JPS6247555A - Scintillation proximity determination method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The development of radioimmunoassay by Berson and Yalow has provided biologists with one of their most sensitive and specific arsenals. As first reported5, this procedure involved equilibration of competing antigens, labeled and unlabeled with antibodies, precipitation of antigen-antibody complexes, centrifugation, and precipitation. Separation of the supernatant from the antigen-antibody complex and measurement of its radioactivity is required. By immobilizing antibodies on solid supports such as plastic beads, radioimmunoassay has been greatly simplified.

Competing antigens are added to antibody-coated beads and incubated under shaking after centrifugation or filtration.
Remove supernatant. Next, all you have to do is count the phase t of the chair nρ).

The term scintillation proximity quantification method was coined by Hart and Wald, who were involved in a new type of quantification method at the time.
(1979). This operation uses the same antigen (
Two different latex beads were used that had previously been covalently bound to human serum albumin.

These 10,000 beads also have a fluorophore immobilized within them.”
fit, the second bead was labeled with tritium (3H). The range of particles emitted during tritium decomposition is only a few micrometers in water, so in ash it is difficult to dilute the 3H beads, which exist close enough that their β-particles can excite the fluorophore beads. There is almost no fluorescence, and therefore only weak fluorescence is observed. Antibodies specific to suspended beads? Upon addition, aggregation occurs and a large number of 3H♂-zes come together within range to excite the fluorophore beads. The emitted fluorescence can be measured in a sensitive photometric scintillation counter. Hart and Grθθnwa
N1 U.S. Patent No. 4.388.296 described by 1a
The operation disclosed in No. ci9ss (registered June 14, 2013) is
Although it has limited sensitivity, it does not require centrifugation or other separation operations. However, this quantitative method requires two different types of coated beads as well as macromolecules that interact with them. I need. The kinetics of collision of the three components is very slow, thus requiring significantly longer incubation times.

Moreover, this conventional technique cannot be applied to the quantification of small molecules such as peptides or therapeutic drugs.

An improved scintillation proximity quantitation method capable of detecting small molecules has been reported by Grunnsr et al. In this method, fluorophore beads were used that were covered with a semi-transparent polyacrylamide layer whose thickness was greater than the average reach of tritium beta electrons. Antibodies or receptors that cannot penetrate the fluorophore bind to the permeable tritiated ligand away from the vicinity of the fluorophore, thus emitting less fluorescence. The change in fluorescence intensity as the ligand is removed from the vicinity of the bead provides a means of quantifying the presence of radioactive ligand.

However, this system generates measurable fluorescence, requires large amounts of tritium for quantification, is difficult to coat beads, has limitations in application to large molecules, and has limitations in its application to large molecules. Since it measures only small changes in the temperature, it has drawbacks such as limited sensitivity and interference.

Therefore, the aim of the invention is to quickly, reliably and
The objective is to provide a quantitative method that uses reagents that are easy to prepare and is applicable to both large molecules and small molecules such as peptides and therapeutic drugs.

The present invention relates to a scintillation near quantitative assay (5PRA) using one type of microbead tray.

The microbeads of the present invention have a suitable fluorophore r@ and are coated with a covalently bonded binding molecule. The binding molecule is selected to bind to the desired ligand. A binding molecule is a component of an antibody-anti-inflammatory response complex or a receptor-ligand binding complex. In the recent literature, the number of reports on receptors is gradually increasing and the range of related biologically active substances is also expanding. Receptor revealed 1
The compounds mentioned in 3) range from simple phenylalkylamines such as amphetamines to very high molecular weight polymers such as protein 1. In other embodiments of the invention, the coating can be a lectin-binding molecule, such as concanavalin A, in which case the beads can bind to glycoproteins in the cell membrane.

Pharmaceutical ligands include narcotic analgesics, hypnotics,
Sedatives, analgesics, antipyretics, anesthetics, hallucinogens, muscle relaxants,
Nervous system stimulants, anticholinesterase agents, parasympathomimetics, sympathomimetics, α-adrenergic neuroleptics, anti-adrenergic agonists, ganglion stimulators and blockers, neuromuscular agents, histamines, antihistamines, 5-hydroxytryptamines and antagonists, cardiovascular agents, antirheumatic agents, antihypertensive agents, vasodilators, diuretics,
Pesticides (antibacterial agents, antiparasitic agents, insecticides, ectocide killers, etc.), antimalarial agents, antibiotics, antimetabolites, hormones, vitamins, sugars, thyroid and antithyroid agents,
Examples include corticosteroids, insulin, oral hypoglycemic agents, and the like.

These agents include alkaloids, steroids, polypeptides and proteins, prostaglandins, catecholamines, xanthines, arylalkyl amines, heterocycles such as thiazine, hyherazine, indoles and thiazoles, amino acids, and the like.

Other interesting ligands besides drugs are industrial contaminants, flavoring agents, food additives such as preservatives, and food contaminants.

Structurally, the ligand can be monomeric or polymeric.
It may be acyclic, monocyclic or polycyclic, carbocyclic or heterocyclic. Ligands can contain a wide range of functional groups, such as halo, oxycarbonyl, non-oxycarbonyl, amino, oxy[
Combinations of hydroxy, aryloxy, alkyloxy, cycloalkyloxy (alkyl means a monovalent aliphatic group), chenooxy, dithio, hydrazo, and the like may also be present.

Ligands can be divided into six different categories based on their biological relationship to the receptor. The first category is antigens, which are introduced into the bloodstream of vertebrates.
il: When an antigen is formed.

The second category is hapten, which binds to an antigenic carrier and, when introduced into the bloodstream of a vertebrate, induces the production of hapten-specific antibodies. Ligand 3rd
The category includes ligands for which there are organisms and receptors that can be isolated in a form specific for the ligand.

Of course, biological substances that are unique to a given animal and have natural receptors within that species can also be used when bound to proteins and introduced into the same or a different species. Therefore, this classification is not fixed in that a diene may have an antigen in one animal species, a hapten in another animal strain, and a natural receptor in a third animal species.

Antigens, in fact mostly proteins or polysancharides, are foreign to the animal into which they are injected.

Haptens are important as ligands.

A substance that does not produce antibodies when injected, but can specifically react with antibodies and cause precipitation, but prevents precipitation, is named pabutene. This definition includes not only simple chemicals that, when conjugated to proteins, are determinants of specificity and also prevent precipitation, but also pneumococcal type-specific polysaccharides that exhibit no antigenicity upon primary injection into rabbits. It has also been used to include substances of natural origin such as and dextran (Kabat et al.: Experi
menta'1work mmuno Ohemi 8 tr7
+ 0harlf380- Thoma81 Bpr
ingfialcl I engineering 11. (1967)]. In the following description, the term hapten is limited to groups artificially introduced into antigenic carriers that promote the formation of antibodies against those groups.

The third group of ligands are those that correspond to the naturally occurring receptor tM. Receptors can be proteins or ribonucleic acids (RNA).
) or a nucleic acid such as deoxyribonucleic acid (DNA), or a membrane associated with a cell. Typical ligands with naturally occurring receptors include thyroxine, many steroids such as nystrodiene, cortisone, corticosterone and estradiol, polypeptides such as insulin and anyaotensin, and other naturally occurring biological There are active compounds (Murphy et al.: J, C11n, Kndo
cra* 24 *187 (1964), Mur
phy:1bid, 27, 973 (1957);
1b1a, 28, 343 (1969).

BBA, 176, 262 (1969), M
cEwen 2): Nature, 226, 2
63 (1970) *Morgan et al.: Diab
etes (1966) e Page et al.: J, C
11n, Kndocr, s 28, 200 (196
See 9)].

Ligands can also be categorized by chemical species that have been accepted in the literature.

The species that are the subject of the present invention include structures that are partially similar to naturally occurring substances, and physiological properties of natural substances. Physiological mimetics that mimic or inhibit may also be included.

Also included are synthetic substances such as barbituric acids and amphetamines.

Additionally, these compounds may be modified such that they can bind to different sites where all biological activity of the enzyme is disrupted. In addition, structural modifications may be made to facilitate synthesis or to control the properties of the antibody. These modified compounds are called pseudoligands.

General categories of ligands of particular interest are drugs and chemically transformed compounds, and metabolites of such compounds. Drugs [The benefits of quantifying are diverse, for example, certain contraband drugs? Quantitative measurements are performed to determine whether the patient has been taking or has constant strength, what drugs the patient is taking, and what the concentration of the drug is in a particular body fluid.

The ligand to be quantified is first labeled with radioactivity. For example, if the radioactively labeled diene r is a radioactive antigen,
This 7"L binds directly to beads coated with specific antibodies, and their proximity ensures that even emitted short-range electrons will excite fluorophores in the beads. Liquid samples, e.g. If non-8A-labeled ligand is present in the tissue extract, it will compete with the labeled ligand for binding to the beads, and the observed fluorescent signal will be proportional to its concentration.

The fluorescence signal observed after addition of the sample is compared with the value obtained with a standard curve using a known amount of ligand:
It is also possible to specifically detect the diene P.
It becomes possible to quantify the total amount of

In another embodiment of the invention, beads can also be coated with a kelectin binding agent such as concanavalin A. These beads can be used to bind whole cells or on membranes and detect radiolabeled compounds inside cells or on membranes. By this method, it is possible to directly monitor the accumulation or depletion of intracellular metabolites such as lactose and glucose, membrane transport properties, and substances that promote or block this transport τ.

The scintillation proximity assay (5PRA) applied in a preferred embodiment of the invention simplifies existing radioimmunoassay methods. For example, no shaking, centrifugation, or separation is required in the practice of the present invention. The simplicity of 5PRA lends itself to automation. Determined values and results can be obtained in less than 1 hour depending on the compound. 5PRA with the antibody-coated beads disclosed herein can be used for both large and small molecules depending on its binding ability to % different antibodies.
It can also be applied to the detection of antigens. Conversely, by binding antigen to beads, it is also possible to quantify specific antibodies. Applications to receptors and their ligands, whole cells, cellular particles and membranes are also possible.

For labeling antigens, antibodies, haptens or other ligands,
Conventional iodination is used. Therefore, the chemistry utilized in the quantitative system is simple and the radioactivity of the labeled ligand decays rapidly.

Broadly speaking, the present invention consists of a new type of scintillation proximity radiometry (5PRA). In certain embodiments, the invention relates to scintillation proximity radiometry. More specifically, the assay method of the present invention provides for determining the presence of a particular drug or molecular species, such as a peptide, hormone, steroid, etc., in a liquid sample by: (a) adding an antibody to the fluorophore-containing beads; (k) The above-mentioned coated beads are attached to the above-mentioned antibodies, antigens, receptors or membranes. or by incubating with a radioactive species wt specific ligand that is capable of binding to cells and whose radioactive label generates fluorescence, such as a fluorophore in the beads; (c) anti-antigen coated beads; To a mixture of radioactively labeled specific ligands is added a synthetic sample containing an unknown amount of the corresponding unlabeled ligand to reduce the fluorescence emitted from the fluorophore. (d) It is a quantitative method that involves measuring the decrease in fluorescence, which is a measure of the unknown amount of ligand in a sample at night.

For example, in one preferred mode of carrying out the invention, the radioactive antigen is bound directly to beads previously coated with a specific 9° C., and the weak electrons released are also in close proximity. Yo! Seven fluorescent groups t1iJJ will be launched.

For example, when unlabeled ligands present in body fluids are added,
Then, it displaces the binding ligand and the fluorescent signal decreases. Fluorescent fern can be measured.

That is, all the requirements of a competitive immunoassay can be performed in one step without the need for separation. It is simple and rapid to operate and can be applied to both small and large molecules. When tritium-labeled antigen was used in this assay, significant fluorescence was emitted upon interaction with beads that had been disrupted with specific antibodies. However, it required a large amount of tritium, and the sensitivity was not high. It was thought that if it were a radioactive isotope that emits high-energy electrons, the sensitivity would be improved and the amount of n1 isotope needed would be smaller. Other commonly used 14q and 35B release particles are +
Energy is too high for 3PRA. Tritium β-
While the electron energy has a range of only 8 μm in water, the corresponding values for 1'C and 35B are:
They are n-rn85 and 89 μm. At such long ranges, the background signal becomes large. 12
Polymers are generally used as r-ray emitters. ρ) while 125 k1, +? ! , 'two f77
N? /J' 7 L 71- 7w-large←l-+4 + Z F 1rQV
The rumored conversion electron has a range of 7 to 60 μm in water.

It has been found that 125 mm can be used very advantageously in 5PRA. The average range of the γ emission of 125 particles is 151, which is much longer than the diameter of the beads, and the energy is also significantly large, so there is almost no absorption of energy by the beads. This n is the same whether the 125 polymer is bound to beads or in the bath solution. A further advantage of 125 is that the ligand can be identified by the iodination procedure used in conventional radioimmunoassays. Is tritium a troublesome way to introduce labels? I need. 125th grade is 5R
In PA, this is the label that should be selected first.

Polyvinyltoluene (Nl1i1Q2A: Nucl
aar Enter-prised Ltd,,Sig
htill,]! ;din'burgh, 5cotl
and ), preferably with a diameter of 1 to 10 μm, are preferred for use as microbeads of the present invention. Other types of fluorophore-containing beads can be made from various polymers, such as vinylbenzene (styrene), vinyltoluene (methylstyrene), and vinylxylene (dimethylstyrene), and can also be used in the present invention. Contains various fluorophores, and reactive groups such as carboxyl casings or amine groups on exposed surfaces! Microbeads are currently commercially available and can be used in the administration of the present invention. Rather than being present in solution during quantification,
Microbeads bound to a solid support, such as dip
Also included within the scope of the invention are microbeads attached to a stick, piece of paper or inside a vial or test.

In the case of polyvinyltoluene beads, the methyl group k[
It is necessary to form an attachment site. In one aspect of this case, the methyl groups on the polyvinyltoluene were converted to carboxyl groups, preferably with potassium permanganate, in the following manner.

Beads 7.1-3% Triton X-100'18
W 70-Jl, shaken to make it hydrophilic, and centrifuged. The bead pellet was suspended in 70* water, shaken, centrifuged, and washed 6 times.

Resuspend the washed beads in water and store in the refrigerator overnight to allow sedimentation. The next morning, the supernatant was aspirated to remove the fine powder and resuspended in 1-sp. water to a final volume of 70 y.

Part 7-z of bead suspension (approximately 700rnli beads)
Transfer to a hlooy round bottom flask and add 22 d of water and 22 d of freshly prepared 0,IN KMnO477i solution.
-jt added. After covering the flask with argon,
'C bath and incubated under stirring for 10 hours. The flask was then removed from the bath and incubated at room temperature for an additional 12
Incubated for hours. After the oxidation was completed, 5* sodium bisulfite solution (40.9/100w) was added with stirring, followed by 30dk of 1NHO. The suspension was then transferred to a centrifuge tube, and after centrifugation, the supernatant was removed, and the bead pellet was washed once with KINI(O)5d. Then, the supernatant of the oxidized peas was centrifuged, suspended, and washed with water repeatedly until the supernatant became neutral. Finally, the oxidized male powder was suspended in 101 Lt of water. The amount of ♂-s in the produced carboxyl suspension was determined by turbidimetry.

A suspension of about 200 beads in l d? Stored in the refrigerator. Samples for coupling with antibodies were collected by shaking and resuspending the bead suspension.

Does antiserum (polyclonal or monoclonal antibody) have a carboxyl group? was attached to the oxidized beads via Coupling can be accomplished using sophisticated coupling operations well known to those of ordinary skill in the art. According to one embodiment, the coupling is performed with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (5tory Ch
chemical 0orpo-ration,Muske
Although it is preferable to use antiserum (
or antigen)? ! - As a method of coupling to reactive groups on microbeads, n'fc is well known in the art.
Any operation? It can be used in the present invention. The coated beads used in the following examples were manufactured by the following procedure. In a 5d polypropylene tube, anti-blood 78% fC was added to approximately 400 to 50 μl of pre-immune serum samples. At capacity 7PfK *1 ') ml-+u('y 1
n mM FTOj! After adjusting to d15.5 from the dropwise addition VC, add 10rn9 of carbodiimide. Cap the tube with kL, rotate and shake on a rotary mixer overnight,
Centrifuged. Supernatant liquid? Remove and antibody-coated beads'l (
It was washed twice with IJ acid buffered saline solution (p87.4) 5, ffi/ to remove uncoupled white matter and unbound carbodiimide. Antibody coated beads t1 after final wash, centrifugation, 0.1% bovine serum albumin and 0.1% Triton x-io.

resuspended in a known volume of sulfate-buffered saline solution.

A portion of this suspension was diluted with water, and after nephelometry, the concentration of antibody-coated beads was adjusted to 20 mf/d. The product was stored at 4° C. and carefully shaken each time two aliquots were taken for quantitative analysis.

The specific free ligands used in the examples herein are leucine-enkephalin and methionine-enkephalin-
Arg'-Phe7(Pen1nsula Labor
a-tories; Bolmont, OA), rabbit anti-cat egg gG (H and L chain specific injection, 0appel
; Malvern, Pa). Morphin I
d 5pector, S., Ph.D. (Hoffmann-D
aRoche, Nutley, NJ). Tyrosyl-125 eloisine-enkephalin, 12
1i items2203μO1/μmol Id New En
grand Nuclear (BoSton, MA)
I bought it. [125 engineering]-methionine-enkephalin-Arg'-Phe7 (specific activity 775μO1/nm
ol) and C'''I]-Y13-259 (specific activity 5
60μO1/nmol) is Pierce Chemi
Cal Co., Ltd. (Rockford, Engineering L.) et al. [125 Komorphin is Roc
hs Diagnostics (NutleyNJ
), and the specific activity is approximately 10 μC1/nmol.
0125 Nithyroxine U Roche C
11nical Labs (BurlingtOn, N
o) 7)+1 et al. supplied f17'c.

Rabbit polyclonal antiserum 61 against leucine-enkephalin and methionine-enkephalin-Arg'-Pb0 was prepared using the stated method.

Titer value A-t'f'L1: 5. Cl 00bzbi 1:15. []OO. Goat polyclonal antiserum to morphine, titer 1:300, from Roche
Diagnostics (Hoffmann-La R
(Oche, Nutly, NJ) 71) et al. Rabbit polyclonal antihematology against thyroxine 19
iJ ROche C11nical Labs (B
urlington, no) (!).

In fact, the use of these 21 reagents in Example 7M is merely for illustrative purposes in the present invention and in no way applies to the scope of the present invention or to the quantification of other ligands (Antibody 1 is an antigen). The present invention is not limited to applications or uses of other dienes 12. As used herein, the term "liquid sample" refers to natural body fluids that have not undergone any manipulation, such as blood, saliva, or urine samples. means. However, this term (2) removes any contaminant proteins or contaminants from the liquid sample that may interfere with the quantitation from the liquid sample prior to performing the quantification by methods well known to those of ordinary skill in the art. It also includes a sample extract.''It also includes a reaction medium,
The quantification of specific metabolites, products, contaminants, toxins, or other test compounds for use as liquid samples such as cell culture media, manufacturing plant effluents, etc. is also within the scope of this invention.

The scintillation proximity quantification method of the present invention is based on f ty in water.
'z −d5 j, e −h: −S l (m n
β-Fishing substance 11. It is preferred to use antigens that are different from each other.

Suspending a small amount of antibody-coated beads in a dilute solution of a radiolabel that is not associated with specific antigen produces only ρ) or ρ・fluorescent signal that is dependent on the concentration of beads and label.

In the presence of a significant amount of labeled specific antigen, even the same amount of antibody-coated beads will generate a large amount of signal because the antigen binds directly to the antibody on the beads.

In the following examples, quantification was performed by polypropylene microcentrifugation of 1.5-%jf (+3arstθdt, n
C1Pr1nceton, NJ), buffer, 125
Add the knee antigen, standard sample or unknown sample, and finally the specific antibody-coated peas in order to make a volume of 50-250μ.
It was carried out in Microcentrifuge tubes were stoppered, spun, placed on a rotary platform mixer, and incubated at a specific temperature for a time appropriate for quantification of a given substance. Early studies showed that antibody-coated peas required constant agitation to remain in suspension, and that (Leu]enkephalin and [uet]enkephalin-Arg'-
The procedure for Phe' (see below) is described using this shaking method. However, it was subsequently shown that the addition of all glycerol to the reaction mixture at a final concentration of 12% kept the beads in suspension, making shaking unnecessary, and at the same time increased the counting efficiency. Morphin and yothyroxine (see below) are described using the chrycerin method. After incubation, each microcentrifuge tube was dropped into a clear glass-split scintillation vial. The vial was placed in a liquid scintillation counter and counted for 2 minutes using a wide open fc3H window. BθckmanLS 7800 counter (
Beckman engineering instruments.

nc,), open the window tO~400,
Packard Trib-Oarb 46Q Liquid Scintillation Counter
ment Co,, window in case of engineering nc+n?
It opened 0-19. Some devices such as Beckman
In the case of the 7800, other adjustments to the counting program are required due to clouding due to suspended beads.

The titer was determined for each antibody-coated bead. This AK is
Various amounts of antibody were used against a constant concentration of [125]-antigen. An amount of antibody-coated beads corresponding to a maximum (125 mm) of 30-50% of antigen binding was considered appropriate for performing sensitive immunoassays.

The reagents used in the 5PRA of the present invention are conveniently packaged in kit form. Each kit has a reagent container,
Each container can contain enough reagents for multiple analyses. A first container of such a kit contains fluorophore-containing microbeads co-encapsulantly coated with a selective binding molecule, and a second container contains a radiolabeled ligand specific for said binding molecule. containing a known concentration of In addition, in the case of a kit used for quantification, if desired, a plurality of containers containing one concentration of the ligand (t), which can be used as a standard to create a standard curve, are attached. The concentration of the ligand in the test sample can be determined from this standard curve ρ.

Although the specific examples below are illustrative of all the applications of the invention, it should be noted that the 5PRA of the invention can be used to detect the presence of many molecules. Further, the method used is not essential to the present invention, and changes in the order of addition of samples and reagents, for example, are naturally included within the scope of the present invention.

Example 1 Based on the titer (Figure 1), it was decided to use antibody-coated beads 0.45In9 for the quantification of (Leu]enkephalin. The final volume of the quantification mixture was 250μ,
[125 engineering]-leucine-enkephalin 20μ, t
(24,0(]Ocpm), Standard(Leul x
y cephalin 20μ) or sample for analysis 10-10
0μ and the antibody coated bead suspension were added at 10μ.

In this case, to adjust the final volume to 250μ, 0.1%
Bovine serum albumin and 0.1% Triton X-
Nonspecific binding was measured using a phosphate-buffered saline solution containing 1[]0 by coupling male to preimmune serum from the same animal. Since peptidases are present in serum and tissue extracts, incubation during immunoassay of peptides is generally carried out using
However, it was revealed that when the peptide was incubated at room temperature for a residence time and then incubated for a long time under cooling, no decomposition of the peptide was observed and the operation time could be shortened. For quantification of Leu-enkephalin, samples were incubated for 15 minutes at room temperature and then for 4 hours at 4"C. With these conditions and the antibody-coated beads and reagent ratios described above, the maximum binding of 125LθU-enkephalin was achieved. About 70% of the unlabeled Leu-
The addition of enkephalin causes the substitution shown in Figure 2? Born friend. From this, engineering C50 is 14 Q fmol
It was evaluated as. This sensitivity is Lahm.

H-W et al. (Arch, Biochem, Biophys
, 2'15, 422-429 (1983)] 1. It is of the same order of magnitude as the standard [:Lau]] leucine-enkephalin R. When two samples of pituitary extract were quantified by both procedures, the results showed 940 fmol and 1Q for R-A.
9 Q fmol, sp engineering with 1000 fmol and 95 [] fmo]- and ivy.

Example 2 Quantification of Heptapeptides A similar procedure was used to develop a specific 5PRA k for the proenkephalin product [Met]]enkephalin-Arg'-phe'. Antibody coated beads 0.
Maximum coupling of C5078fmox in 4 hours using 161Vk? It was possible to obtain. In the case of (Met) Encare 71J-Arg6-Phe7 (heptapeptide), the titer measurement in a volume of 50μ was determined to be V (Figure 6), and antibody-coated beads from 0.16 to approximately 30 μL binding of the labeled ligand. This suggests that it is ideal for high-sensitivity quantification.

The final quantitative components were 20 μg of either buffer standard or unknown sample, 20 μg of (12511-hebutapeptide) (approximately 20,000 cpm) and 20 μg of antibody coated beads (0,16In9). did.

For Leu-enkephalin, incubation was performed for 15 minutes at room temperature followed by 4 hours in the cold (4°C). Maximum binding of heptapeptide was also achieved under these conditions. A late incubation in the cold room does not increase maximum binding and improves reproducibility during repeated quantification.
I thought. From the displacement curve (Figure 4), the engineering C50 is 73
It was measured as fmol.

In the case of standard RA with the same antiserum, it is 59 fmol.

Example 6 Morphin Quantification Morphin SPA was coupled with Beadke goat anti-morphin serum. From the titer (Figure 5) it was clear that the antibody coated beads 0.4In9 bound 60% of the label and were therefore eligible for quantification. The quantitative mixture was [125th - morphine 100 μt (30,
000 Cpm), standard morphine 40 μm.

Or unknown sample for analysis 10-50μ, glycerol 60μ and antibody coated heath (0,4r19) 4
0μノ? Total volume 2 in phosphate buffered saline bath M (pH 7-4)
It was set to 50μ. Maximum binding was obtained when antibody-coated beads were incubated at room temperature between 72# and 1n
i (Figure 5). In competitive studies, incubation is 6
It was carried out for 1 hour at 7°C, at which point 69% of maximal binding had been achieved'n-fco standard displacement-! 1 (Figure 6)
26 pg of 5PRA was obtained (Fig. 6). This 5PRA is commercially available for measuring morphine in urine.
fman-La Rochs Engineering nc,).

Since the sensitivity of 5PRA is high, urine was diluted 50 times to collect a 10μ sample for quantitative analysis. When the concentration of added Mu morphine in six types of urine samples was measured, R
IA Tri 568.205>yo CF295 amy
zt-C'', SP upper level is 506.126 and 245nσd.

Example 4 T 4Cl) 5PRA VC was mixed with Peas T 4 serum. As shown in Figure 7,
The titer indicated that 0.51 ng of the antibody-coated resin bound approximately 60% of the added labeled T4 and was suitable for quantifying tube sensitivity. The quantification mixture was prepared using serum or serum standards of less than 2 Gμ [1250An-T].

100 μi (approximately 50,000 guns-rC, p, m,),
Glycerol 53% PBS-EDTA
1-1 (7,4 buffer 90 μ), 2-produced antibody-seed peas (0,5 ~) suspension 640 μ, total volume 250
It was set as μ.

After rotating the test tube, it was incubated at 67° C. for 1 hour.

Standard displacement curve 2 is shown in FIG. TC5o is T41.5n
It was g.

Increasing the incubation time increased total binding and therefore sensitivity, but the factor remained virtually unchanged. −

[Brief explanation of the drawing]

Figure 1 is a graph showing the effect of (125)-leucine-enkephalin binding when the amount of leucine-enkephalin antibody-coated beads is varied. Figure 2 is a graph showing the effects of unlabeled leucine-enkephalin binding. FIG. 6 is a graph illustrating the substitution of (125)leucine-enkephalin when the amount is varied. FIG. FIG. 4 is a graph showing [125
]-methionine-enkephalin~Arg6-Phθ
7 is a graph showing an example of replacement basin. Figure 5 shows [125 units] when the amount of morphine antibody-coated beads is varied.
Graph showing the influence of morphine on binding. Figure 6 shows antibody-coated beads binding to 0.41n9 [12
5 is a graph illustrating the substitution of morphine with a series of dilutions of standard morphine. Figure 7 shows [125 engineering] -T, when the amount of T4 antibody beads was changed.
It is a graph showing the shadow #r for the combination of. Figure 8 shows the amount of non-standard RT4? [125 works] when changing 7'c -
This is a constant graph showing seven examples of permutation of T4. FIG. 9 ID is a graph showing the rate of binding of 5H (3G) to antibody-coated beads.

Claims (23)

[Claims] (1) In quantifying the presence of a ligand in a liquid sample, (a) a fluorophore-containing microbead has a selective binding molecule covalently attached to it; A fluorophore-containing microbead configured such that the radioactive label causes a fluorophore in the microbead to fluoresce when a possible radioactively labeled ligand binds to its selective binding molecule; scintillation proximity, characterized by preparing a mixture with a liquid containing an amount of unlabeled ligand, and (b) measuring the fluorescence produced by binding of the labeled ligand, corresponding to the concentration of unlabeled ligand in the sample. Quantitative method. (2) The quantitative method according to claim 1, wherein the microbeads are polyvinyltoluene. (3) The quantitative method according to any one of claims 1 and 2, wherein the microbeads have a diameter of about 1 to 10 microns. (4) The quantitative method according to any one of claims 1 to 3, wherein the quantitative method is a radioimmunoassay. (5) The quantitative method according to claim 4, wherein the radioactive label is I^1^2^5. (6) Claim 1, wherein the selective binding molecule is an antigen.
Quantitative method described in any one of paragraphs to paragraphs 5 to 5. (7) Claim 1 that the selective binding molecule is an antibody
Quantitative method described in any one of paragraphs to paragraphs 5 to 5. (8) The quantitative method according to any one of claims 1 to 4, wherein the selective binding molecule is a receptor or a membrane. (9) The quantitative method according to any one of claims 1 to 8, in which a small amount of glycerol is further added to the quantitative mixture. (10) Claim 1: The liquid sample is a body fluid.
The quantitative method described in any one of paragraphs 9 to 9. (11) The quantitative method according to any one of claims 1 to 10, wherein the measurement is performed using a scintillation device. (12) (a) a first container comprising a fluorophore-containing microbead coated with a covalently bound selective binding molecule; and (b) a radioactivity capable of binding to the selective binding molecule coating the microbead; A kit for performing scintillation proximity quantitation of a selected ligand, comprising a second container containing a labeled ligand. (13) The kit according to claim 12, further comprising containers containing different known concentrations of the selected ligands suitable for preparing a standard curve for the ligand according to the quantitative method. (14) Fluorophore-containing microbeads coated with covalently bound selective binding molecules. (15) The microbead according to claim 14, having a diameter of about 1 to 10 microns. (16) The microbead according to claim 14, wherein the selective binding molecule is an antigen, antibody, receptor, or membrane. (17) The microbead according to claim 14, wherein the selective binding molecule is a lectin binding molecule. (18) The microbead according to claim 17, wherein the lectin-binding molecule is concanavalin A. (19) The microbead according to claim 18, wherein the microbead is bound to the cell membrane by a lectin-binding molecule. (20) Attachment of fluorophore-containing microbeads coated with covalently bound lectin-binding molecules to cells or membranes and the presence of radioactive labels in the cells or membranes results in the presence of radioactive labels in the microbeads. characterized in that a cell or membrane is provided with a radioactively labeled ligand that generates an amount of fluorescence proportional to the concentration of the radioactive label,
A method of monitoring the presence of specific ligands in cells or membranes. (21) The method according to claim 20, wherein the lectin-binding molecule is concanavalin A. (22) The method according to claim 20, wherein the radioactive label is ^1^2^5I. (23) The method according to claim 20, wherein the specific ligand is lactose.