FR2523311A1 - Conjugates of specific ligand and albumin - for use in immunoassay - Google Patents

Abstract

The following are claimed: (A) an alburmin/specific ligand coupling prod. which is soluble in aq. media; (B) a process for immunoassoy of a biological substance (I), comprising (a) immobilising a substance (II) having binding affinity for (I), (b) incubating with a medium contg. (I), (c) washing the resulting reaction mixt. and incubating with an albumin/specific ligand coupling prod. in soln. in an aq. medium, where the ligand is capable of reacting specifically with (I) or (II), (d) washing the resulting reaction mixt. and incubating with a labelled anti-albumin antibody, and (e) detecting the label; (C) an immunoassay test kit comprising an albumin/ specific ligand coupling prod., a labelled anti-albumin antibody and reagents for detecting the label. Coupling with albumin increases sensitivity, esp. in the base of enzyme immunoassays for antigens, haptens or antibodies.

Description

 The present invention relates to a coupling product between an albumin and a specific ligand, its production and its applications in biological techniques, in particular of localization, detection and assays of antibodies or antigens. The coupling product according to the invention is particularly suitable as a universal reagent in the various types of biological and in particular immunological assays.

 In immunology, we have already used, in particular, products for coupling antibodies or antigens with different substances, as markers, such as, for example, enzymes, radioisotopes, ferritin, red blood cells, fluorochromes. Such coupling products are used as reagents according to various immunological methods for the localization, the demonstration or the determination of the humoral or cellular constituents.

For more details on these known coupling products, reference may be made to the following work: "Handbook of experimental immunology", 3 vol. 3 Ed. DM Weir (1978)
Blackwell Scientific. Increasingly, enzyme immunoassays are used. However, they are very expensive insofar as they require significant quantities of eftzymefe
It has also been proposed to use, as dosing reagents, coupling products between a lectin and a specific ligand. To this end, reference may be made to patent application FR 80.11.470 (filed on May 24, 1980). to the names of the applicants, entitled: "Coupling product between a lectin and a specific ligand, obtenton and applicatioals in the biological field".

 We have now found a new series of coupling products which can be used in a large number of fields, these are the coupling products between an albumin and a specific ligand.

 It should be noted that the use of albumin has already been proposed as a stabilizing agent in reagents for immunoenzymatic assays. To this end, mention may be made of US Pat. No. 4,002,532 which describes enzymatic conjugates consisting of an enzyme, a polyfunctional coupling agent, a polyamine, such as albumin and a non-protein macromolecule. - enzymatic. In this con jugue, polyamine, for example albumin, plays the role of conditioning agent to stabilize the macromolecule-enzyme complex. This polyamine does not intervene directly in the immunoenzymati assay (eu in which such a conjugate is used.

 The present invention relates, as a new product, to a coupling product between an albumin and a specific ligand, suitable as an assay reagent, in which albumin intervenes directly in the conjugate.

 By specific bone ligand denotes in the present description any soluble substance which can react in a specific way with another biological or particulate substance.

 By soluble substance is meant any substance soluble in the media commonly used for biological reactions. They can be aqueous media, such as physiological media, or mixtures of aqueous and organic media.

 In addition, the specific ligand used according to the invention must be such that the albu mine-ligand coupling product according to the invention is soluble in an aqueous medium.

 The term "aqueous medium" means, according to the invention, aqueous media, tarnished or not, commonly used in the biological field, such as phosphate buffer solutions; buffer solutions containing a detergent, such as "Tween", or gelatin , bovine lactalbumin and other substances usually used in such fields.

 The specific ligands corresponding to such a definition are in particular antibodies, macromolecular antigens or haptens, hormones and their receptors, enzymes and their inhibitors, lectins and similar substances. Among the specific ligands mentioned above, antibodies and antigens are most commonly used.

 For the purposes of the invention, a serum albumin (main protein present in the serum) is generally used, which can come from different animal species (beef, sheep, rabbit, man, etc.). Advantageously, bovine serum albumin is used.

 The product according to the invention is obtained by coupling an albumin with the ligand via an appropriate coupling agent, such as for example glutaraldehyde and benzoquinone. The coupling is advantageously carried out by covalent bonds.

 The specific albumin-ligand coupling is carried out according to a process analogous to those used for protein coupling.

 For example, the specific albumin-ligand coupling can be carried out by a process similar to that described for obtaining antibody-enzyme conjugates by S.

Avrameas et al. in Scand. 3. Immunol., Vol. 8 suppl. 7, pages 7-23, 1978. Such a coupling process consists in bringing into contact, in one or more stages, a coupling agent with the substances to be coupled.

 When benzoquinone is used as coupling agent, it is equally possible to activate albumin or the specific ligand first. As a document illustrating the coupling technique with benzoquinone, mention may also be made of the patent FR 75.37.392 (publication NO 2.334.107).

 When glutaraldehyde is used as the coupling agent, the albumin and the specific ligand are advantageously mixed and glutaraldehyde is added to the mixture in appropriate amounts to effect the coupling.

However, this coupling can also be carried out in two stages, by bringing one of the reagents into contact with glutaraldehyde, eliminating the excess glutaraldehyde and adding the other reagent.

 In many cases, glutaraldehyde is suitable as a coupling agent, for example with antigens or antibodies. The following table illustrates the preparation of specific albumin-ligand conjugates (antiserum, immunoglobulins, antibodies) using glutaraldehyde as a coupling agent. However, these examples are not limiting.

TABLE 1 specific ligand quantity quantity of albumin concentration of
glutaraldehyde
Antiserum 50 1 5 mg 0.2 X
Immunoglobulins 2.5 mg 5 mg 0.2 X
Antibody 2.4 mg 6 mg 0.2%
The albumin and the specific ligand are dissolved in a phosphate buffer pH 6.8 0.1 M; to this mixture is added glutaraldehyde at a concentration of 0.2%. The total volume of the reaction medium is 1 ml.

 During the coupling between albumin and the specific ligand, it is possible to use a large excess of albumin so as to obtain a high value of the albuminelligand molar ratio. It is not essential to use a highly purified form of the ligand. For example, total serum may be used, as well as serum purified by suitable conventional means, such as saline quarries, ion exchange columns, affinity chromatography.

The specific albumin-ligand coupling product according to the invention can be characterized by its ability to react with a specific substance and at the same time with a specific albumin marker, such as an anti-albumin antibody. For example, if the specific albumin-ligand coupling product is an albumin-an-
tigene, it can be characterized by its ability to react
with the corresponding specific substance, namely the corresponding antibody.

 If the specific albumin-ligand coupling product is an albumin-antibody product, it can be characterized by its ability to react with the specific substance, namely the corresponding antigen. In all cases, these conjugates must react with the anti-albumin antibody labeled with a suitable marker such as an enzyme, a fluorescent, radioactive substance or red blood cells coupled by appropriate means as described in "Handbook of experimental immunology" DM Weir 1978 , Blackwell Scientific.

 The coupling product according to the invention can advantageously be stored in aqueous solution at low temperature, for example at 40C after addition of an adequate volume of glycerol.

 The specific albumin-ligand coupling product according to the invention is suitable as a reagent in various immunological methods for localizing, detecting and assaying biological substances, such as the immunological assays of the non-competitive type, with double antibody or by competition. It is advantageously used in the form of an aqueous solution, buffered or not.

 The albumin-ligand coupling product according to the invention in the form of an aqueous solution is particularly suitable as a reagent in immunological techniques, in which one of the reagents or the substance to be identified is insoluble or immobilized on an insoluble support. It is therefore particularly suitable for all types of assays using an insoluble support. However, the product of the invention can also be used in the soluble phase.

 By way of illustrative example, the diagram attached to a method for detecting an immobilized antigen has been indicated in the attached single figure (diagram A).

 To demonstrate the antigen (2) immobilized on a specific immunoadsorbent (1), add the albumin-ligand coupling product (3) according to the invention, dissolved in the aqueous medium, the ligand in this particular case being a antibody. The antibody part of the coupling product (3) reacts with the immobilized antigen, while the albumin of said coupling product remains accessible.

 After washing the immunoadsorbent to remove the excess antibody-albumin conjugate, a compound (4) consisting of anti-albumin antibodies linked with a marker, that is to say a substance which is easy to demonstrate, is added.

 In the method for assaying an antigen described above, albumin acts as a marker acceptor.

 By "marker" is meant, according to the invention, any substance of any kind, which allows an identification or a determination. Examples of markers1 include enzymes, fluorescent derivatives (fluorochromes), radioactive elements, red blood cells, lectins, particulate matter and the like.

 The anti-albumin antibody used in the assay method is obtained by conventional means.

 The anti-albumin antibody is linked with the substance which plays the role of marker by suitable conventional coupling means. It constitutes another aspect of the invention being a means for implementing the dosing method according to the invention. .

 Each of the above markers corresponds to an immunological technique which is respectively the immunoenzymatic technique (immunoenzymatic assay or histochemical detection), immunofluorescence (immunofluorimetric assay or histochemical detection) radioimmunological technique, hemagglutination or erythroadsorption, as it is. defined for example in patent application FR 80.15.293 in the name of the applicants and entitled "Method for detection and determination of a biological substance by erythroådsorption" filed on July 9, 1980.

 By way of nonlimiting example, there will be described below an assay method using an albumin-ligand coupling product according to the invention.

This assay method for determining a given biological substance consists of
1) to immobilize a substance having an affinity
fixing tee with respect to the organic substance
logical to dose;
2) to incubate this substance with the medium
containing the biological substance to be assayed;
3) to incubate, after washing, the medium
resulting share with a coupling product
age specific albumin-ligand, in solution
in an aqueous medium, said ligand being ca
able to react specifically with ladi
the substance with an affinity for sub
biological stanza to be dosed or with said sub
biological stanza itself;
4) washing the resulting reaction medium and
incubate with anti-al antibody
bumine carrying a marker;
5) to reveal the marker by an appropriate means.

 This method is suitable for assaying antigens, antibodies, immunoglobulins and other substances of biological interest.

 The substance having a binding affinity towards the biological substance to be assayed can be any substance capable of specifically binding with said biological substance. For example, if the biological substance to be assayed is an antibody, the substance having a binding affinity will then be an antigen and vice versa.

 Any marker can be used according to this method, such as those mentioned above, in particular enzymes and red blood cells, in which case step 5) is a step of enzymatic determination or erythroadsorption.

Advantageously, microplates are used to immobilize the antigen or the antibody used according to the above method, for example polystyrene microplates. You can also use supports
in the form of gels, preferably magnetic gels, such
than those described in patent FR 75.36.889 (published under
NO 2,334,106) and in patent application FR 79.21.343.

As a support, you can use any im
which insoluble support is presented in a challenge form
denies, such as a plate or sheet, or in a form
particulate. As a constituent substance of such sup
ports, we can use cellulose and its derivatives, po
lyacrylamide, polymethyl alkyl methacrylates and others
lymers of natural or synthetic origin as well as the worm
re.

Note that the coupling product according to
the invention, used in the form of a solution, provides
considerable advantages in the field of immunoassays
logical in the sense that its implementation requires no
separation steps, centrifugation, filtration, other than
that imposed by the immobilization support of one of the
reagents, for example, washing the support or separation at
using a magnet in the case of magnetic gels.

The sensitivity of the challenge dosing process
neither above is magnified compared to that of the processes
conventional immunoassays, in effect for the same
enzymatic reaction time, the sensitivity of the process of the invention is of the order of 3 to 5 times greater than that
conventional methods.

We think the amplification is provo
as albumin, because you can attach more marker
since albumin at multiple sites available
to fix said marker. The coupling product according to
the invention makes it possible to carry out assays with more than
sensitivity without using large amounts of enzyme
mes for example.

The subject of the invention is also a cof
freight for immunoassay, including
1) a coupling product between an albu
mine and a specific ligand,
2) an anti-albumin antibody carrying a
marker pen,
3) means to reveal the marker.

The invention will now be described in more detail in the illustrative but nonlimiting examples below.
EXAMPLE 1:
Preparation of the albumin-antibody coupling product using glutaraldehyde.

Anti sheep antibodies were used
Pure rabbit Ig isolated by affinity chromatography.

 Antibodies and albumin (bovine serum albumin) were dialyzed against 0.1M phosphate buffer pH 6.8 overnight at 40C. The dialyzed antibodies (3 mg) and albumin (6 mg) were mixed in a 0.1 M phosphate buffer solution, pH 6.8. The total volume was 1 ml, 0.2 ml of a 1% aqueous glutaraldehyde solution was added to the mixture, which was then incubated for 3 hours at room temperature.

EXAMPLE 2
Antigen assay
In this example, bovine serum albumin (BSA) coupled with anti IgE antibodies was used as coupling product of the invention according to the procedure described in Example 1 above. This coupling product will be referred to below: anti IgE-BSA,
The anti-albumin antibody carrying the marker used in this product was the anti-BSA-P-galactosidase conjugate, obtained by coupling of -galactosidase with the anti BSA antibody by means of glutaraldehyde.

Determination of human IgE
The assay of human IgE was carried out by a "sandwich" technique following the protocol below:
Adsorption of anti IgE antibodies on a polystyrene plate (Elisa type);
Washing the plate;
Incubation (overnight at 40C) with standard serum (0 - 100 IU / ml) and with patient sera;
Washing the plate;
Incubation (two hours at 370C) with the anti IgE - BSA conjugate (obtained according to Example 1);
Washing the plate;
Incubation (two hours at 370C) with the anti BSA - * galactosidase conjugate;
Washing the plate;
Enzymatic reaction (30 minutes);
stopping the enzymatic reaction and measuring the optical density at 405 m.

 The results obtained by following this protocol are given in Table 2 compared with the results obtained by the conventional technique using an anti IgE-g galactosidase conjugate.

The results clearly show that the sensitivity of the assay is better when the anti IgE-galactosidase conjugate is replaced by the anti conjugate.
IgE - BSA according to the invention.

EXAMPLE 3
Determination of a hapten
The hapten assay was performed using the competition technique and a hapten conjugate
BSA. The assay of Adenosine - Mono - Cyclic Phosphate (cAMP) was used as an experimental model.
The protocol below was followed
Passive adsorption of anti cAMP antibodies on a polystyrene plate;
Washing the plate;
Incubation (one hour at 370C) of the standard solution containing known concentrations of dlAMPc or of the solution to be titrated;
Addition of the cAMP-BSA conjugate;
Incubation (two hours at 370C) of the melanue
Washing the plate;
Incubation (three hours at 370C) with the anti BSA - t galactosidase conjugate;
Washing the plate;
Enzymatic reaction (16 hours at 200C);
Stopping the enzymatic reaction and measuring the optical density at 405 nm.

 The result of a cAMP calibration range obtained by following this protocol is given in Table 3.

EXAMPLE 4:
Titration of group B anti-streptococcus type III antibodies
The titration is carried out as follows
Adsorption of the polysaccharide antigen on a polystyrene plate;
Washing the plate;
Incubation with the serum to be titrated;
Washing the plate;
Incubation with the anti IgC-BSA conjugate;
Washing the plate;
Incubation with the anti-BSA-Bga-lactosidase conjugate;
Washing the plate;
Enzymatic reaction;
stopping the enzymatic reaction and measuring the optical density at 405 nm.

 The titration results reported in Table 4 show that the process according to the invention gives better results. In fact, it can be seen that the sensitivity is significantly increased compared to the conventional method (method 2).

EXAMPLE 5
Titration of anti-Echinococcus granulosus antibodies
60 sera from patients with hydatidosis were tested by the method described above, and the results were compared with those obtained by the conventional Elisa method. It follows from this study that there is a good correlation between the two methods and that the results are obtained more quickly with the technique using the anti IgG-BSA conjugate (enzymatic reaction 20 minutes) than with the technique using the anti IgG conjugate -phospatase (enzyme reaction 18 hours).

 As an example, Table 5 gives the. result of the titration of a patient serum and a normal serum.

TABLE 2
Determination of human I9E. The assay is carried out in the same way either using the anti IgE-BSA conjugate of the invention (method 1), or using the anti IgE-pga-lactosidase conjugate (method 2).

(IgE Optical density in IU / ml method 1 method 2 100 2.08 0.59
50 1.47 0.36
25 0.80 0.15
13 0.34 0.04
6 0.15 0.02
3 0.07 0.00
TABLE ss
CAMP assay using a cAMP-BSA conjugate (cAMP) Density Percentage in optical nM of activity 10,000 0.084 11
3.333 0.237 32
1,111 0.379 51
0.370 0.525 70
0.123 0.670 90
0.041 0.700 94
0.014 0.783 105
0.000 0.745 100
TABLE 4
Titration of an anti stretococcus type III group B serum.

The titration is carried out either with the anti IgG-BSA conjugate (method 1) or by way of comparison, with the anti IgG-p galactosidase conjugate (method 2).

Optical density
Dilution of serum method 1 method 2 1 / 200,000 1.25 0.32 1 / 400,000 0.88 0.20 1 / 800,000 0.60 0.14 1 / 1,600,000 0.33 0.06 1 / 3,200,000 0.20 0.03 1 / 6,400,000 0.13 0.03 1 / 12,800,000 0.07 0.01 1 / 25,600,000 0.04 0.00
TABLE 5
Titration of human anti-Echinococcus granulosus antibodies using the anti IgC-BSA conjugate
Optical density
Dilution of serum normal serum patient serum 1/1600 0.109 1.106 1/3200 0.070 0.983 1/6400 0.024 0.840 1 / 12.800 0.031 0.670 1 / 25.600 0.014 0.483 1 / 51.200 0.000 0.281 1 / 102.400 0.000 0.159 1 / 204.800 0.000 0.057
It goes without saying that the above examples are purely illustrative. The products of the invention are applicable in the most varied assay techniques. If necessary, a person skilled in the art can refer to the works below.

- Affinity techniques: E myme Purification
WB 3acoby and M. Wilchek, eds Methods in Enzymology - Vol 34, part B - Academic Press NY 1974,
- Affinity chromatography and Molecular
Interactions, 3.M. Egly ed. INSERM - Vol. 86.

which describe techniques which can be used for the purposes of the invention.

Claims (14)

 1. As a new product, the coupling product between an albumin and a specific ligand, said product being soluble in an aqueous medium.  2. Coupling product according to claim 1, characterized in that the albumin is of animal or human origin.  3. Product according to one of claims 1 or'2, characterized in that the specific ligand is an antigen or a hapten, an antibody, a hormone or its receptor, an enzyme or its inhibitor, an iectin.  4. Product according to any one of claims 1 to 3, characterized in that it is obtained by coupling using glutaraldehyde.  5. Product according to any one of claims 1 to 4, characterized in that it is obtained by coupling using benzoquinone in two stages, the first consisting in activating albumin or the ligand with benzoquinone and the second to incubate the resulting product with the second product to couple.  6. Immunoassay reagent, characterized in that it consists of a product for coupling an albumin with a specific ligand, said coupling product being soluble in an aqueous medium.  7. Assay reagent according to claim 5, characterized in that the albumin is bovine serum albumin and in that the specific ligand is an antigen or an antibody.  8. Application of the coupling product according to any one of claims 1 to 7, for the localization, the detection and the assay of an antigen or an antibody by fluorimetry, radioimmunology, immunoenzymology, erythroadsorption.  9. A method of immunological assay of a biological substance, characterized in that it consists  1) to immobilize a substance having a binding affinity for the biological substance to be assayed;  2) to incubate this substance with the medium containing the biological substance to be assayed;  3) to incubate, after washing, the resulting reaction medium with a specific albumin-ligand coupling product, in solution in an aqueous medium, said ligand being capable of reacting specifically with said substance having an affinity for the biological substance to to dose or with said biological substance itself;  4) washing the resulting reaction medium and incubating it with an anti-albumin antibody carrying a label;  5) to reveal the marker by an appropriate means.  10. Method according to claim 9, characterized in that the marker is an enzyme, a substance carrying a radioactive isotope or a fluorochrome or a particulate substance coupled with an anti-albumin antibody.  11. Method according to claim 9, characterized in that the marker consists of red blood cells.  12. Means for implementing the method according to any one of claims 9 to 11, characterized in that it consists of an anti-albumin antibody coupled with a label.  13. Means according to claim 12, characterized in that the marker is an enzyme, a substance carrying a radioactive isotope or a fluorochrome or a particulate substance.  3) means to reveal the marker.  2) an anti-albumin antibody carrying a marker  1) a coupling product between an albumin and a specific ligand  14, Reagent kit for the immunoassay, characterized in that it comprises