DE69432897T2 - METHOD FOR DETERMINING MORE THAN AN IMMUNOLOGICAL LIGAND AND DETERMINATION REAGENT AND METHOD THEREOF - Google Patents

Abstract

An assay reagent and a kit whereby nonspecific capture of a labeled substance by a solid phase can be suppressed and more than one antibody or more than one antigen can be detected via a simple assay by using one reagent. An immunological ligand assay reagent comprising components selected from the following groups (A) and (B) at the same time is reacted with immunogical ligands to thereby form complexes, which are captured by separate solid phases (C) and analyzed based on the labeling substance contained in each complex: group (A): more than one type of immunological antiligand-nucleotide conjugates wherein antibodies having specific avidities respectively for immunological ligands of different types are bonded to nucleotides which are specific respectively for said immunological ligands and each has an arbitrarily selected base sequence; group (B): labeled substances having specific avidities respectively for the immunological antiligands of different types which are to be assayed; and group (C): solid phase-nucleotide conjugates wherein nucleotides having base sequences capable of complementarily binding respectively to the nucleotides of the group (A) are bonded to a water-insoluble carrier. Either antigens or antibodies are applicable to said immunological ligands and immunological antiligands. <MATH>

Description

TECHNICAL TERRITORY

The present invention relates to a method for the immunological determination of biological substances using an antigen-antibody reaction. More specifically The present invention relates to a method for determining one or more types of antigen or one or more Types of antibodies, characterized in that the method is capable of an almost infinite number of combination of a or more types of antigen or one or more species of antibodies to determine a determination reagent that uses the method and equipment, which applies the method.

STATE OF THE ART

Immunoassays were in the field clinical diagnosis for determining and detecting a lane biological substances, and it has become a variety of methods designed for it. Because immunoassays are non-radioactive substances, such as fluorescent Substances, luminescent substances and enzymes as markers Substances of antibodies use and therefore do not need the specific equipment that is needed when a radioactive substance is used as a marker substance, Such provisions have been made in terms of ease of use the reagents and processability of a large number of more widely used samples for determining biological substances used.

Such immunoassays can be used in the Generally only a single antigen of a single clinical Sample is determined or detected. Are several types of antigen present in a sample which, e.g. B., defined as antigen A, B and C. then a specific reagent should be used to selectively detect the Antigens A may be required, as is the case for the antigens B and C is. These antigens also require their corresponding specifics Reagents. In general, there are a number of clinical test results Integrally required to clinically treat the illness of a patient to diagnose. To the appropriate treatment under a suitable Therefore, one patient should generally receive several be subjected to clinical tests. That's why that takes Volume of sample taken from the patient in most cases proportional to the required number of clinical tests for this patient to what a physical one Burden for represents the patient. In order to reduce such burden, However, there is currently no satisfactory method of determination, that is simple and very sensitive.

As an immunoassay method, to simultaneously the presence and / or amount of one or more Types of antibodies in a sample, the dot blot was ting, z. B., from the JP-OS Hei 4-232465 (1992). Dot blotting includes the previous one Applying different types of antigens to a nitrocellulose membrane, Reacting a sample that may be several antibodies to be detected contains with the antigens on the nitrocellulose membrane and subsequent Binding of a labeling substance to the specified antibodies the nitrocellulose membrane to increase the presence or amount of antibodies determine. Such dot blotting to detect one or more Types of antibodies however, is problematic in that respect as the whole process a long time and a larger volume requires a sample. There is also a problem with dot blotting that the antigens to detect antibodies on a solid phase should be immobilized and the immobilized antigens at Camps affected become.

Alternatively shows 1 the scheme of a general method of a different from dot blotting other immunoassay method, as a conventional immunoassay method, the sandwich assay called and performed by means of a labeled compound.

Now, conventional sandwich assays using a labeling compound will be described with reference to FIG 1 described in the 1 represents a solid phase of a water-insoluble carrier, 2 represents an antibody immobilized on the solid phase, 3 represents an antigen to be detected which has reacted with the antibody and is then bound to the antibody and 4 represents a labeled antibody.

By first putting a test sample in contact with on the solid phase 1 immobilized antibody 2 should, the antigen to be detected in the test sample to the antibody 2 ( 1 (a) ). To remove the unreacted impurities in the reaction system, a washing is carried out ( 1 (b) ). The labeled antibody 4 reacts with the solid phase antibody-antigen complex to form a solid phase antibody-antigen-labeled antibody complex ( 1 (c) ). A washing is carried out to remove the impurities such as unreacted labeled antibody and the like in the reaction system according to the above method ( 1 (d) ). By detecting the label, the washed solid-phase antibody-antigen-labeled antibody complex is determined ( 1 (e) ).

By such general immunoassay procedures will the for the determination responsible labeled antibodies from the solid phase antibody antigen-labeled Antibody complex won. Sometimes a so-called non-specific binding occurs of the labeled antibody directly on the solid phase, and in such a case is the Finally, assay sensitivity disadvantageously reduced.

Many of the pathogenic factors, such as specific hormones, tumor markers and bacteria, are present in a trace amount in samples. Therefore, a highly sensitive assay system was required for the assay or detection and was developed (see, e.g., Enzyme Immunoassay, Eiji Ishikawa, Ed., Igaku Shoin, 1987; Micrbiol Immunol., Y. Oku et al , 32 , pp. 807-816, 1988). The main problem in designing a very sensitive assay system is the non-specific binding of the labeled antibody to the solid phase.

For the purpose of eliminating such non-specific binding, the immunocomplex transfer assay (J. Biochem., S. Hashida et al., 108 , pp. 960-964, 1990) and the like were developed to provide practically higher sensitivity than the to achieve conventional assays. The immune complex transfer assay comprises the following steps. More specifically, the assay involves simultaneously mixing an antibody conjugated to a dinitrophenyl group (termed "DNP" group) and biotin, a specific antigen-recognizing antibody, a test sample, and an enzyme-labeled antibody that recognizes the antigen to inhibit the reaction of the antibody antigen contained in the test sample with each antibody to form an immune complex (hereinafter referred to as "IC"). Alternatively, by immobilizing an antibody against the DNP group on a solid phase, bringing the previously prepared solid phase IC into contact to promote an antigen-antibody reaction between the DNP group contained in the IC and the antibody the DNP group on the solid phase, the IC on the solid phase.

To the effects of non-specific Binding of the reaction system contained in the solid phase Enzyme-labeled antibody ruled out The retained IC is added by adding an excess amount of a connection with a DNP group released from it. After that is the liberated IC of the reaction with another solid phase, immobilized the avidin, subjected to the IC again on the solid Phase, and then the activity of the enzyme is on the solid Phase captured IC measured in a test tube, causing the Effects of non-specific binding of the enzyme-labeled antibody the solid phase can be eliminated to high sensitivity to achieve. None of the conventionally known immune complex transfer assay However, there is evidence and determination of the presence of a or more types of antigen or one or more types of antigen antibodies by means of a single reagent.

Japanese Laid-Open Patent Publication Sho 63-188399 (1988) a method for determining a target molecule as a biological binding pair in a sample. Specifically, the publication describes that the determination method bringing a sample containing a target molecule into contact with one first anti-ligand probe and a second labeled anti-ligand probe, to bind the target molecule to form a complex and a recoverable carrier in are capable of, then essentially separating the recoverable carrier from the sample to recover an isolated product containing the target molecule and the first and second probe in the presence of the target molecule in the Sample includes, and further determining the presence of the target product that the Presence of the target molecule indicates includes. The assay method described in JP-OS Sho 63-188399 does not describe the detection and determination of presence one or more types of antigen or one or more Types of antibodies by means of a single reagent.

JP-OS Hei 4-273065 (1992) describes a method for detecting an antigen contained in a sample with high sensitivity, including prior immobilization an antibody through a nucleic acid on a solid phase, capturing one contained in a sample Antigen by the antigen-antibody reaction on solid phase, then holding a marker substance on it before washing; selective splitting of the nucleic acid for Separating and determining the separated labeling substance. The However, the method described in JP-OS Hei 4-273065 describes not the detection and determination of the presence of one or more several types of antigens or one or more types of antibodies by means of a single reagent, it still contains a hint to that effect.

As described above, the entire Method of conventional dot blottin for detecting an or several types of antibodies requires a long time, and in addition it required a relative time larger volume a sample.

Because the reaction to retain the labeled antibody on the solid phase, an antigen-antibody reaction in the in 1 As shown in the conventional immunoassay method, the time required to retain the labeled antibody on the solid phase is relatively long in units of hours. The duration during which the labeling substance of the reaction system is exposed to the solid phase is prolonged so much that a so-called non-specific binding of the labeling substance occurs directly on the solid phase, which is problematic because of the decrease in the assay sensitivity.

The conventional immune complex transfer assay method, the one higher Sensitivity should require a larger number complex assay procedure takes a long time for such reactions, which is the Main disadvantage is.

It is thus a first task of present invention, the occurrence of nonspecific binding a labeling substance to be added to the reaction system which causes a decrease in assay sensitivity to a minimum bring to. additionally to the first object, it is a second object of the present invention, an assay reagent provide one or more types of antibodies or one or more types of antigens using a single reagent can prove by a simple procedure, further equipment below Application thereof and an assay method using the same provide.

DISCLOSURE OF THE INVENTION

To the problems described above to overcome, The present invention provides a determination reagent for determination one or more types of immunological ligands that simultaneously contains the following substance groups (A) and (B): Substance group (A): one or several types of immunological anti-ligand nucleotide conjugates, in each of which nucleotides having a specific base sequence, independently selected dependent on of the types of an immunological ligand, to an immunological one Antiligands with a specific immunological affinity for one immunological ligands, as distinct types of subjective Substances are bound; and

Substance group (B): marked substances, each having a specific affinity for one of the immunological Have ligands, as the different types of subjective Substances.

In addition, the present presents Invention a determination equipment for determining one or more types of immunological ligands ready, comprising a determination reagent for determining a or several types of immunological ligands, the same at the same time Substance groups (A) and (B) and the following solid phase (C) contain:

Substance group (A): one or more Types of immunological anti-ligand nucleotide conjugates, wherein in each of which nucleotides having a specific base sequence independently selected in dependence from the types of an immunological ligand, to an immunological one Antiligands with a specific immunological affinity for one immunological ligands, as distinct types of subjective Substances are bonded;

Substance group (B): marked substances, each having a specific affinity for one of the immunological Have ligands, as the different types of subjective substances; and

Solid phase (C): solid phase nucleotide conjugate, wherein nucleotides having a base sequence complementary to the Nucleotides of the above group of substances (t connects, on a water carrier are immobilized.

According to the present invention the term "immunological Ligand "a molecule in one immunologically formed couple while the term "immunological Antiligand "that other molecule in the immunologically-formed pair. This immunological Ligand or immunological anti-ligand specifically means either Antigen or antibody.

As the nucleotides, a complementary sequence according to the present Invention can have DNA or RNA are used. For such nucleotides can both synthetic nucleotides and naturally occurring nucleotides The nucleotides may be oligonucleotides or polynucleotides his.

An antibody-nucleotide conjugate or an antigen-nucleotide conjugate is attached to nucleotides that through the complementary Pairing of the base sequences these nucleotides on a solid Phase are immobilized. Their complementary base sequences can be partial or completely be complementary, as long as the nucleotide molecules bind each other.

In general, the complementary pairing has of nucleotides characteristically has a high specificity, so the for such a complementary one Pairing is considerably shorter than education an antigen-antibody complex required time.

According to the present invention, the binding of an antibody-nucleotide conjugate or an antigen-nucleotide conjugate to a solid phase to which other nucleotides are immobilized (solid-phase immobilized nucleotides) is much shorter than the time for binding a labeled substance to a complex of an antibody immobilizing solid phase and an antigen (a solid phase-bound antibody-antigen complex) according to the conventional immunoassay is required. The reason for this is that according to the conventional method for determining an immunological ligand, namely an antigen or an antibody, a subject substance conjugated to a labeled substance is retained on a solid phase by an antigen-antibody reaction requiring a longer time while for the method of determining immunological ligands according to the present invention Invention relates a subject substance conjugated to a labeled substance to the complementary pairing of nucleotides, which advantageously takes much less time than that for the antigen-antibody reaction, to capture it on a solid phase. Because a labeled substance, eg. A labeled antibody, according to the present invention, can not be given sufficient time to directly attach to a solid phase, non-specific binding can be reduced to achieve a highly sensitive assay system.

According to the present invention is a single reagent capable of one of several types of immunological Ligands, namely Antigens or antibodies, or to determine, so that for their purpose or their Proof required time is noticeably shortened, compared with the conventional method.

With regard to the solid-phase nucleotide conjugate, as the solid phase (C), can the nucleotides at the 5 'end or 3'-end or another optional position than the final position to a water-insoluble carrier be bound, directly or through a functional group, in the water-insoluble Carrier is introduced to a solid phase nucleotide conjugate to form C as a solid phase. A functional phase introduced into a solid phase Group can be covalently bound to a functional group, which is introduced into a nucleotide-forming base to form a covalent one Bond to form.

As another method of tying from nucleotides to a solid phase, instead of covalent bonding a physical adsorption can be used. Spezfischer are nucleotides covalent at the 5'-end or 3'-end or another optional position than these ends to a linking ligand bonded directly or through a functional group in the binding ligands introduced to form a binding ligand-nucleotide conjugate, which is then physically to a water-insoluble carrier is adsorbed to a solid phase nucleotide conjugate than the solid Phase (C) to form. Preferably, the binding ligand may be a protein.

For into a labeled substance in the assay reagent for determination immunological ligands according to the present invention Invention to be introduced Labeling, this may be from an enzymatic atomic group, biotin, Avidin, digoxigenin, nucleotides, a metal colloid particle, a fluorescent substance, a luminescent substance, a metal compound, a ligand having a specific binding affinity or a Be prepared radioisotope. For according to the present The invention to be used solid phase may preferably polystyrene to be used.

The method for determining one or more types of immunological ligands according to the present invention will now be described with reference to 2 to 10 in which an antigen is shown as the immunological ligand to be determined.

2 FIG. 12 shows an example of the case where the assay reagent for determining one or more types of immunological reactants is a detection reagent for determining one or more kinds of antigens and thereby schematically the detection reagent for determining one or more kinds of antigens, antigens A, B and C, images. In 2 all components within the scope of the components contained in a single reagent. In 2 represent α-A, α-B and α-C antibodies against the antigens A, B and C.

The antibodies α-A, α-B and α-C, each with a label are thus prepared to modified labeled substances, so the reagent labeled three types of substances at the same time contains. Together with the aforementioned three kinds of labeled substances, the reagent contains three more Types of antibody-nucleotide complexes, composed of the antibodies α-A, α-B and α-C, which are independently with different sequences of the nucleotides ON1, ON2 and ON3, in this order, are connected; The nucleotide ON1 has a base sequence, which differs from those of the other nucleotides ON2 and ON3. The nucleotide ON2 has a base sequence different from that of the different nucleotides ON1 and ON3. The nucleotide ON3 has a base sequence different from those of the other nucleotides ON1 and ON2 is different.

Nucleotides of an identical base sequence should be bound to an identical type of antibody. According to the invention are defined by the term "one identical type of antibody "also polyclonal antibody includes.

3 shows an example of solid phases to which nucleotides are attached for use in combination with the assay reagent for determining one or more types of antigens according to the present invention; 3 (a) shows a solid phase to which nucleotides (ON) adhere to a base sequence that is complementary to the nucleotide ON1 (solid phase for antigen A); 3 (b) shows a solid phase to which nucleotides (ON) adhere to a base sequence that is complementary to the nucleotide ON2 (solid phase for antigen B) and 3 (c) shows a solid phase to which nucleotides (ON) adhere to a base sequence that is complementary to the nucleotide ON3 (solid phase for antigen C). These different types of single solid phases are independent and available separately. Used in combination with the assay reagent to determine one or more types of antigens 2 These single solid phases represent an example of the determination equipment according to the present invention.

If the assay reagent is the 2 to a test sample containing antigens A, B and C, then complexes of (antibody-nucleotide conjugate) antigen-labeled substance individually corresponding to antigens A, B and C are formed in the reaction solution as in 4 shown.

Bringing the reaction solution containing the complexes of (antibody-nucleotide-conjugate) antigen-labeled substance in contact with the solid phase for antigen A, the solid phase for antigen B and the solid phase for antigen C, all of them Phases are independently and separately present, then the nucleotides contained in the complexes are complementarily paired by hybridization with the nucleotides of the solid phases for antigens A, B and C, which have base sequences complementary to those of the nucleotides described in the above Complexes are included, as in 5 . 6 or figure shown.

By subsequent washing, impurities that do not adhere to one of the solid phases, eg, labeled substances and the like can be removed. figure 8th 9 and 10 show the state of the complex carrying antigen A complementarily paired on the solid phase for antigen A, the state of the complex carrying antigen B mated complementarily on the solid phase for antigen B, and the state of the complex carrying antigen C, which is complementarily paired on the solid phase for antigen C, respectively after washing. The labels contained in the complexes attached to each solid phase are determined next. be, for. For example, enzyme reactions are promoted in each independent reaction system to determine the level of labeling over individual colors and the like. If the labels are fluorescent substances, dyes, metal colloids or the like, then the determination can be carried out without such enzyme reactions.

As described above, this explains Method for determining one or more types of antigens the case where three types of antigens are to be determined. According to the present Invention can by reacting a test sample with one or more types of antibodies those with different nucleotide properties of nucleotides different Sequences are provided, each of which antibodies to bind one of the subjective Subtanzen is capable, along with labeled antibodies, each recognize one of the subjective substances formed immune complexes and reacting the nucleotides with antibodies solid phases attached to groups of nucleotides that bound to groups of nucleotides (solid phases with it bound nucleotides), wherein each of the groups is a partial or completely complementary Sequence to that of the antibody having bound nucleotides to the immune complexes on the solid Establish phases, subsequent washing of the solid phases with the immune complexes bound to it, determining or detecting the Levels of markers in immune complexes, one or more Types of subjective substances in the test sample determined or proven become.

The above example illustrates a method for determining antigens in a test sample, but the present invention also includes the method for determining antibodies in a test sample. With reference to the 11 - 19 For example, the method of determining one or more types of antibodies as the immunological ligand to be determined is explained. 11 schematically shows the assay reagent for determining one or more types of antibodies, ie antibodies A, H and C.

12 Fig. 12 illustrates an example of solid phase-bound nucleotides to be used in combination with the assay reagent for determining one or more types of antibodies according to the present invention; 12 (a) shows a solid phase having attached thereto nucleotides having a complementary base sequence to that of nucleotide ON1 (solid phase for antibody A); 12 (b) shows a solid phase to which nucleotides having a complementary base sequence are bonded to that of nucleotide ON2 (solid phase for antibody B) and 12 (c) shows a solid phase to which nucleotides having a complementary base sequence are bonded to that of nucleotide ON3 (solid phase for antibody C). These different types of solid phases are independent and separate. Used in combination with the assay reagent to determine one or more types of antibodies 11 ; then these single solid phases form an example of the determination equipment for determining one or more types of antibodies according to the present invention:

Will the assay reagent be used to determine one or more types of anicons, as described in U.S. Pat 11 is added to a test sample containing antibodies A, B and C, then, individually for the antibodies A, B and C, complexes of (antigen-nucleotide-conjugate) antibody-labeled substance are formed in a single reaction solution. 13 shows the level.

The reaction solution containing the complexes of (Antigen-nucleotide conjugate) antibody-labeled substance is in contact with the independent and sepärat existing Solid phases for antibody A, B and C to complementary To prepare pairs by hybridization between the nucleotides, contained in the complexes; and the nucleotides of the solid Phases of antibodies A, B and C, the complementary Base sequences to which the nucleotides in the complexes have.

14 . 15 and 16 show the complexes by hybridization on the individual solid phases are recorded; 14 shows the complex carrying antibody A; recorded via the complementary nucleotide pairing on the solid phase for antibody A; 15 shows the complex carrying antibody B, recorded via the complementary nucleotide pairing on the solid phase for antibodies B and 16 shows the antibody-carrying complex captured via the complementary nucleotide pairing on the solid phase for antibody C.

By subsequent washing of the complexes adhering to the individual solid phases, it is possible to remove impurities which do not adhere to the solid phase, for example marked substances. In such a manner, complexes adhering to the three kinds of solid phases from which impurities have been removed, that is, the antibody A-bearing complex adhering to the solid phase for antibody A, as in 17 shown carrying the antibody B complex, which is recorded on the solid phase for antibody B, as in 18 and antibody C-bearing complex adhering to the solid phase for antibody C, as shown in FIG 19 shown to be won individually.

The ones contained in the complexes Marks that adhere to the individual solid phases should be determined become. So should, z. B., enzyme reactions and the like are carried out in independent reaction systems, about the levels of the marks above colors To determine. Are the markings fluorescent substances, Coloring and metal colloids and similar, then the determination can be carried out without such an enzyme reaction.

The solid phase-nucleotide conjugate as the solid phase C for use in the assay reagent for Determining one or more types of immunological ligands according to the present invention Invention, is labile in the dry state, while the conjugate itself in solution can be stably stored in the presence of EDTA. The conjugate can thus be kept stable in the state of the reagent.

BRIEF DESCRIPTION OF THE DRAWING

1 shows the scheme of the general method of conventional immunoassay method using labeled compounds;

2 shows an example of the assay reagent for determining one or more types of antigens, schematically illustrating the assay reagent for determining one or more types of antigens, antigens A, B, and C;

3 shows an example of a solid phase to which a nucleotide is bound to be used in combination with the assay reagent for determining one or more kinds of antigens according to the present invention;

4 shows how the complex of (antibody-nucleotide-conjugate) antigen-labeled substance can be formed for one or more kinds of antigens according to the present invention contained in a single reaction solution;

5 Fig. 12 shows the state in which an antigen-bearing complex is retained by the complementary pair of nucleotides on the solid phase for antigen A according to the present invention;

6 Fig. 12 shows the state in which an antigen B-bearing complex is retained by the complementary pair of nucleotides on the solid phase for antigen B according to the present invention;

7 Fig. 12 shows a state in which an antigen C-bearing complex is retained by the complementary pair of nucleotides on the solid phase for antigen C according to the present invention;

8th shows the state of the antigen A-bearing complex which is complementarily paired on the solid phase for antigen A, after washing;

9 shows the state of the antigen B-bearing complex complementarily matched on the solid phase for antigen B after washing;

10 shows the state of the antigen C-bearing complex which is complementarily paired on the solid phase for antigen C after washing;

11 Fig. 12 shows an example of the assay reagent for determining one or more types of antibodies, wherein the assay reagent for determining one or more types of antibodies, antibodies A, B and C, is shown schematically;

12 shows an example of the solid phase to which nucleotides are bound to be used in combination with the assay reagent for determining one or more types of antibodies according to the present invention;

13 Figure 4 shows how the complex can be formed as an (antigen-nucleotide-conjugate) antibody-labeled substance individually for antibodies A, B and C in a single reaction solution according to the present invention;

14 Fig. 11 shows the state in which an antibody A-bearing complex is retained by the complementary pair of nucleotides on the solid phase for antibody A according to the present invention;

15 shows the state in which an antibody B carrying complex by the complementary Retaining pairs of nucleotides on the solid phase for antibody B according to the present invention;

16 Fig. 11 shows the state in which an antibody C-bearing complex is retained by complementary pairs of nucleotides on the solid phase for antibody C according to the present invention;

17 shows the state of the antibody A-bearing complex complementarily paired on the solid phase for antibody A, after washing;

18 shows the state of the antibody B-bearing complex complementarily paired on the solid phase for antibody B, after washing;

19 shows the state of the antibody C-bearing complex complementarily paired on the solid phase for antibody C, after washing;

20 shows the absorbance of the labeled substances retained on solid phases A, B and C by the method of determining one or more types of immunological ligands in Example 1; and

21 shows the absorbance of the labeled substances retained on solid phases A, B and C by the method of one or more types of immunological ligands in Example 2.

BEST TYPE THE EXECUTION THE INVENTION

The present invention will now be explained in detail using examples.

example 1

The following three types of oligonucleotides having an amino group at the 5'-end were synthesized using an automatic DNA synthesis apparatus, type 391A, manufactured by Applied Biosystems:
Amino group GAA TTC CCG GGG ATC CGT CG (referred to as "nucleotide pair 1 (+)");
Amino group-GCC AAG CTT GGC TGC AGG TC (referred to as "nucleotide pair 2 (+)");
Amino Group AAG CTT GCA TGC CTG CAG GT (referred to as "Nucleotide Pair 3 (+)").

Using glutaraldehyde were these oligonucleotides are individually covalently bound to polystyrene beads, introduced into the one amino group The beads were then dissolved in 10 mM sodium phosphate buffer and 0.1 M sodium chloride, pH 7.0, containing 0.1% skimmed milk, 0.1% Sodium azide and 5 mM EDTA (ethylenediaminetetraacetic acid). The following are the solid phases to which the nucleotide pairs individually 1 (+), 2+) and 3 (+) are bonded, referred to as solid phases A, B and C, respectively.

Rabbits were immunized individually with antigens, cholera toxin (CT); Generated from Vibrio cholerae, thermostable direct hemolysin (TDH), generated from Vibrio parahaemolyticus and Campylobacter jejuni to produce antibodies to the individual antigens. From each of the individual antibodies, the F (ab ') 2 was prepared from the IgG to give the Fab' _n according to the method of Y. Oku et al., Microbiol. Immunol., 32 , pp. 807-816, 1988. The nucleotide pair 1 (-) having a base sequence complementary to that of nucleotide pair 1 (+) was covalently attached to the anti-CT Fab '. Similarly, nucleotide pair 2 (-) having a base sequence complementary to that of nucleotide pair 2 (±) was covalently attached to the anti-TDH Fab '. Further, nucleotide pair 3 (-) having a base sequence complementary to that of nucleotide pair 3 (+) was also covalently attached to the anti-CJ Fab '. According to the method of Y. Oku et al., Microbiol. Immunol .; 32 , pages 807-816, 1988, horseradish peroxidase (HRPO) was introduced into each of the Fab's on the SH group of their joints.

Using 10mM bicine buffer, 0.3 M sodium chloride, 0.1% bovine serum albumin, 0.002% thimerosal and 5 mM EDTA, pH 8.3, became a mixture solution containing six types of the following Complexes contained, prepared; 20 pmol / ml nucleotide pair 1 (-), bound on anti-CT Fab ', 20 pmol / ml nucleotide pair 2 (-), bound to anti-TDH Fab ', 20 pmol / ml nucleotide pair 3 (-), bound to anti-CJ Fab ', 800 ng / ml HRPO bound to anti-CJ Fab ', 800 ng / ml HRPO bound to anti-TDH Fab' and 1600 ng / ml HRPO, bound to anti-C-Fab '.

1.5 ml of the solution was separated into three test tube given. Sample 1 (1.5 ml) containing 10 ng / ml CT was placed in a the tube given; Sample 2 (1.5 ml) containing 10 ng / ml TDH was placed in other tube and Sample 3 (1.5 ml) containing CJ at 0.005 turbidity 600 nm, was added to the remaining tube. Then subdued these tubes the reaction at 37 ° C for one hour.

The solution in each of the tubes was subdivided into 0.5 ml portions in six tubes for the subsequent reaction at 37 ° C for one hour; solid phase A was added to two of the tubes; Solid Phase B was added to two other tubes and Solid Phase C was added to the remaining two tubes. After discarding the reaction solution, the remaining solid phases became in 0.3 M sodium chloride solution (3 × 5 ml). After washing, the individual solid phases were independently transferred to other test tubes. The HRPO adhering to the individual solid phases was prepared according to the method of Y. Oku et al., Microbiol. Immunol., 32 , pages 807-816, 1988. The results are in Table 1 and 20 shown.

Table 1

As in Table 1 and the graph of 20 shown, CT-containing sample 1 reacts clearly with the solid phase A to determine CT; Sample 3 containing CJ clearly reacts with solid phase C to determine CJ. It has been shown that even a single reagent can determine one or more types of subjective substances using the substances and method described in this example.

Example 2

Method 1

The following four types of 5'-amino group-containing oligonucleotides were synthesized using an automatic DNA synthesizer, Type 391A, manufactured by Applied Biosystems:
Amino group GAA TTC CCG GGG ATC CGT CG (referred to as "nucleotide pair 1 (+)");
Amino Group AAG CTT GCA TGC CTG CAG GT (referred to as "Nucleotide Pair 3 (+)").
Amino Group GGC GAC TGT CGA ACC GGA AA (referred to as "nucleotide pair 5 (+)") and
Amino group CCA CCC CTA CTC CTA ATC CC (referred to as "nucleotide pair 6 (+)").

Using glutaraldehyde were these oligonucleotides are individually covalently bound to polystyrene beads, introduced into the one amino group The beads were then dissolved in 10 mM sodium phosphate buffer and 0.1 M sodium chloride, pH 7.0, containing 0.1% gelatin, 0.002% Timerosal and 5 mM EDTA (ethylenediaminetetraacetic acid) were stored.

Method 2:

It became a sulfhydryl group before introduced into different types of allergens. The allergens, d. h, wheat flour, Protein, Soybean and rice for clinical trials purchased from Torii Pharmaceutical Kabushiki Kaisha, were under cooling concentrated in ice by means of a YM-2 ultrafiltration membrane. Thereafter, these allergens were challenged with 0.1 M sodium phosphate buffer, pH 7.0, dialyzed. After dialysis, an excess of N-succinimidyl-S-acetylthioacetate was found (SATA, manufactured by Pierce, Co., Ltd.) with the resulting Allergens for one hour at 37 ° C implemented. Upon completion of the reaction, 1 M Tris-HCl buffer, pH 7.0, and 1M hydroxylamine, pH 7.0, independently of the individual allergen reaction products to final concentrations of 0.1 M for reaction for 15 minutes at 37 ° C added to promote the removal of the protection. After Completion of the reaction, the reaction products were to a Gel filtration carrier, SEFADEX G-25 (trade name of the Pharmacia Biotechnology Group for microscopic Beads of synthetic compounds of dextran), applied, with 0.1 M sodium phosphate buffer, pH 6.0, containing 5 mM EDTA, equilibrated, to collect fractions; the protein corresponded. The fractions were concentrated using the YM-2 UItrafiltration membrane to four species to gain concentrated allergens, each containing a sulfhydryl group contained.

Method 3:

By the same procedure and in the same manner, oligonucleotides having sequences complementary to those of the oligonucleotides synthesized in Method 2 were synthesized to obtain four kinds of oligonucleotides each having an amino group at its 5 'end. One oligonucleotide complementary to nucleotide pair 1 (+) is referred to herein as nucleotide pair 1 (-) and the others complementary oligonucleotides were designated nucleotide pair 3 (-), nucleotide pair 5 (-) and nucleotide pair 6 (-), respectively.

Method 4:

An excess amount of N- (ε-maleimide-caproyloxy) succinimide (Abbreviation: EMCS) was compared with each of the four types of complementary oligonucleotides, which were prepared in the above process 3, at 37 ° C for one hour reacted to introduce the maleimido group into the 5 'end of each oligonucleotide. After Completion of the reorganization were the four types of oligonucleotides with imported Maleimide group purified by ethanol precipitation according to a routine method.

Method 5:

The four types of concentrated Allergens with imported Sulfhydryl group as prepared in Method 3 were used with the four kinds of maleimide group-introduced oligonucleotides as described in U.S. Pat Method 4, mixed for reaction for one hour at 37 ° C, to prepare an allergen mixture into which the oligonucleotides introduced were. The wheat flour allergen was linked to the nucleotide pair 1 (-); the soybean allergen was at nucleotide pair 3 (-) bound; the protein allergen was at nucleotide pair 5 (-) and the rice allergen was bound to nucleotide pair 6 (-).

Method 6:

The allergen mixture to which the four types of oligonucleotides were bound, prepared in the process 5, was supplemented with 5 mM sodium phosphate buffer, pH 7.0, containing 0.1%. Gelatin, 0.3 M sodium chloride and 5 mM EDTA, to a final protein concentration of 1 μg / ml and a final concentration of 100 mg / ml anti-human IgE, labeled with horseradish peroxidase, diluted. The resulting diluted allergen mixture was defined as reagent A. Reagent A (7.2 ml) was placed in a Test tube poured, followed by the addition of patient serum (2,4 ml) for reaction at 37 ° C for one Hour. The resulting reaction solution was defined as reagent A mixture solution. The patient serum became independent won by three patients.

Method 7:

After completion of the implementation were Nucleotide pair 1 (+) bound to solid phase, nucleotide pair 3 (+), bound to solid phase, nucleotide pair 5 (+) bound to solid phase and nucleotide pair 6 (+) bound to solid phase individually in test tubes followed by the addition of the reagent A mixture solution (400 μl) after the Finish the reaction to each other at 37 ° C for one hour implement.

Method 8:

After the conversion, the resulting solution Washed three times with 0.3 M sodium chloride solution. After that were transferred the individual solid phases into fresh test tubes, where the activity of the solid phase adherent enzyme with 3,3 ', 5,5'-tetramethylbenzidine was determined. The determination was carried out in duplicate.

The results are in the bar chart in 21 shown. In the graph of the 21 The abscissa represents the type of allergen and the identity of the patient serum for each allergen, while the ordinate represents the absorbance at 450 nm.

21 shows that the use of proteins into which oligonucleotides are introduced; Simultaneous detection of several specific allergen IgE's possible.

INDUSTRIAL APPLICABILITY

According to the present invention is the time required for the reaction of a mixture containing one with an immune complex labeled substance, with nucleotides bound to solid phase shorter as the reaction time for the antigen-antibody reaction using binding on a solid phase according to conventional methods is required. The so-called non-specific binding of a marked Substance directly on a solid phase can be reduced, causing a very sensitive determination system is achieved.

According to the present invention, a single reagent may detect or detect one or more types of immunological ligands, one or more types of antigens or one or more antigens or more types of antibodies, so that the time required for detection or determination can be greatly reduced.

Theoretically, the number of combinations the base sequences between complementary nucleotides almost infinite. According to the present Invention is therefore an almost infinite number of proven Combination of either one such immunological pair, namely one or more types of antigen or one or more species of antibodies, possible.

Claims (20)

A determination reagent for determining one or more types of immunological ligands in a group of preselected ligands comprising a single mixture: (i) a multi-conjugate group capture reagent including a conjugate for each of the plurality of preselected ligands, each of which comprises a plurality of different conjugates ( 1 ) an anti-ligand that specifically binds to its corresponding preselected ligand and ( 2 ) a preselected nucleotide sequence, each conjugate of the group-capture reagent comprising a different preselected nucleotide sequence, and (ii) a group-labeling reagent comprising a labeled anti-ligand for each of the preselected ligands, each comprising the labeled anti-ligand ( 3 ) an anti-ligand that specifically binds to its corresponding preselected ligand and ( 4 ) a detectable mark. A determination reagent according to claim 1, wherein each of the a plurality of different conjugates has an anti-ligand, the itself from the anti-ligands of other of the several different conjugates different. A determination reagent according to claim 1 or 2, wherein the detectable label is selected from the group consisting from an enzyme, biotin, avidin, digoxigen, a nucleotide, a Metal colloid, a fluorescent compound, a luminescent Compound, a metal compound and a radioisotope. Determination reagent according to one of claims 1 to 3, wherein the nucleotides are oligonucleotides. Determination reagent according to one of claims 1 to 5, wherein the anti-ligand antigens are. Determination reagent according to one of claims 1 to 5, wherein the anti-ligand antibodies are. Determination kit for determining one or more types of immunological ligands in a group of a plurality of preselected ligands, the identification kit comprising: (i) a group capture reagent comprising a plurality of conjugates in a single mixture, including a conjugate for each of the plurality of preselected ligands , wherein each of the several different conjugates comprises ( 1 ) an anti-ligand that specifically binds to its corresponding preselected ligand and ( 2 ) a preselected nucleotide sequence, wherein each conjugate of the group capture reagent comprises a different preselected nucleotide sequence; (ii) a group-labeling reagent comprising, in a single mixture, a labeled anti-ligand for each of the preselected ligands, wherein each of the labeled anti-ligands ( 3 ) an anti-ligand that specifically binds to its corresponding preselected ligand and ( 4 ) comprises a detectable label; and (iii) a plurality of solid phase reagents each comprising a water-insoluble solid phase and a specific nucleotide sequence immobilized on the water-insoluble solid phase, each of the immobilized nucleotides being a complement of one of the nucleotide sequences of the conjugates in the group capture Reagent is. The assay kit according to claim 7, wherein the group-capture reagent and the group-labeling reagent combined in a single mixture are present. The assay kit according to claim 7 or 8, wherein the anti-ligand is any of several different conjugates from the anti-ligands of other of the several different conjugates and wherein the water-insoluble solid Phase another solid-phase substrate for each of the immobilized thereon Includes nucleotides. The identification kit of any one of claims 7 to 9, wherein each of said immobilized Nucleotide is covalently bound to the water-insoluble solid phase through its 5'-end or its 3'-end. The assay kit according to any of the claims 7-10, wherein each of the immobilized nucleotides is at another Position as its ends covalently attached to the water-insoluble solid Phase is bound The assay kit according to any of the claims 7 to 11, wherein each of the immobilized nucleotides is covalently attached a binding ligand is attached and the covalently linked nucleotide and the binding ligand physically on the water-insoluble solid phase are absorbed. The assay kit according to any of the claims 7 to 12, wherein the binding ligand is a protein. The assay kit according to any of the claims 7 to 13, wherein the detectable label is selected from the group consisting of an enzyme, biotin, avidin, digoxigen, a nucleotide, a metal colloid, a fluorescent compound, a luminescent compound, a metal compound and a Radioisotope. The assay kit according to any of the claims 7 to 14, wherein the nucleotides are oligonucleotides. The assay kit according to any of the claims 7 to 15 wherein the anti-ligand antigens are. The assay kit according to any of the claims 7 to 16, wherein the anti-ligands are antibodies. An immunological assay for simultaneously determining the presence or amount of one or more types of immunological ligands in a group of preselected ligands in a fluid sample comprising: (a) providing (i) a group-capture reagent comprising a plurality of different conjugates in a single mixture include a conjugate for each of the plurality of preselected ligands, wherein each of the several different conjugates comprises ( 1 ) an anti-ligand that specifically binds to its corresponding preselected ligand and ( 2 ) a preselected nucleotide sequence, wherein each conjugate of the group-capture reagent comprises a different preselected nucleotide sequence and (ii) a group-labeling reagent comprising in a single mixture a labeled anti-ligand for each of the preselected ligands, wherein each of the labeled anti-ligands includes ( 3 ) an anti-ligand that specifically binds to its corresponding preselected ligand and ( 4 ) a detectable label; (b) contacting the sample with the group-capture reagent and the group-labeling reagent in a reaction solution to form a corresponding immune complex comprising a conjugate-ligand label for each of the ligands present in the sample ; (c) separately contacting the reaction solution of step (b) with each of a plurality of different water insoluble solid phase substrates, each of the solid phase substrates immobilized thereon having only a single type of complementary nucleotide sequence aligned with its corresponding preselected nucleotide sequence hybridizing to separate any corresponding immune complex from the reaction solution of step (b); and (d) separately and independently measuring the detectable label bound in each of the separated corresponding immune complexes by the presence or amount of each of the one or more types of ligands to be determined in the sample. The method of claim 18, wherein the anti-ligands Antigens are. The method of claim 18, wherein the anti-ligands antibody are.