a) Antibody conjugate and
• b) a hapten conjugate which consists of one or more, bound to a specifically bindable carrier Hapten molecules exist and
• c) with the binding part present in the insoluble phase ner for the carrier contained in the hapten conjugate in cubed, the liquid separated from the solid phase and the marking in one of the two phases will.

This procedure is suitable for quick and one fold determination of haptens, whereby for many ver different compounds of the same in the insoluble phase present binding partner can be used if an appropriate carrier in the respective hapten conjugate is available. This procedure simplifies the determination of haptens, as no longer for each one Connection a special solid-phase anti body must be made available, but there with a uniform matrix for many different haptens can be worked.

The method according to the invention can be used both as a step reaction, as well as a two-step reaction be performed. In the one-step reaction Antibody conjugate, hapten conjugate and binding partner for the carrier contained in the hapten conjugate brought and incubated in one step, whereby after Separation of the solid from the liquid phase the con concentration of the hapten in one of the two phases he follows. It is also possible to first find the soluble ones Reactants in a homogeneous phase with each other incubate and then incubate with the insoluble Phase in a second stage. Prefers is the inventive method as a two-step ver driving done.

To determine the hapten, a marked anti Hapten antibody, which here as an antibody conjugate is used. It is used as an antibody either the complete, specific against that to be determined hapten-directed antibodies or an anti body fragment used that does not match the specific  bindable carrier of the hapten cross-reacted. It can be monoclonal or polyclonal antibodies or Antibody fragments can be used. Is preferred a Fab fragment used as an antibody.

The antibody is labeled in itself known way. The antibody is preferably by Coupling with an enzyme, a fluorescent, chemiluminescent or radioactive substance mar kiert. Methods for labeling antibodies are known to the expert.

The antibody conjugate is used in the invention Process used in a known amount. The amount can vary depending on the specific test. So can for example a deficit of antibody conjugate, based on hapten and hapten conjugate used will. The amount of antibody conjugate cannot be determined generally determine, but can be by some prer search can be easily determined.

The hapten conjugate used according to the invention exists from a specifically bindable carrier to which one or several hapten molecules are bound. The spec fish-binding carrier forms insoluble with that Phase present binding partner a specific bindable pair of substances. As suitable substance pairs are z. B. antigen antibodies; Protein A immunoglobulin G; Avidin-biotin; Concanavalin A lectin; DNA-DNA (hybrid) to call. Any partner in such a couple can thereby as a carrier for the hapten or the hapten molecules used, the other partner of the substance couple then as a binding partner that is insoluble in  Phase is present, is used. For example the hapten is bound to protein A, while in insoluble phase the immunoglobulin is present. Prefers antigens are used as carriers of the hapten conjugate. In a particularly preferred embodiment, as specifically bindable carrier in the hapten conjugate Ig fraction used. In this case the is in binding partner an insoluble phase Ig directed antibody. The binding of the hapten or of the hapten molecules to the specifically bindable Carrier is made according to known methods, such as for example in Methods in Enzymology (Vol. 73B, page 203 to page 244 (1981)).

The binding partner present in the insoluble phase for the carrier contained in the hapten conjugate from a solid phase to which the binding partner of the specifically bindable carrier is bound. As a festival phase can be the usual solid phases such as polystyrene, other plastics, activated carbon, cellulose etc., against also used in derivatized form.

This solid phase is carried out in a manner known per se Binding partner for the contained in the hapten conjugate Carrier tied. The other part of the specifically bindable substance pair used. Preference is given as a pair of substances capable of binding the pair of antigen-antibody or antibody fragment used. It is particularly preferred if the hapten is on an Ig fraction is bound as in the insoluble phase present binding partner uses an antibody, that against the Fc part of as the carrier of the hapten conjugate used Ig’s is directed. As antibodies you can both polyclonal and monoclonal antibodies or Antibody fragments can be used.  

In a particularly preferred embodiment, a solid-phase complete polyclonal antibody from an animal species used against the Fc portion of immunoglobulin G from a second animal species is directed. As a specifically bindable carrier in this case immunoglobulin G of the second animal species used. Then as a labeled antibody conjugate an antibody fragment-enzyme conjugate from the first or another animal species, but not the second animal species used.

To carry out the method according to the invention the sample containing hapten, antibody conjugate and Hapten conjugate and simultaneously or if necessary then the bin in the insoluble phase incubated. The incubation times depend on essential of the properties of the used Reagents. In particular, the Inkuba are determined times according to the affinity constants of the use antibodies and the concentrations of the analytes. The higher the affinity constant, the shorter it is the incubation period.

The process according to the invention can be carried out in a suitable manner Vessels are performed. Examples are suitable disposable containers known as disposables, the Contain reaction and measuring chambers as well as reagents, Microtiter plates or tubes. Are also suitable Dry reagent carriers such as B. Test strips. When used With disposables, the method is preferred performed that in a first step the sample with the hapten conjugate and the antibody conjugate  becomes. The antibody conjugate is in deficit in front. The incubate then becomes the solid phase admitted. After another incubation step the solid and the liquid are separated Phase. Then the amount of labeled antibody perkonjugat either in the liquid or in the solid phase can be determined. The determination is made in in a known manner and is dependent on the as Marking substance used. From the received The measured value can then be in a known manner in the sample existing hapten can be calculated.

In a further preferred embodiment of the the method according to the invention are tubes or microtitres plates used. In this case, rehearsal, hapten conjugate, antibody conjugate and solid phase at the same time incubated. Then the separation takes place and the liquid phase and determining the amount of labeled antibody conjugate. Also preferred as Reaction medium is a dry reagent carrier, which in is built up of several layers. The top layer the sample is placed on contains the marked one Antibody conjugate. The second layer contains that Hapten conjugate. The third layer contains the in binding partner for the insoluble phase carrier contained in the hapten conjugate and a fourth Layer contains the chromogenic reagent. Here occurs the sample with the antibody conjugate, then with the hapten conjugate and finally with the binding partner present in the insoluble phase in contact.

The method according to the invention enables many to determine different haptens using one and the same solid phase, the unity matrix can be designated.  

Another object of the invention is a reagent for the determination of hapten containing labeled anti Hapten antibodies in a certain amount and in solid Phase present specifically bindable substance, the is characterized in that it is a soluble Hapten conjugate of one or more hapten molecules and a specific bindable carrier in certain Quantity contains and that the solid phase specific bindable substance the carrier of the hapten conjugate binds.

The reagent according to the invention preferably contains as Carriers of the hapten conjugate and as solid-phase bound specifically bindable substance capable pair of substances that u. a. Antigen antibody; Protein A immunoglobulin G; Avidin-biotin; Concanavalin A- Lectin; DNA-DNA hybrid can be. Particularly preferred contains the reagent as a carrier of the hapten conjugate Antigen or an antibody and as a solid phase the specific substance capable of binding the corresponding Antibodies or the corresponding antigen.

It is particularly preferred as the antigen-antibody complex forming an Ig fraction and the thus corresponding anti-Ig antibodies are used. In a preferred embodiment contains the reagent as an anti-Ig antibody, the antibody against the Fc part of the Ig fraction is directed.

The reagent component according to the invention for the determ This results in the formation of a number of different haptens characterized in that they consist of a solid inert carrier material to which an against the Fc part of an Ig-Frak tion is directed antibody exists.  

Known per se as inert carrier material Materials such as plastics, cellulose and others macromolecular substances are used. Especially is preferred as a carrier for the antibody Nonwoven used.

The antibody can either be covalent to the support or adsorptively bound. The is preferred Antibodies to the carrier by immunoprecipitation bound.

The inventive method and the inventive Reagent and the reagent component according to the invention enable simple and quick determination many different haptens because the medium in which the immunological reaction takes place, for many ver divorced haptens remains the same.

The invention is further illustrated by the following examples are explained:

Example 1

The method described in EU-A 17 35 13 and the insert element described there in FIGS. 1a and 1b for an automatic centrifugal analyzer were used to carry out the method. This insert element contains 7 interconnected chambers, each containing a fleece and metered in succession under the influence of centrifugal force for liquid.

Following fleeces and watering solutions for the fleeces were applied:

Fleece 1: filter paper impregnation solution: 3 ‰ wetting agent (Tween 20) fleece 2: filter paper impregnation solution: 100 mmol sodium phosphate buffer pH 7.2, 5 mmol EDTA, 1% bovine serum albumin, 0.75 ‰ wetting agent Tween 20. fleece 3: filter paper (antibody conjugate) Impregnation solution: anti-T4 antibody from sheep, labeled with β- galactosidase, 3 mU / test activity determined with ortho-nitrophenylgalactoside as substrate, 1% bovine serum albumin, 4 mmol magnesium aspartate, 50 mmol hepespuffer pH 7.2. Fleece 4: filter paper (hapten conjugate) impregnation solution: 50 ng / test rabbit immunoglobulin G, which was covalently derivatized with T4 according to EU-A 01 08 400. Fleece 5: filter paper (insoluble binding partner)
5 mg / g sheep antibody, directed against the Fc part of rabbit IgG covalently linked to the carrier via glutardialdehyde. (Meth. In Enzymol., Vol. 73B, pp. 203-245, 1981). Fleece 6: filter paper impregnation solution: 15 mmol chlorophenol red galactoside, produced according to DE 33 45 748.

The insert element chambers were as follows busy:

• Chamber 1: 1 fleece 1
  Chamber 2: 1 fleece 2
  Chamber 3: 1 fleece 3
  Chamber 4: 1 fleece 4
  Chamber 5: 2 fleeces 5
  Chamber 6: 1 fleece 6.

The serum used as a test serum was 1:10 with Diluted 0.9% NaCl solution. 50 µl of the so obtained Solution were used in the sample application chamber pipetted element and then the following centrifugation program carried out:

25 sec 250 rpm detachment of detergent, buffer, conjugate and hapten conjugate; Start of first incubation 20 sec 2000 rpm
300 sec 600 rpm Incubation of sample, antibody conjugate and hapten conjugate 300 sec 0 rpm Incubation with carrier-fixed hapten 15 sec 2000 rpm End of the second incubation 15 sec 0 rpm
5 sec 100 rpm cuvette 50 sec 720 rpm measurement at 578 nm

The calibration curve shown in FIG. 1 was thus obtained.

Example 2

A dry reagent carrier, according to FIG. 2, is used. Here mean:

• 1 conjugate paper,
  2 hapten conjugate paper,
  3 membrane with binding partner bound to the solid phase,
  4 color reagent film,
  5 network
  6 carrier foil.

Construction and manufacture of such reagent carriers is in the DE 21 18 455 described.

Be used:
Conjugate paper ( 1 )

Long fiber paper No. 6776 (Schoeller and Hoesch), impregnated with 400 U / ml antibody conjugate (Fab fragment, directed against theophylline and bound to β -galactosidase).
Hapten conjugate ( 2 )

Long fiber paper No. 6776 (Schoeller and Hoesch), impregnated with 35 µg polyhapten in 1 ml phosphate buffered saline (PBS). Theophylline was introduced as a hapten conjugate IgG coupled used.

Matrix with binding partner bound to the solid phase ( 3 ) nitrocellulose membrane (company Biorad), impregnated with sheep IgG, directed against the Fc part of rabbit IgG, 1 mg / ml PBS. After soaking, the membrane is washed twice with PBS and subsequently soaked in PBS with the addition of 1% RSA, 0.1% Tween 20.
Test paper ( 4 )

Tea bag paper No. 212 from Schoeller and Hoesch soaked in 1 mg / ml indolylgalactoside
Carrier film ( 6 )

A polycarbonate film (Pokalon, Lonza) used.

The size of the test field is 6 × 6 mm.

Hapten is determined in such a way that a serum sample is applied and the one formed Color is evaluated visually. Likewise, reflection be measured photometrically. The calibration takes place about a standard that is 0 to 40 µg theophylline / ml contains.

Example 3

Determination of phenytoin (diphenylhydantoin, DPH)

To carry out the method, the method described in EU-A 17 35 13 and the insert element described there in FIGS . 1a and 1b were used for a centrifugal analysis machine. This insert element contains seven interconnected chambers, each of which contains a fleece and which are successively passed under the influence of the centrifugal force of liquid.

Following fleeces and watering solutions for the fleeces were applied:

Fleece 1: filter paper impregnation solution: 3 ‰ wetting agent (Tween 20) fleece 2: filter paper impregnation solution: 100 mmol sodium phosphate buffer pH 7.2, 5 mmol EDTA, 1% bovine serum albumin, 0.75 ‰ wetting agent Tween 20. fleece 3: filter paper (antibody conjugate) Impregnation solution: sheep antiphenytoin antibody, labeled with β- galactosidase, 2 mU / test activity, determined with ortho-nitrophenylgalactoside as substrate, 1% bovine serum albumin, 4 mmol magnesium aspartate, 50 mmol Hepes buffer pH 7.2. Fleece 4: filter paper (hapten conjugate) impregnation solution: 10 ng / test rabbit immunoglobulin G, which has been covalently derivatized with phenytoin in accordance with EU-A 01 08 400. Fleece 5: filter paper (insoluble binding partner)
5 mg / g sheep antibody, directed against the Fc part of rabbit IgG, covalently linked to the carrier via glutardialdehyde (Methods in Enzymology, Vol. 73b, pp. 203-245). Fleece 6: filter paper impregnation solution: 15 mmol chlorophenol red galactoside (CPRG), produced according to DE 33 45 748

The chambers of the insert element were like follows as follows:

• Chamber 1: 1 fleece 1
  Chamber 2: 1 fleece 2
  Chamber 3: 1 fleece 3
  Chamber 4: 1 fleece 4
  Chamber 5: 2 fleeces 5
  Chamber 6: 1 fleece 6

The serum used as a sample solution was Diluted 1: 1500 with 0.9% sodium chloride solution. 50 ul of the solution thus obtained were in the Sample application chamber of the insert element pipetted and then the following centrifugation program carried out:

25 sec 250 rpm detachment of detergent, buffer, conjugate; Start first incubation 20 sec 200 rpm
300 sec 600 rpm Incubation of sample, antibody conjugate and hapten conjugate 300 sec 0 rpm Incubation with carrier-fixed hapten 15 sec 2000 rpm End of the second incubation 15 sec 0 rpm
5 sec 100 rpm Transport of the liquid in a cuvette 50 sec 720 rpm measurement at 578 nm

The calibration curve shown in FIG. 2 was thus obtained.

Example 4 Test principle

Microtiter plates or plastic tubes are included Hapten conjugate (anti-rabbit IgG phenytoin; coupling of phenytoin on rabbit IgG, manufactured according to DE 26 31 656) coated. The sample is in a first Reaction in an inert vessel with antibody enzyme conjugate and with hapten conjugate for 5 to 60 minutes beers, then this solution is in the microtiter pipetted plates or plastic tubes. After 5 to The solution is removed for 60 minutes and the enzyme acti vity of either the solid phase or the distant over status determined.

Coating buffer

0.2 M NaHCO ₃ , pH 9.5 + 0.01% bovine serum albumin, coating with 200 µl coating buffer + 100 µg / ml sheep anti-rabbit IgG.

After 10 minutes of incubation at room temperature suction filtered and coated for 10 minutes with 50 mmol Sodium phosphate, pH 7.2, 100 mmol sodium chloride, 1% RSA and vacuumed again.  

5 µl sample are mixed with 200 µl buffer (50 mmol sodium phosphate, pH 7.2, 100 mmol sodium chloride, 1% RSA) and 80 µl buffer + 2 mU antibody conjugate and 0.05 µg Hapten conjugate mixed and 30 minutes at room temperature incubated. Then 200 µl are in the micro Pipette titer plates or plastic tubes. To 30 minutes incubation at room temperature is suctioned off, washed with 200 µl buffer and after repeated suck with 800 µl substrate (5 mmol / l CPRG, 50 mmol Hepes, pH 7.2). At λ = 578 nm the Zu The adsorption was monitored for 5 minutes. The Slope is the measurement signal, the determination of concentration measurement is carried out via a calibration curve.

Claims (16)

1. A method for determining a hapten according to the principle of competitive heterogeneous immunoassay using a labeled anti-hapten antibody (antibody conjugate), characterized in that the sample containing hapten with certain amounts of

• a) Antibody conjugate and
• b) a hapten conjugate which consists of one or more hapten molecules bound to a specifically bindable carrier and
• c) incubated with the binding partner present in the insoluble phase for the carrier contained in the hapten conjugate, the liquid phase is separated from the solid phase and the marking is measured in one of the phases.

2. The method according to claim 1, characterized characterized that antibody conjugate a), hapten conjugate b) and insoluble Phase present binding partner c) simultaneously are incubated with each other. 3. The method according to claim 1, characterized characterized that antibody conjugate a) and hapten conjugate b) with each other cubed, then with the insoluble phase lying partner c) brought together and be incubated again.   4. The method according to any one of claims 1 to 3, characterized, that the antibody conjugate a) an antibody Fab Contains fragment. 5. The method according to any one of the preceding claims, characterized, that the antibody conjugate a) a determinable Contains enzyme. 6. The method according to any one of the preceding claims, characterized, that the hapten conjugate as a carrier is an Ig fraction contains. 7. The method according to any one of the preceding claims, characterized, that the insoluble phase c) as a binding partner contains antibodies directed against Ig. 8. The method according to claim 7, characterized characterized that the unsolvable phase against the Fc part of Ig Contains antibodies. 9. Reagent for the determination of hapten containing labeled anti-hapten antibodies in certain Quantity and in the solid phase, specific bindable substance, thereby characterized that it is a soluble hapten conjugate of one or more Hapten molecules and a specifically bindable Contains carriers in a certain amount and the solid phase-bound, specifically bindable substance binds the carrier of the hapten conjugate.   10. Reagent according to claim 9, characterized characterized that the carrier of the hapten conjugate and the solid phase bound specifically bindable substance from each other an antigen-antibody complex forming sub punching exist. 11. Reagent according to claim 10, characterized characterized that the with form an antigen-antibody complex the substances from an Ig fraction and the corresponding anti-Ig antibodies exist. 12. Reagent according to claim 11, characterized characterized that the anti Ig antibodies against the Fc part of the Ig fraction is directed. 13. Reagent component for the determination of a multiple number of different haptens according to the procedure according to claims 1 to 8, characterized characterized that they are from a solid inert carrier material to which one against the Fc part of an Ig fraction teter antibody is bound. 14. reagent component according to claim 13, characterized, that the antibody is covalently attached to the support or is adsorptively bound. 15. reagent component according to claim 13, characterized, that the antibody is attached to the carrier by immunoprecision pitation is bound.   16. reagent component according to claim 13, 14 or 15, characterized, that the carrier consists of a non-woven fabric.