CH635438A5 - Test process operating using a specific bond for the investigation of a liquid medium on a ligand and reagent for carrying out the process - Google Patents

Description

The present invention can be applied to the discovery of any ligand for which there is a specific binding partner. The ligand is usually a peptide, protein, carbohydrate, glycoprotein, steroid or other organic molecule for which a specific binding partner is present or can be synthesized in biological systems. The ligand usually consists of antigens or antibodies to it, haptens or antibodies to it or hormones, vitamins, metabolites and pharmacological agents, their receptors and binding substances. Specific ligands that can be detected according to the invention are hormones such as insulin, chorionic gonadotropin, thyroxine, liothyronine and estriol; Antigens and haptens such as ferritin, bradykinin, prostaglandins and tumor-specific antigens, vitamins such as biotin, vitamin Bu, folic acid, vitamin E and ascorbic acid, metabolites such as 3 ', 5'-adenosine monophosphate and 3', 5'-guanosine monophosphate, pharmaceuticals such as Dilan -tin, digoxin, morphine, digitoxin and barbiturates, antibodies such as microsome antibodies and antibodies against hepatitis and allergens, and specific binding receptors such as thyroxine, binding globulin, avidin, intrinsic factor and transcobalamin.

In the conjugate according to the invention, the reagent is bound to a specific binding substance or coupled to it.

11 635 438

pelt, which consists of the ligand, a specific binding response in different categories, since log of the ligand or a specific binding partner that can act in several ways depending on the chemi-ligand, depending on the test method chosen, where see surroundings. However, it is the activity that - a measurable amount of the activity of the reagent obtained ser substance in the respective monitor reaction that remains. The bond between reagent and specific binding identity of this substance in the context of the present substance is usually determined under the test conditions.

Versible in that the monitor reaction, in which the reagent is active, preferably the reagent is an enzymatic reagent, should not chemically destroy this bond, such as, for example, an enzyme substrate, a coenzyme or, for example, in the above luminescent and cyclic reac - active modification or a derivative thereof. An enzyme substrate system. In certain cases, however, the bond io is a compound or a residue which, catalyzed by an enzyme as intended by the monitor reaction chosen, undergoes a chemical conversion. If a sub-first is disturbed or otherwise influenced, using strat as the conjugated reagent in this way, this is before the change in the activity of the reagent is determined. molecular weight less than 9000 and preferably one such case are the reaction systems with enzymatically less than 5000. Substrates of this size are for use with the fluorescent substrate mentioned above. is the cheapest in the preparation of the conjugate since it does not have any. The reagent can have molecules directly on the specific binding sub-complex. The activity of these substrates, punch-coupled so that the molecular weight of the coupled with a specific binding substance, is slightly influenced by conjugate equal to or less than the sum of the molecular- the reaction of the conjugate with a specific binding weight of reagent and specific binding substance. Examples of enzyme substrates. The reagent and specific use according to the invention are usually those which are cleaved by enzymes, however, via a bridge member composed of 1 to 50 and bare fluorescent substrates of the aforementioned preferably 1 to 10 carbon atoms or heteroatoms, such as, for example, the fluorescein and umbelliferone species Nitrogen, oxygen, sulfur, phosphorus or the like, pH indicators and spectrophotometric indicators linked together. Examples of bridges made of dyes, especially chromogenic compounds.

One atom is the methylene group (1 carbon atom) 25 For the above reasons and because of the versatility and or the amino group (1 hetero atom). The bridge member adaptability, coenzymes are particularly preferred for use as usually having a molecular weight of no more than reagent in the conjugate. A coenzyme is 1000 and preferably less than 200. It consists of a non-protein molecule that migrates from one chain of carbon or heteroatoms or an enzyme protein to another and the catalytic radio combination of both and is reagent and specific bin - 30 tion of the enzyme increases. All known coenzymes have an additive or an active derivative thereof bound via molecular weights less than 9000 and the preferred one connecting group, which usually consists of an ester, coenzymes have molecular weights less than about

Amide, ether, thioester, thioether, acetal, methylene or 5000. Examples of useful coenzymes are the nucleo amino group. tid coenzymes, especially those with adenine groups like that

The reagent in the conjugate according to the present invention 35 adenosine phosphates (i.e. mono-, di- and tri-phosphate) can be any substance which has a given (i.e. nicotinamide-adenine dinucleotide and its reduced forms fixed or known) activity as Part of a and nicotinamide adenine dinucleotide phosphate and its reduced

specific monitor response. Under «reagent» or decorated forms. Other useful coenzymes are the guanosine "substance with reactivity" is in the present phosphate, flavin mononucleotide and their reduced form

Description understood any chemical substance that a 4Q men, flavin adenine dinucleotide and its reduced forms, finite measurable chemical conversion are subject to coenzyme A and its thioester including succinyl, in which one or more of the starting material coenzyme A, 3 ' , 5'-adenosine diphosphate and adenosine-3'-phos-different products are formed and because of the single-phosphate sulfate.

Reaction-initiating agents are used, for example. Useful coenzyme-active conjugates contain nucleo-

a chemical substance (that is, another reactive coenzyme with an adenine group to which the specifics, a catalyst or material which participates in such a binding substance, ie a ligand, undergoes a specific chemical transformation), electromagnetic binding -Analogon of a ligand or a specific bin radiation, thermal or acoustic energy. The class of binding partner of a ligand in direct binding or via a punch, which are thus referred to as “reagents”, includes a bridge member of the type mentioned above. Therefore, conventional inorganic and organic rea- 50 These coenzyme-active conjugates, which contain an adenosine phosphate, genes and various biochemical substances, but not nicotinamide adenine dinucleotide or its reduced form, substances such as catalysts including enzymes and radioak - or nicotinamide adenine DinucIeotidphosphat or its active isotopes, which contain no reagents of the monitor reaction reduced form, have the following general requirements. Of course, a certain chemical submei can:

635 438

where R

00

1 <-0-P-0

0

12

R1-CH

j2 / -0

r *

oh r

O® O0

I I Q

-0-p-0-p-0u ii ii 0 0

0 ° 0 0 °

I 1 I 0

-0-p-0-p-0-p-0u

II II II 0 0 0

0e o0

, or -o-p-o-p-o-ch where R =

where R =

0 '

, 0

0

ï ©

-oh o -o-p-o ^ and h

il

2 ^ 0

R

oh oh nh2

r-

1 year

r *

nh.

© M

('ii n

VU «'

fi

<

N-

nhr *

n nh2 5

| Z R *

n-

n

©

N

where R ^ =

\

N.

n-

nh n ^!

mean,

® \\ //

conh ^

and R5 = -Y-Z, where Y is a bond or a bridging member and Z is a ligand, a specific binding analog of a ligand or a specific binding partner of a ligand. The above formula represents the ionized form of the coenzyme-active conjugate, but protonated or acidic forms are equally useful. The extent of the protonation depends on the pH of the environment. Given

conh2

otherwise the salts of these conjugates can also be used.

In addition to the above compounds, useful coenzyme-65 active conjugates are the adenosine phosphates to which the specific binding substance is bound via the phosphate group. These compounds correspond to the general formula:

13

635 438

oh oh 0 © 0 ©

where R

-o-p-o-r

è

0 © o © 0 ©

I l 1

O-p-o-p-o-p-o-r

II!

-o-p-o-p-o-r

I! Il

0 0

or

0

A fi where R2 = -Y-Z and Y is a bond or a bridging member and Z is a ligand, a specific binding analogue of a ligand or a specific binding partner of a ligand. The protonated or acid form and salts can optionally also be used.

According to one embodiment of the invention, the components of the specific binding reaction which are to be combined with the liquid medium in which the ligand is suspected are in liquid or solid form. In the preferred homogeneous test system, the components are usually in solution or in a solid form that easily dissolves in the liquid medium. Since the liquid medium to be tested is usually aqueous, the components are generally water-soluble, that is to say they are present as an aqueous solution or in a water-soluble solid form, for example as a powder or resin. The test method can be carried out in a conventional laboratory vessel such as a test tube, the components of the specific binding reaction and the components of the reaction system being added in solid or liquid form.

One or more components of the specific binding reaction and / or one or more components of the monitor reaction with a carrier can also be added. The carrier may be a liquid-containing vessel such as a reagent tube or a capsule containing the component (s) inside, these components being in the form of a liquid or a loose solid or coating on the inside surface of the vessel. Furthermore, the carrier can have the form of a matrix which is insoluble and porous and preferably absorbs the liquid medium to be tested. The matrix can consist, for example, of absorbent paper, polymer film, membranes, nonwovens or blocks, gels and the like. In this form, the corresponding device provides a convenient means of making contact with the liquid medium to be tested, carrying out the specific binding reaction and / or the monitor reaction and observing the resulting changes.

The liquid medium to be tested can be a naturally occurring or artificially formed liquid in which the ligand is suspected. It is usually a biological fluid or a fluid that results from dilution or treatment thereof. Biological fluids that can be tested according to the invention are, for example, serum, plasma, urine, amniotic fluid, cerebral and spinal fluid. Other substances, such as solids, for example tissue, or gases can also be tested by returning them to a liquid form, for example by dissolving solid or gas in a liquid or liquid extraction of the solid.

In contrast to previous homogeneous test systems, biological fluids which have a reaction activity which is similar or identical to that of the conjugated labeling substance can be tested on the ligand 30 without background interference. The activity of the endogenous background can be easily eliminated in various ways. The biological fluid can be treated to selectively destroy endogenous activity. Such treatments include exposure to a clarifier that chemically destroys endogenous activity, followed by treatment to inactivate the destructive effect of the clarifier.

For example, enzymes that degrade the reagent are often found in biological fluids, especially if the reagent is a coenzyme such as NAD, NADP or ATP. There are numerous inhibitors of coenzyme-degrading enzymes, for example chelating agents, which deprive the enzymes of the essential metal ion activators. As a specific example, NAD-degrading enzymes are found in normal serum 45 which have sufficient enzymatic activity to remove essentially all endogenous NAD activity from isolated serum within a few hours. The . degrading effect of these enzymes can be effectively inhibited by adding a chelating agent such as ethylenediamine-5o tetraacetic acid. The degradation activity can also be eliminated by adding a specific enzyme inhibitor. For example, one can inhibit ATP-degrading enzymes by adding β-methylene-ATP or aß-methyl-ATP.

55

Example 1: Preparation of a conjugate from nicotinamide-adenine dinucleotide-biotin

6o A. Nicotinamide-6- (2-aminoethylamino) purine dinucleotide

2 g of nicotinamide adenine dinucleotide (NAD) are dissolved in 10 ml of water and 0.6 ml of ethylene imine are added dropwise,

wherein the pH is kept below 7 by adding 1 m perchloric acid. When the addition of ethyleneimine 65 has ended, the pH is adjusted to 4.5 and the reaction is incubated at 20 to 25 ° C. At intervals of 24 hours, 0.6 ml of ethylene imine is added and the pH is adjusted to 4.5 again. After 96 hours the solution is poured into 10

635 438 14

mina poured acetone from -10 ° C. The oil which forms 264 nm and a ratio of optical density at 340 nm is collected, washed with ether and combined in about 50 ml of water to 264 nm of more than 0.18 and the water is dissolved in a flask. material obtained is concentrated to 4 to 5 ml and as follows

The resulting solution is purified with 1 m sodium hydroxide solution by electrophoresis:

solution to pH 7.0 to 7.5 and then 1 g of sodium bis. The concentrated material is added to a sheet of what carbonate. Then the solution is bubbled through 4MM nitrogen for 4 to 5 minutes through 3MM paper (Reeve Angel, Clifton, New Jersey) and 1 g of sodium hydrosulfite is added. The is applied in the form of a 1 to 2 cm wide strip, the flask is tightly closed and at 45 ° is perpendicular to the direction of flow. The paper is then left to stand for minutes. The solution is then wetted for 15 minutes with 0.02 molar sodium phosphate solution at pH 6.0. The aerated and adjusted to pH 11.3 with sodium hydroxide. Then "> electrophoresis is heated according to the hanging method according to Durrum, S. for 1 hour at 75 ° C. Thereafter, the reaction science 121: 829 (1955) is cooled for 4 to 7 hours in a mixture to room temperature and with 0.6 g potential gradients of about 8.5 volts / cm

Tris (hydroxymethyl) aminomethane is added and then with the location of the desired pyridine nucleotide derivative is on

5N hydrochloric acid adjusted to pH 7.5. The resulting solution is determined on the basis of the fluorescence which is obtained after spraying 1000 test units of alcohol dehydrogenase 15 test strips with 0.5 m sodium cyanide solution after the in J. and 1 ml of acetaldehyde. The decreasing optical density of the reaction mixture described at Biol. Chem. 191: 447 (1951) is followed at 340 nm and kelt. The area containing the desired derivative is cut out as soon as no further decrease is observed, and the area is extracted three times with 50 ml of water each time. The pH is adjusted to 3.5. The solution is combined in 10 volumes of resulting extracts which pour the nicotinamide-6- (2-aminoethyl-acetone at -10 ° C. The oil which forms will contain 20 amino) -purine dinucleotide, are combined to 3 to and washed with ether and then concentrated in 10 to 15 ml 4 ml and stored at - 20 ° C.

Water dissolved.

The resulting solution is applied to a 2.5 x 90 cm column of B. nicotinamide-adenine-dinucleotide-biotin conjugate from Sephadex G-10 (Pharmacia AB, Uppsala), which are mixed with water-16 mg biotin in 1 ml of water , which is equilibrated with 20 mg nicotine ser. Fractions of 12 ml each are collected 25 amid-6- (2-aminoethylamino) -purine dinucleotide (see Part A). The wavelengths containing are suspended for each fraction. In order to support the solution of the biotin-maximum optical absorption in the ultraviolet range and zen, a few drops of 0.1 n sodium hydroxide solution are added to determine the optical density at this wavelength. Furthermore sets. Then the resulting solution 240 mg of 1-cyclo-the optical density of each fraction at 340 nm is determined after the hexyl-3- (2-morpholinoethyl) carbodiimide-metho-p-toluenesulfo reduction with alcohol dehydrogenase. Those 30 nat were added, solution by dropwise addition of 0.1 n hydrochloric acid fractions which have an absorption maximum at 264 nm. The reaction mixture is left for 5 hours and a ratio of optical density at 340 nm to 264 nm at room temperature and then in 10 ml of acetone of more than 0.05 are combined. This combined material is poured in at -10 ° C. The oil which forms is reduced to 15 to 20 ml on a rotary evaporator, washed twice with 5 to 10 ml of ether and in 1 to 2 ml and through a 2.5 x 28 cm column filled with Dowex 1-X8 35 water dissolved. The resulting material was purified as described above (Bio-Rad Laboratories, Richmond, California) using paper electrophoresis. After water had been equilibrated. With additional spraying with sodium cyanide appearing 2 fluorescent water, the combined material becomes bands through the column, one of which faces the cathode and the other, collecting 10 ml fractions. The Frak has migrated to the anode. The latter band, which contains the NAD bio-ion with an absorption maximum at 264 nm and a 40 tin conjugate, is washed with water and stored at a ratio of optical density at 340 nm to 264 nm of -20 ° C.

more than 0.1 are combined.

This combined material is determined by a Doweg Example 2: Homogeneous specific binding test; Effect of

50-X2 filled column of 5x45 cm (sales Bio-Rad Laborato-Avidin and Biotin to the enzymatic circulatory system, Richmond, California), which had been equilibrated with water, led to 45% of NAD and NAD-biotin conjugates. The material is washed with additional water. The circulation system used in this example is based on the column, whereby 20 ml fractions are collected on the following reactions:

the. The fractions with an absorption maximum at

/ \ t. » j. r lactic acid -

(a) NAD ligand + lactate de hydrogenase ^

NADH ligand + pyruvate

(b) NADH ligand + thiazolyl blue (oxidized) diaPhorase ^

NAD ligand + thiazolyl blue (reduced)

8 reaction mixtures were prepared, of which incubation was carried out at room temperature for 2 to 3 hours,

each had a total volume of 0.5 ml and 0.12 m. Each reaction mixture was washed with an aqueous

N, N-bis-2-hydroxyethylglycine hydrochloride buffer of pH 7.8 enzyme-substrate mixture contacted by addition contained. Concentrations or activities of NAD, NAD- of 0.1 ml of 1 M lithium lactate, 0.05 ml of 10 mM thiazolyl blue in biotin conjugate (see Example 1), biotin and avidin, which last- 65 oxidized form and one for filling up to 1 ml reaction

res has binding affinity for biotin, shows Table 1. A lumen sufficient amount of 0.12 m N, N-bis-2-hydroxy-

The unit of avidin activity is that amount of avidin which forms the ethylglycine hydrochloride buffer with a pH of 7.8 and 0.38 interatio-

Binding of 1 p, g biotin is capable. The reaction mixtures naie units bovine heart lactic acid dehydrogenase and 1.5

15 635 438

international units contained pig heart diaphorase. Density at 570 nm during a 24-minute period

The relative production rate of the reduced form half the first hour after adding the enzyme substrate

The thiazolyl blue was then determined in all reaction mixtures. The entire procedure was determined in duplicate by performing the total change in optical and the mean values are shown in Table 1.

Table 1

reaction

concentration

Concentration

concentration

Avidin activity mitti. increase

to NAD (nM)

NAD-biotin conjugate (nM)

to biotin (nM)

(Units)

optical density (570 nm)

1

0.002

2nd

220

-

-

-

0.103

3rd

-

360

-

-

0.079

4th

-

-

360

-

0.003

5

220

-

-

0.11

0.103

6

-

360

-

0.11

0.025

7

-

360

360

0.11

0.069

8th

-

-

-

0.11

0.001

Reactions 1, 4 and 8 are comparative experiments, the drawing decreases proportionally to the concentration of the biotin gene in the absence of NAD and the NAD-Biotin-Kon- in the reaction mixture.

jugat essentially no cycle takes place. The result thus shows that the activity of NAD in the reaction of reactions 2 and 3 shows that the NAD-biotin conjugate-25 NAD-biotin conjugate has a significant coenzyme activity with respect to the native stems in relation to the circulatory reaction syjugate Presence of avidin is decreased and the Aus-NAD exerts. From the results of reactions 3 and 6 due to this reduction due to the additional presence, it can be seen that the presence of avidin in the reaction mixture of biotin is reduced.

mixed inhibits the formation of thiazolyl blue (reduced form) when the existing NAD is conjugated with biotin. 30 Example 3: Homogeneous Competing Binding / Biolumines-by comparing the results of reactions 6 and 7 zenztest; Influence of different amounts of biotin on the maxi shows that the presence of free biotin shows the overall light intensity due to the inhibition of thiazolyl blue (reduced form). The reaction system of this example is based on the following

Reactions:

(c) NAD ligand + ethanol ^ ^ - ^ ohol-->

Dehydrogenase

NADH ligand + acetaldehyde

(d) NADH ligand + FMN * + H® ^ v *? ADH.

Dehydrogenase "

NAD ligand + FMNH ^

(e) FMNH2 + long chain aldehyde + 02 Luciferas e— ^

J? MN + long chain acid + H20 + hV

Flavimaononucleotide

A light-generating solution to carry out the reac suspension was centrifuged at 1500xg for 10 minutes, and the ions (d) and (e) were prepared as follows: A 0.13 m Phos cake was discarded. The light generating solution was made up of phate buffer pH 7.0.0.67% by weight bovine serum albumin, then used within 5 minutes until use 15.7 (xM flavin mononucleotide (FMN) and 13.3 mM sodium-60 by combining 75 μl of the Reagent mixture containing 5 μl or acetate was stored in the dark with a dodecanal emulsion and 20 μl of the luciferase solution at −20 ° C. An emulsion of 5 μl of dodecanal in 5 ml Was- Um the amount of light generated by reaction (s) on the day on which the light-generating solution came together, a photometer was built, which was to be used from a photodetector, consisting of Photobacterium fisheri tor and a 6 x 50 mm cuvette, which consisted of inside (distribution Worthington Biochemical Corp., Freehold, New York-65 a light-integrating sphere was attached such that sey), extracted, lyophilized luciferase was converted to 0.013-m-the light generated in the cuvette onto the photodetector phosphate buffer of pH 7 , 3 was reflected in a concentration of. The electrical generated by the photodetector

20 mg / ml added. After 30 minutes the resulting tronic signal was passed on to a line recorder

635 438

16

tet. The maximum light intensity was taken from the writer's notes and arbitrary units attached to the strip.

7 reaction mixtures were prepared, each of which had a total volume of 0.2 ml and 0.1 m tris (hydroxymethyl) aminomethane hydrochloride buffer with a pH of 8.0.0.6 m ethanol, 0.01 m semicarbazide hydrochloride, 343 nM NAD-biotin conjugate (see Example 1), 0.025 international units of alcohol dehydrogenase and 0.055 units of avidin. Biotin was added to 6 of the 7 reaction mixtures, that is to say mixtures 2 to 7 of Table 2, in the concentrations indicated.

The reaction mixtures were incubated at room temperature for about 30 minutes. 10 μl of each reaction mixture was injected into individual cuvettes, which were arranged in a photometer as in the previous example and contained 100 μl of the above light-generating solution, which had been preincubated at 28 ° C. for 2 to 3 minutes. The entire procedure was carried out in duplicate, the mean values are shown in Table 2.

Table 2

Reaction mixture

Concentration of biotin (nM)

maximum

Light intensity

(Average)

1

0

36

2nd

25th

44

3rd

50

57

4th

100

79

5

150

90

6

200

97

7

300

104

gene medium using a competitive binding reaction in a bioluminescence test.

Example 4: Preparation of 2,4-dinitrophenyl-fluorescein-Kon-5 jugate; Fluorescein-3 '[6- (2,4-dinitroanilino) hexanoate]

In the synthesis, the acid chloride of 6- (2,4-dinitro-anilino) -hexanoic acid is reacted with the disodium salt of fluorescein. The 6- (2,4-dinitroanilino) -hexanoic acid was after the in Biochem. J. 42: 287-294 (1948).

1.5 g (5 millimoles) of 6- (2,4-dinitroanilino) hexanoic acid was converted into the acid chloride in solution by reaction with 10 ml of warm thionyl chloride for 15 minutes; it was then cooled and diluted with 20 ml of hexane. The solid acid chloride thus formed was filtered off and carefully dried and then added to 600 mg of the disodium salt of fiuorescein in 10 ml of dry acetone. After refluxing for 5 hours, the reaction was stopped by adding 2 ml of water and 5 ml of acetone. After 30 minutes at 25 ° C the mixture was evaporated to dryness and the residue was partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The organic phase was separated and washed with 1% aqueous sulfuric acid, dried over anhydrous magnesium sulfate and concentrated. The red oil was chromatographed on 60 g of silica gel 60 (E. Merck, Darmstadt, Germany) using 20% by volume of acetone in carbon tetrachloride as the eluent. The 1.2 g of impure bis-ester obtained in this way were again chromatographed on 60 g of silica gel with 10% by volume of acetone in 30 ° carbon tetrachloride. Appropriate fractions were pooled and concentrated to give 180 mg of a yellow glassy solid.

Analysis: Ber. for GwHssNsChs: C: 59.33; H: 4.30; N: 9.43; Found: C: 60.92; H: 4.35; N: 6.65.

35 The infrared spectrum showed the expected ester carbonyl absorption at 1765 cm-1.

This example thus shows that the peak of the light intensity that the bioluminescence reaction produces, and thus the activity of the NAD in the NAD-biotin conjugate, is a direct function of the amount of biotin present in the reaction mixture. The invention therefore provides a test mixture and a method for the quantitative determination of the ligand biotin in a liquid. Example 5: Homogeneous specific binding test for derivatives of 2,4-dinitrobenzene and antibodies against it using an enzyme substrate (modified fiuorescein) as a labeling substance

The test systems used were based on the reaction of Scheme 1.

17th

2, 4 - Dinitrophenyl-fluorescein K. conjugate

635 438

+ other products

(maximum fluorescence at 510 nn)

r = -

- (ch2) 5

nh

0 "N

C.

r

//

.no,

A. Homogeneous direct binding / fluorescence test for antibodies against 2,4-dinitrophenyl; Influence of different amounts of the antibody on the reaction rate

7 specific reaction mixtures were prepared and analyzed. For each mixture, 20 μl 1 μl M 2,4-dinitro-phenyl-fluorescein conjugates (see Example 4) in dimethyl sulphoxide with a volume of antiserum against 2,4-dinitrophenyl according to Table 3 and with a volume of 2, 0 ml of sufficient volume of 0.1 m bis-hydroxy-ethylglycine hydrochloride buffer of pH 7.0 combined. The background hydrolysis rate of the ester bond in the conjugate was followed for 3 minutes for each reaction mixture by measuring the increase in fluorescence intensity at 510 nm with a fluorometer set at 470 nm for excitation. Then 10 ml of 0.1 M bis-hydroxyethylglycine hydrochloride buffer of pH 7.0, the 0.54 units of type I esterase (EC No. 3.1.1.1, sales Sigma Chemical Co, St. Louis, Missouri) contained, admitted. The resulting overall rate was measured as in the case of the background hydrolysis rate, the results are shown in Table 3.

Table 3

Reaction

amount

Background-

Total react

mixture

Antiserum (jil)

Hydrolysis rate

speed speed

1

0

0.02

2.68

2nd

5

0.07

2.48

3rd

10th

0.11

1.91

4th

20th

0.17

1.08

5

30th

0.21

0.63

6

40

0.22

0.45

7

60

0.26

0.30

This part of the example shows that the net reaction rate of hydrolysis is an inverse function of the amount of antibody to ligand, 2,4-dinitrophenyl present in the reaction mixture. It could also be shown that the reaction rate of the background hydrolysis reaction is a direct function of the amount of antibody in the reaction mixture. The invention therefore represents a test mixture and a method for determining an antibody against 2,4-dinitrophenyl (ligand) in a liquid medium using a direct binding test (fluorescence method).

45

B. Homogeneous test (competitive binding / fluorescence) for derivatives of 2,4-dinitrobenzene; Influence of different amounts of 2,4-dinitrophenyl-ß-alanine on the reaction rate.

10 reaction mixtures were prepared, each of which had a total volume of 2.0 ml and 0.1 M bis-hydroxyethylglycine hydrochloride buffer of pH 7.0 and 2,4-dinitrophenyl-β-alanine (preparation see J. Amer. Chem. Soc. 76: 1328 (1954) in the concentrations given in Table 4. 9 55 of the 10 reaction mixtures, that is to say Samples 2 to 10, were treated with a large amount of antiserum against 2,4- Dinitrophenyl was added, which was sufficient to inhibit the speed of the esterase-catalyzed reaction by 60% in mixture No. 1. After mixing, 20 μl of 1 nm 2,4-dinitrophenyl-fluo-60 rescein conjugate (see Example 4) were added Dimethyl sulfoxide was added to each reaction mixture, followed by 10 (0.1 ml 0.1M bis-hydroxyethylglycine hydrochloride buffer of pH 7.0 containing 0.54 units of type I esterase (EC No. 3.1.1.1, sales Sigma Chemical Co, St. Louis, Missouri) The resulting 65 reaction rate was measured as in Part A. For reactions 2 to 10, the reaction rate is calculated as a percentage of the rate of reaction No. 1 (no antibody present), the results are shown in Table 4.

635 438

18th

Table 4

Reaction

Concentration reaction

% of the mixture of speed

Reaction area

2,4-Dinitrophic speed

nyl-ß-alanine (nM)

of reaction 1

1

0

2.78

2nd

0

1.04

37

3rd

5

1.01

36

4th

10th

1.04

37

5

20th

1.24

45

6

30th

1.51

54

7

50

1.54

56

8th

75

1.80

65

9

100

1.85

67

10th

150

2.33

84

This part of the example thus shows that the rate of hydrolysis is a direct function of the amount of 2,4-dinitrophenyl-β-alanine in the reaction mixture. The invention therefore provides a test mixture and a method for the determination of ligands such as derivatives of 2,4-dinitrobenzene in a liquid medium using a competing binding reaction / fluorescence.

C. Homogeneous spectrophotometric test with competing binding for derivatives of 2,4-dinitrobenzene; Influence of different amounts of 2,4-dinitrophenyl-ß-alanine on the reaction rate

8 reaction mixtures were prepared, each of which had a total volume of 1.0 ml and 0.1 M tris (hydroxymethyl) aminomethane hydrochloride buffer of pH 7.0 and 2,4-dinitrophenyl-β-alanine in contained the concentrations given in Table 5. To 7 of the 8 reaction mixtures, namely mixtures 2 to 8, an amount of antiserum against 2,4-dinitrophenyl was added which was sufficient to inhibit the rate of the esterase-catalyzed reaction in mixture No. 1 by 82%. After the mixing, 10 (il 0.1 mM 2,4-dinitrophenyl-fluorescein conjugate (see Example 4) in dimethyl sulfoxide were added to each reaction mixture, then 20 p.1 0.1 M tris (hydroxymethyl) aminomethane than hydrochloride pH 7.0 buffer containing 2.16 international units of type I esterase (EC No. 3.1.1.1, Sigma Chemical Co., St. Louis, Missouri) was added tracked at 489 nm per minute with a Gilford 2000 spectrophotometer, the results are shown in Table 5.

Table 5

Reaction mixture concentration of change in

2,4-dinitrophenyl-ß-alanine absorption (UM)

1

0

0.0261

2nd

0

0.0047

3rd

1.25

0.0118

4th

2.5

0.0131

5

5.0

0.0185

6

7.5

0.0202

7

10.0

0.0192

8th

12.5

0.0223

This part of the example shows that the reaction rate is a direct function of the amount of 2,4-dinitrophenyl-β-alanine in the reaction mixture. The invention therefore provides a test mixture and method for determining the presence of ligands such as derivatives of 2,4-dinitrobenzene in a liquid medium using a spectrophotometric test and a competing binding reaction.

D. Homogeneous competitive binding / fluorescence test for derivatives of 2,4-dinitrobenzene; Using a non-enzymatic monitor response

The test system corresponded to the system shown in Scheme 1, but no esterase was added to catalyze the hydrolysis of the ester bond in the conjugate.

8 reaction mixtures were prepared, each of which had a total volume of 2 ml and 0.1 M tris (hydroxymethyl) aminomethane hydrochloride buffer of pH 7.5 and 2,4-dinitrophenyl-ß-alanine in the in Contained concentrations given in Table 6. To each reaction mixture was added 50 (il antiserum against 2,4-dinitrophenyl, and after mixing, 20 (12 jiM 2,4-dinitrophenyl-fluores-cein conjugate (see Example 4) in dimethyl sulfoxide) was added, followed by the resulting Reaction rate was measured as described in Part A of this example, Table 6 shows the results.

Table 6

Reaction mixture

Concentration

Reaction rate

2,4-dinitrophenyl-ß-alanine acidity

(nM)

1

0

0.96

2nd

12.5

0.94

3rd

31.2

0.84

4th

62.5

0.78

5

94.0

0.70

6

125

0.59

7

187

0.57

8th

250

0.53

This part of this example shows that the background hydrolysis rate in the absence of esterase is an inverse function of the amount of 2,4-dinitrophenyl-ß-alanine in the reaction mixture. The present invention therefore provides a test mixture and method for determining the presence of ligands such as derivatives of 2,4-dinitrobenzene in liquid medium using competing binding / fluorescence, the binding partner after binding to the ligand in the conjugate on the monitor reac- tion participates.

Example 6: Preparation of 2,4-dinitrophenyl-ATP conjugate (derivative in 6-position)

N6- [2- (2,4-dinitrophenyl) aminoethyl] adenosine 5'-triphosphate.

A. N6- (2-aminoethyl) adenosine 5 'monophosphate

2 g (7 millimoles) of 6-chloropurine riboside (Sigma Chemical Co, St. Louis, Missouri) are stirred with 17 ml of triethyl phosphate and with phosphoryl chloride in the presence of water according to the instructions from Chem. Scrip. 19: 165-170 (1972). After hydrolysis of the phosphodichloridate, 9.5 ml (140 millimoles) of ethylenediamine are added and the mixture is left to react at room temperature for 3 hours. The reaction mixture is diluted to 4 liters with water and adjusted to pH 12 with sodium hydroxide. This solution is passed through a 5x30 cm column filled with Dowex 1 x8 (Bio-Rad Laboratories, Richmond, California) in acetate form. The column is washed with 310.01 m ammonium chloride solution and the chromatogram is analyzed with a linear graph.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

19 635438

were developed, which is tet from 31 water and 311-m-acetic acid and the chromatogram is generated with a linear gradient. An isolated peak of UV-absorbing material from 31 water and 310.5 m ammonium carbonate solution ent-rial, which eluates between 1800 and 2050 ml of the gradient. The first eluted peak of yellow material is called, is concentrated in vacuo to about 25 ml. When standing the diphosphate identified. A second peak of yellow at 7 ° C white crystals form overnight, the collected material, which is eluted between 4.15 and 4.41 of the gradient and dried, becoming 65% yield of product, is evaporated to dryness and gives the desired get. A sample is crystallized from hot water for analysis in 20% yield. The analysis shows 3.0 phospha-installed. residue per ribose residue.

Analysis: Ber. for C12H19N6O7P.2H2O: C: 33.8; H: 5.45; N:

19.7; Found: C: 34.3; H: 5.22; N: 19.7. 10 Example 7: Preparation of 2,4-dinitrophenyl-ATP conjugate

(Derivative in 8-position); 8- [2- (2,4-dinitrophenyl) aminoethyl] ami-

Thin-layer chromatograms are developed with 2 solution noadenosine 5'-triphosphate by means of systems, the first consisting of 4 parts of 0.5 m

Ammonium acetate to 1 part of ethanol and the second consists of 3 parts of A. 8- (2-aminoethyl) aminoadenosine-5'-monophosphate isobutyric acid to 5 parts of 1 m ammonium hydroxide. In 15 A mixture of 2.2 millimoles of 8-bromadenosine-5'-mono-two cases showed a component that the fluoresis phosphate (production see Biphys. 163: 561-159 (1974)), 66 milli-zenz steamed and reacted with ninhydrin. The compound in mol of ethylenediamine and 25 ml of water is heated in an oil bath for 2 hours at 0.14N hydrochloric acid with an absorption maximum at 264 nm at 140 ° C. The cooled mixture is sodium with the millimolar extinction coefficient was 17.7. The spectral hydroxide was adjusted to pH 11.5 and the properties on a 2.5 x 55 cm column thus correspond to the characteristics of 20 from Dowex 1x8 (particle size 0.074 to 0.037 mm, bicarbo-N6-alkylated adenosine derivatives. Natform). The column is filled with 300 ml of water and then with a linear gradient of 31 water and 31

B. N6- [2,4-dinitrophenyl) aminoethyl] adenosine-5 '-monophos- 0.5 m ammonium bicarbonate solution. The absorption of the leaking solution at 25 nm is followed and a

250 mg (0.65 millimoles) of N6- (2-aminoethyl) adenosine-5'-mono-25 main peak of absorbent material is between 4.6 and phosphate according to Part A of this example in 20 ml of water-5.81 of the gradient eluted.

dissolved at pH 8. Then 168 mg of sodium bicarbonate. The ammonium bicarbonate is

and then 0.2 ml of l-fluoro-2,4-dinitrobenzene (1.58 milliseconds in vacuo to dryness (5 times, in each case 20 to 30 ml mol, dissolved in 2 ml of ethanol) added. The reaction mixture water) removed and the residue is stirred in 20 ml of water in the dark at room temperature for 4 hours, then 30 addition of ammonium hydroxide to pH 8.0 is dissolved. The solution is then filtered with 0.1 ml of 1-fluoro-2,4-dinitrobenzene in 1 ml of ethanol, adjusted to pH 5.0 with formic acid and given one. The reaction mixture is stirred overnight, then left to stand at 5 ° C. for a day. The crystals that form are adjusted to pH 21.0 with hydrochloric acid and collected in 200 ml of methanol, dissolved at pH 8.0 and re-cast at pH 5.0. The precipitate that forms is installed in 200 ml of water. The yield of intermediate is 27%. Dissolved in water and the solution is adjusted to pH 8.0 with sodium hydroxide. 35 Thin layer chromatogram, the product migrates in a set and on a 2.5 x 30 cm column of DEAE cellulose in solvent from 4 parts of 0.5 m ammonium acetate solution to bicarbonate form (Reeve Angel, Clifton, New Jersey) chromato- 1 part ethanol as a ninhydrin positive stain that fluoresces. The chromatogram is damped with a linear limit. The absorption maximum in 0.02-n-acetic acid served from 1.51 water and 1.510.7-m-ammonium bicarbonate - is at 275 nm and the millimolar extinction coefficient solution developed. A main peak made of yellow material, the 40 is 17.5. These spectral properties are characteristic of UV light, is between 1200 and 1500 ml of Gra- for alkylated 8-aminoadenosine derivatives.

served eluted. The ammonium bicarbonate is again analyzed by: Analysis. for C12H20N7O7P.H2O: C: 3.40; H: 5.33; N: 23.2. Holte evaporation to dryness, with 40% Ausgef .: C: 34.1; H: 5.28; N: 23.9.

loot of the desired product. This product migrates as a yellow spot in thin-layer chromatograms, 45 B. 8- [2 <2,4-dinitrophenyl) aminoethyl] aminoadenosine-5'-monodie developed with the solvent systems mentioned in part A, and on epichlorohydrin / triethanolamine -Anion- 0.64 millimole of 8- (2-aminoethyl) aminoadenosine-5'-monophos-

Exchanger paper, which is developed with 0.25 m sodium acetate / acetic acid phate (see Part A) in 20 ml water with the addition of buffer of pH 5.0. Ammonium hydroxide dissolved in 0.02 N hydrochloric acid has a pH of up to 8.0. Then 168 mg the product absorption maxima at 264 and 363 nm, milli- 50 sodium bicarbonate and then 0.2 ml l-fluoro-2,4-dinitro-molar extinction coefficients 21.8 and 14.2, respectively. benzene (1.58 millimoles in 2 ml ethanol) was added. The reaction mixture is stirred at room temperature for 18 hours,

C. 2,4-dinitrophenyl-ATP conjugate then 0.1 ml of l-fluoro-2,4-dinitrobenzene in 1 ml of ethanol

0.3 millimoles of N6- [2- (2,4-dinitrophenyl) aminoethyl] adenosine was added. After stirring for a further 4 hours, the 5'-monophosphate (see Part B) is adjusted to a pH of 2.0 in the pyridinium salt 55 mixture with hydrochloric acid and transferred to 200 ml by chromatography on a 1.5 x 20 cm acetone (- 10 ° C) poured. The resulting yellow low column made of Dowex 50 x 2 in pyridinium form (Bio-Rad Laboratolag is filtered off, dissolved in 200 ml of water and poured onto a ream, Richmond, California). The yellow outflowing solution 2.5 x 45 cm column made of DEAE cellulose in bicarbonate form is evaporated to dryness and treated with 15 ml of dimethylformamide. A linear salt gray and 0.3 millimole tri-n-butylamine is added to the chromatogram. This mixture is eo served from 21 water and 21.0.7 m ammonium carbonate solution, evaporated to dryness and the residue is redeveloped by. A peak of yellow material further dried with an absorptive evaporation. The intermediate maximum at 275 nm is between 2 and 31 of the gradient obtained monophosphate is then according to the in J. Amer. Chem. Ten eluted. The ammonium bicarbonate is made from this Mate Soc. 87: 1785-1788 (1965) described in the triplet by evaporation in vacuo, the yield of phosphate converted. The desired intermediate product, which is soluble in dimethylformamide, is 37%. Optical absorption products are added to 250 ml of water, which then appear maxima in 0.02 n hydrochloric acid at 275 and 373 nm, adjusted to pH 8.0. This solution is characterized by a millimolar extinction coefficient of 21.8 and 15.5 respectively. The additional 2.5 x 58 cm column made of DEAE cellulose in bicarbonate form analysis shows 1.07 phosphate residues per ribose residue.

635 438

20th

C. 2,4-Dinitrophenyl-ATP conjugate

0.5 millimoles of the monophosphate are converted to the tri-n-butylammonium salt by adding 0.8 millimoles of tri-n-butyla-min. The mixture is dried by repeated evaporation from dry dimethylformamide (4 times, 10 to 15 ml each) 5. The last residue is dissolved in 1 ml of dimethylformamide and mixed with 2.0 millimoles of carbonyldiimidazole, which is also present in 1 ml of dimethylformamide, and left to react for 4 hours at room temperature. Excess carbonyldiimidazole is destroyed by reaction with 15 ml of methanol for an hour. Finally, 3 millimoles of tri-n-butylammonium pyrophosphate in 4 ml of dimethylformamide are added and allowed to react with it for 20 hours. The solid residue which forms is centrifuged off and washed twice with 5 ml each of dimethylformamide. The combined supernatant solutions are added to 200 ml of water, then the pH is adjusted to 8 and the mixture is chromatographed on a 2.5 × 25 cm column of DEAE cellulose in bicarbonate form. The chromatogram is developed with a linear gradient of 21 water and 210.5 m ammonium bicarbonate solution. A peak of 20 yellow material with absorption maxima at 275 and 363 nm is eluted between 2.0 and 2.91 of the gradient. The ammonium bicarbonate is removed by evaporation, the desired conjugate being obtained in a 22% yield. The analysis values show that this product has 3.2 phosphate residues per 25 ribose residues.

Example 8: Homogeneous test with competing binding / bioluminescence for derivatives of 2,4-dinitrobenzene; Use of ATP as a labeling substance

The reaction system used in this example was based on the following implementation:

B. Test using the 8-position bound ATP derivative

4 reaction mixtures were prepared, each of which had a total volume of 100 μl and 20 mM tris (hydro-xymethyl) aminomethane hydrochloride buffer of pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 p.1 antiserum against 2,4- Dinitrophenyl, 594 nM 2,4-dinitrophenyl-ATP conjugate (derivative bound in the 8-position, preparation see Example 7) and 2,4-dinitrophenyl-ß-alanine in the concentrations given in Table 8. After incubation at 25 ° C for 2 hours, 10-fil samples of each reaction mixture were tested in duplicate as in Part A and the average of the maximum light intensity was calculated. The results are shown in Table 8.

Table 8

Table 7

Reaction

Concentration on maximum mix

2,4-Dinitrophenyl-ß-alanine light intensity

(AROUND)

(Average!)

1

0

7

2nd

25th

14

3rd

2500

185

Reaction

Concentration on maximum mix

2,4-Dinitrophenyl-ß-alanine light intensity

ftiM)

(Average)

1

0

18th

2nd

25th

51

3rd

250

191

4th

2500

190

ATP ligand + reduced luciferin luciferase;

AMP ligand + oxidized luciferin + h 0

A. Test with the ATP derivative bound in the 6-position

3 reaction mixtures were prepared, each of which had a total volume of 100 | xl and 10 mM tris (hydro-xymethyl) aminomethane hydrochloride buffer of pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 μl antiserum against 2,4-dinitrophenyl , 512 mM 2,4-dinitrophenyl-ATP conjugate (derivative in 6-position, preparation see Example 6) and contained the amounts of 2,4-dinitrophenyl-ß-alanine given in Table 7. After incubation for 2 hours at 25 ° C., 10 μl samples of each reaction mixture were tested in duplicate by injection into 0.1 ml of the light-generating solution described above, which had previously been incubated at 25 ° C. for at least 2 minutes and was in a reagent tube, which was built into a Dupont Model 760 bioluminescence photometer (EI duPont de Nemours, Willmington, Delaware). The maximum light intensity was read on the photometer. Both the average of the maximum light intensity and the relative intensity (100% x ratio of the maximum light intensity of the sample to the maximum light intensity in the absence of the antiserum) were calculated. The results are shown in Table 7.

35

40

45

55

60

65

The results of this example show that the marking substance ATP, after the formation of a derivative, is used in various positions to produce a conjugate which can be used in the test method according to the invention.

Example 9: Preparation of biotin-isoluminol conjugate. 6- (3-

Biotinoylamido-2-hydroxypropylamine) -2,3-dihydrophthalazine-

1,4-dione

A.4- (3-chloro-2-hydroxypropylamino) -N-methylphthalimide

25 g (0.142 mol) of 4-amino-N-methylphthalimide (preparation see J. Chem. Soc. 26: (1937)) and 20.7 g (0.21 mol) of 1-chloro-2,3-epoxypropane were added 150 ml of 2,2,2-trifluoroethanol were added and the reaction mixture was heated under reflux for 48 hours with stirring. 70 to 80 ml of the 2,2,2-trifluoroethanol were distilled off, and a heavy yellow precipitate formed when the remaining solution cooled to room temperature. This precipitate was triturated with ethyl acetate, filtered off and dried, giving 29.5 g (77% yield) of the desired intermediate from the F.

136-138.5 ° C were obtained.

Analysis: Ber. for C12H13CN2O3: C: 53.64; H: 4.88; N: 10.45. Found: C: 53.87; H: 4.85; N: 10.81.

B. 4- [3- (N-Phthalimido) -2-hydroxypropylamino] -N-methyl-phthalimide

13.5 g (0.05 mol) of the intermediate shown in Part A and 15.7 g (0.085 mol) of potassium phthalimide are refluxed for 24 hours with stirring in 150 ml of dimethylformamide. The dimethylformamide is removed and the residue is washed with water and filtered off. The yellow filter cake is recrystallized from acetic acid / water, 12.8 g of product (67% yield) of mp 247-248.5 ° C. are obtained.

Analysis: Ber. for C20H17N3O5: C: 63.32; H: 4.52; N: 11.08. Found: C: 63.16; H: 4.38; N: 10.93.

C. 6- [3-Amino-2-hydroxypropylamino] -2,3 dihydrophthalazine-1,4-dione

5.0 g (13.2 millimoles) of the intermediate according to Part B, 90 ml of absolute ethanol and 35 ml of 95% hydrazine are refluxed with stirring for 4 hours. The solution

21st

635 438

medium is removed in vacuo and the resulting solid is dried in vacuo at 120 ° C. for 24 hours. This material is then stirred for 1 hour with 70 ml of 0.1 N hydrochloric acid. The insoluble 2,3-dihydroxyphthalazin-1,4-dione is filtered off and the filtrate is adjusted to pH 6.5 with saturated sodium bicarbonate solution. The white precipitate which forms is filtered off and dried, giving 2.2 g of product (67% yield). After recrystallization from water, the compound decomposes at 273 ° C.

Analysis: Ber. for ChHmNjOî: C: 52.79; H: 5.64; N: 22.39. Found: C: 52.73; H: 5.72; N: 22.54.

D. Biotin-isoluminol conjugate

0.29 g (1.2 millimoles) of biotin and 0.17 ml of triethylamine are dissolved in 20 ml of dry dimethylformamide under anhydrous conditions and cooled to -WC. A solution of 0.141 ml of ethyl chloroformate in 2.86 ml of ether is then slowly added and the reaction mixture is stirred for 30 minutes. A precipitate that separates is filtered off. A suspension is quickly added to the filtrate, 20 which consists of 600 mg (2.4 millimoles) of the intermediate according to Part C, 20 ml of dry dimethylformamide and 1 ml of dry pyridine. This mixture is stirred for 30 minutes at -10 ° C and then overnight at room temperature. A solution will be obtained during this period. The dimethylformamide is distilled off at 60 ° C. and 0.10 mm Hg, the oily residue is stirred with 50 ml of n-hydrochloric acid for 1 hour. A white solid which forms is filtered off and washed with 0.1N hydrochloric acid and then with water. After drying in vacuo overnight at room temperature, 0.55 g of product (97% yield) of mp 170-173 ° C.

Analysis: Ber. for C21H28N6O5S: C: 52.92; H: 5.92; N: 17.64. Found: C: 51.69; H: 5.90; N: 17.63.

Example 10: Homogeneous competing binding / chemiluminescent test for biotin

The reaction system used in this example was based on the following implementation:

Biotin isoluminol + KO2 - *

Biotin aminophthalate + N2 + H 0

16 reaction mixtures were prepared, each of which had a total volume of 150 μl and 0.1 m tris (hydro-xymethyl) aminomethane hydrochloride with a pH of 8.0.42 nM bio-tin-luminol conjugate (preparation see Example 9) and biotin in the concentrations given in Table 9 and 0.12 units / ml avidin (added at the last position). After 5 minutes of incubation at 25 ° C., 10 μl of dimethylformamide which contains 0.15 M potassium superoxide (KO2) (Alpha Products, Beverly, Massachusetts) and 0.10 M 1,4,7,10,13,16-hexaoxy-cyclooctadecane ( Aldrich Chemical Co., Milwaukee, Wisconsin) is injected into each reaction mixture. After a further 2 minute incubation at 25 ° C., 10 (xl 0.95 mM hydrogen peroxide in 10 mM tris (hydroxymethyl) aminomethane hydrochloride buffer of pH 7.4 are injected into each reaction mixture and the maximum light intensity is measured with a Dupont Model 760 bio-luminescent photometer (EI duPont de Nemours, Willmington, Delaware) was measured and the results are shown in Table 9.

60

Table 9

Reaction area

Maximum concentration of biotin mixed

(nM)

Light intensity (mean)

1

0

38.5

2nd

13

38.5

3rd

27th

34.3

4th

40

36.1

5

53

35.2

6

67

36.2

7

101

34.0

8th

133

31.7

9

166

29.1

10th

200

24.2

11

267

22.8

12

333

20.5

13

400

13.4

14

534

8.6

15

667

8.3

16

800

7.0

25th

30th

35

45

50

It has thus been shown that the level of maximum light intensity which is formed by the chemiluminescent reaction system is the opposite function of the amount of biotin present in the specific binding reaction mixture. The invention therefore provides a test mixture and method for determining the presence of the ligand biotin in a liquid medium using a competing binding chemiluminescence technique that does not use an enzyme-catalyzed monitor response.

Example 11: Preparation of biotin-umbelliferone conjugate;

(2-oxo-2-H-l-benzopyran-7-yl) -5- [cis-hexahydro-2-oxo-lH-

thieno- (3,4-d -) - imidazole] valeric acid ester

A solution of 300 mg (1.2 millimoles) of anhydrous biotin in 20 ml of dry dimethylformamide is stirred at -10 ° C. under nitrogen gas and 0.17 ml (1.2 millimoles) of rocked triethylamine are added. Then the solution of freshly distilled ethyl chloroformate (0.141 ml in 3 ml dry ether) is added dropwise. After incubation for one hour with stirring, the resulting precipitate is filtered off in a dry nitrogen atmosphere and immediately cooled to -10 ° C. A solution of 197 mg (1.2 millimoles) of anhydrous 7-hydroxycoumarin in 3 ml of pyridine is added to the residue,

then the mixture is stirred at -10 ° C. for 1 hour and at 25 ° C. for 20 hours. The solvents are distilled off at 40 ° C. in a high vacuum. After cooling, the resulting solid is filtered off and recrystallized from methanol, giving the desired product of mp 216-218 ° C.

Analysis: Ber. for C19H20N2P5S: C: 48.75; H: 4.19; N: 7.21. Found: C: 58.4; H: 5.12; N: 6.86.

Example 12: Heterogeneous test with competing binding / bioluminescence for biotin; Influence of different amounts of biotin on the maximum of light intensity

The reaction system of this example was based on the reactions given in example 3.

A. Preparation of the light generating solution

The light-generating solution was prepared according to the instructions in Example 3.

65 B. Preparation of an Insolubilized Binding Partner Avidin, which has a binding affinity for biotin, was made insoluble by being bound to a water-insoluble polymer as follows: Sepharose 4B (Pharmacia AB, Upp-

635 438 22

sala, Sweden) was made according to the method of March et al., NAD, NAD-biotin conjugate, avidin-bound Sepharose

Analytical Biochemistry 60: 149 (1974) for binding to avidin 4B suspension (preparation see part B) and Sepharose activated. About 4 ml of the activated Sepharose 4B was packed in 4B suspension (obtained by suspending 1 ml

8 ml of 0.1 m citrate buffer of pH 7.0 suspended. For suspension of Sepharose 4B in 60 ml 0.1 m tris (hydroxymethyl) aminome-

6 mg of avidin with an activity of 9.9 units per s than hydrochloride buffer (pH 8.0) were contained. The reaction

mg added in 3 ml of water. One unit of avidin activity is mixed. For 15 minutes at room temperature, the amount of avidin that has been shaken to bind 1 (ig biotin is mild. Then 0.22 international units of alcohol

is. The resulting reaction mixture was added 6 hours hol dehydrogenase to each reaction mixture

stirred at 7 ° C. Then the Sepha- bound to avidin was used to initiate the reduction reaction. Compound semicarbazide

Rose 4B filtered off, washed with 100 ml of 0.1 m sodium bicarbonate buffer with the acetaldehyde produced in reaction (c) under pH 9.0 and again in 240 ml of 0.1 M tris (hydro- formation of a semicarbazone whereby reaction (c) is forced in the xymethyl) aminomethane hydrochloride buffer of pH 8.0 desired direction.

suspended. The reaction mixtures were again at 15 minutes

Shaken at room temperature. Then 10 ixl's in each

C. Comparative experiments 15 reaction mixture supernatant liquid in the cuvette

9 reaction mixtures were prepared, each of which was a DuPont Model 760 bioluminescence photometer (E.I.

had a total volume of 0.19 ml and 0.1 m Tris (hydro-duPont de Nemours, Willmington, Delaware) injected, the xymethyl> aminomethane hydrochloride buffer of pH 8.0.0.6 M already 100 jxl previously produced light-producing

Ethanol, 0.01 M semicarbazide hydrochloride and contained in solution, which pre-incubated at 25 ° C for 2 to 3 minutes

Amounts or concentrations of 20 given in Table 10. The results are shown in Table 10.

Table 10

reaction

concentration

Concentration

Avidin-bound

Sepharose maximum

to NAD (nM)

NAD-biotin con-

dene Sepharose 4B suspension

Light intensity

jugat (nM)

4B suspension (ixl)

00)

(Average)

1

-

-

_

_

1.2

2nd

21st

-

-

-

159

3rd

21st

-

20th

-

147

4th

-

10th

-

-

45.9

5

-

21st

-

-

110

6

-

21st

20th

-

28.5

7

21st

-

-

10th

154

8th

-

21st

-

10th

114

0

-

-

20th

-

1.6

40

The results of comparative reactions 1 and 9 show that very little light is generated in the absence of NAD and NAD-biotin conjugate. Reactions 2 and 3 show that the light-generating reaction takes place when free NAD is added, and that this reaction is essentially unaffected by the presence of avidin-bound Sepharose 4B. The results of reactions 4, 5 and 6 show that the NAD-biotin conjugate is active in the light-generating reaction, that the maximum light intensity increases with an increasing amount of NAD-biotin conjugate and that the presence of avidin-bound Sepharose 4B is used to generate light inhibited. A comparison of the results of reactions 3 and 5 with the results of reactions 7 and 8 shows that the light-generating reaction is not influenced by the presence of simple Sepharose 4B.

D. Test procedure

5 further reaction mixtures were prepared, each of which had a volume of 0.19 ml and 0.1 M tris (hydroxymethyl) aminomethane hydrochloride buffer of pH 8.0.0.6 M ethanol, 0.01 M semicarbocide and contained the amounts or concentrations of NAD-biotin conjugate, free biotin and avidin-bound Sepharose 4B suspension listed in Table 11. Each reaction mixture was treated in the same manner as the comparative mixtures from Part C of this example. The results are shown in Table 11.

Table!!

Reak-45 tion

, 10th

11

12

13

14

Concentration concentra- Avidin-bound maximum ion of biotin light-in

NAD-Biotin (nM) Sepharose sity

Conjugate (nM) 4B suspension (mean)

(nD

21 21 21 21 21

79 158 158

20 20 20

79.1 17.4 43.1 59.9 79.7

55

The results of reactions 11, 12 and 13 show that free biotin and NAD-biotin conjugate successfully compete for the binding sites on the insolubilized avidin, since the maximum light intensity generated depends on the free amount of biotin. Reactions 10 and 14 show that in the absence of insolubilized avidin, the maximum light intensity is constant at widely different concentrations of free biotin.

This example thus shows that the amount of NAD-biotin-65 conjugate in the liquid phase is inversely related to the amount of free biotin present, so that the test method according to the invention is useful for determining a ligand in an unknown liquid sample.

23

635 438

Example 13: Heterogeneous specific binding test for avidin and biotin using an enzyme substrate as a labeling substance

The test system used in this example was based on the following reactions:

-C- (CH2) 4 ~

X

Si N

/

Esterase.

H20, pH 8.0 p

Bi ot in-IJmb e 11 if ermn conjugate

20th

+ eats +

Biotin

(max.fluorescence at 448 nm)

A. Preparation of the Insolubilized Binding Partner

Avidin was made insoluble by covalent binding to a water-insoluble polymer using the procedure of Example 12, Part B with the exception that after washing with 100 ml 0.1 n sodium bicarbonate buffer pH 9.0 the avidin bound Sepharose 4B in 12 ml of 0.1 M tris (hydroxymethyl) aminomethane hydrochloride buffer of pH 8.0 and then in a 1: 1 ratio with 0.1 M bis-hydroxyethylglycine hydrochloride buffer of pH 7.0 was diluted.

At room temperature, the fluorescence intensity generated in each reaction mixture 30 at 448 nm with excitation at 364 nm was measured with an Amico-Bowman spectrophotofluometer. The results are shown in Table 12.

35

Table 12

40

B. Competing Biotin Binding Assay; Influence of different amounts of biotin on the amount of Umbelli-feron released

Eight reaction mixtures were prepared, each of which had a total volume of 0.2 ml and 0.1 m bis-hydroxyethylglycine hydrochloride buffer with a pH of 7.0.0.3 μl Biotin-45 umbelliferone conjugate (preparation see example 11), 15 | il of the avidin-bound Sepharose 4B suspension (preparation see Part A) and biotin in the amounts given in Table 12. The reaction mixture was incubated at room temperature with gentle shaking for 20 minutes. Each reaction mixture was centrifuged and 100 µl of the supernatant was combined with 2 ml of 0.1 M bis-hydroxyethylglycine hydrochloride pH 8.2 buffer containing 1.08 units of porcine esterase. After 5 minutes of incubation at

Reaction mixture

Concentration of biotin

(Tim)

intensity

Fluorescence-

1

0.00

0.355

2nd

0.10

0.495

3rd

0.20

0.469

4th

0.30

0.503

5

0.40

0.547

6

0.50

0.502

7

0.75

0.580

8th

1.00

0.688

In this example it could thus be shown that the amount of NAD biotin in the liquid phase is directly proportional to the amount of free biotin present, so that the test method is useful for determining a ligand in an unknown liquid sample.

Claims (22)

635438 PATENT CLAIMS 1. A specific binding test method for examining a liquid medium for a ligand, the test method comprising the following steps: a) the liquid medium is brought into contact with a reagent which contains a conjugate of a labeling substance which has a predetermined characteristic and a specific binding substance, the reagent and the ligand forming a binding reaction system which i) form a bound phase of the forms a labeling conjugate in which the specific binding part is bound to it by a specific binding partner, and ii) forms a free phase of the marking conjugate in which the specific binding part is not bound to it by a specific binding partner, b) determination of the characteristic mentioned either in the bound phase or in the free phase as a measure of the ligand mentioned in the liquid medium mentioned, characterized in that the characteristic of the labeling substance in the conjugate mentioned proves to be active in the following reaction systems: 1) ^ l as substrate in an enzyme-catalyzed reaction, 2) as a reactant in a cyclic reaction system, 3) as a reactant in a chemiluminescent reaction system, or 4) as a coenzyme in an enzyme-catalyzed reaction. 2. Test method according to claim 1 of the homogeneous type, characterized; that said labeling substance in the bound phase is measurably different from that in the free phase and that the characteristic is determined without physical separation of the bound phase from the free phase. 3. The test method according to claim 1 of the heterogeneous type, characterized in that said labeling substance in the bound phase is essentially the same as in the free phase and that the bound phase and the free phase are subjected to a physical separation and that said Characteristic is determined in one of the separate phases. 4. Test method according to claim 1, characterized in that the labeling substance is a reaction participant in an enzyme-catalyzed reaction, by which a product is obtained which has a detectable property, by which it differs from said conjugate. 5. Test method according to claim 4, characterized in that the detectable property is fluorescence. 6. Test method according to claim 1, characterized in that the labeling substance is a cyclic reactant in said cyclic reaction system. 7. The test method according to claim 1, characterized in that the labeling substance is a reactant in a chemiluminescent reaction system and said characteristic is determined by measuring the total amount of light produced or the peak intensity of light produced. 8. Test method according to claim 7, characterized in that said labeling substance is luminol or iso-luminol or a derivative thereof. 9. Test method according to claim 1, characterized in that the labeling substance is a nucleotide coenzyme. 10. The test method according to claim 1, characterized in that said labeling substance is a nicotinamide dinucleotide, which may be in reduced form, an adenosine triphosphate or an elavin adenine dinucleotide. 11. Test method according to one of claims 1 to 10, characterized in that the ligand from the following Group is selected: antigens and corresponding antibodies, haptens and corresponding antibodies, hormones, vitamins, metabolites and pharmacological agents as well as their receptors and their binding substances. 12. Reagent for use in the test method according to claim 1 in the determination of a ligand in a liquid medium containing a conjugate of a labeling substance which contains a predetermined characteristic and a specific binding substance, and wherein the reagent and the ligand are a binding reaction Form a system that generates a bound phase of the labeled conjugate, wherein the specific binding substance contained therein is bound to a specific binding partner and forms a free phase, wherein the specific binding substance contained therein is not bound to a specific binding partner and the said characteristic, either in the bound phase or in the free phase, is a function of the amount of ligand in the said liquid medium, characterized in that the said characteristic of the labeling substance in the said conjugate proves to be active in the following reaction systems: 1) as a substrate in an enzyme-catalyzed reaction, 2) as a reactant in a cyclic reaction system, 3) as a reactant in a chemiluminescent reaction system, or 4) as a coenzyme in an enzyme-catalyzed reaction. 13. A reagent according to claim 12, characterized in that it is said labeled conjugate (1), wherein the specific binding substance contained therein is said ligand or a specific binding analogue of said ligand and (2) a specific binding partner of said Contains ligands. 14. Reagent according to claim 12, characterized in that it contains an incorporated carrier matrix. 15. Reagent according to one of claims 12 to 14, characterized in that the ligand is selected from the following group: antigens and corresponding antibodies, haptens and corresponding antibodies, hormones, vitamins, metabolites and pharmacological agents as well as their receptors and their binding substances . The present invention relates to a multi-stage test method for the examination of a liquid medium for a ligand and a reagent which is used to carry out the method according to the invention, which works with a specific binding. Because of the safety risk and the difficulties in handling radioactive materials, numerous attempts have been made to develop test systems which work with specific bindings and which are as sensitive and fast as radioimmunoassays, but use properties other than radioactivity to detect the binding reaction. As will be explained in more detail below, the substances already used as labeling substances instead of radioactive atoms or molecules include, for example, enzymes, fluorescent molecules and bacteriophages. The test methods initially discussed, which are based on specific binding, belong to the "heterogeneous" type, in which bound and free phases are separated. This separation is necessary if the marked material in the bound phase cannot be distinguished from the marked material in the free phase. Examples of heterogeneous methods with enzymes as mar- 2nd 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 3,635,438 of the substance are described in US Pat. No. 3,654,090,3,791,932, the difficulty of the preparation of useful enzyme-linked 3,839,153,3,850,752 and 3,879,262 in Journal of Immunological conjugates. Methods 1: 247 (1972) and in Journal of Immunology 109: 129. The present invention provides a very convenient, adaptable (1972). All of these methods provide a solution-capable and sensitive improved test method Enzyme chemically either on the ligands 5 to be found, which is based on the fact that a sub- or its binding partner coupled and a suitable punch used as part of a predetermined heterogeneous specific binding reaction scheme is shown on monitor system responsiveness and as below, where after incubation with the sample the amount of "the reagent" is referred to. The inventive Enzymatic activity that is either bound to the insoluble or kier substance with either a new homogeneous portion of the soluble portion is a function of the amount of the io test technique or in the conventional heterogeneous test ligand in the sample. The problems used in the synthesis schemes. and characterization of the enzyme conjugates are difficult. The present invention thus relates to a test method which works with significant disadvantages of the above methods GB-PS 1 392 403 and FR-PS 2 201299 describe examination of a liquid medium for a ligand, a heterogeneous specific binding test which is inactive, the test method comprising the following steps: precursor of a spectrophotometrically active substance as a) the liquid medium is mixed with a reagent which Marking substance used. After incubation of the sample, a conjugate of a labeling substance that with the specific binding reaction system, unresolved characteristics are exhibited, and a specific binding Liche and liquid fractions separated from each other and the amount of substance brought into contact, the reagent of labeling substance in the liquid portion, which form a Funk- and the ligand a binding reaction system, which is tion of the amount of ligand in the sample, is ermit- i) form a bound phase of the labeling conjugate by performing reaction steps that marten the inactive ones, in which the specific binding fraction is determined by a specific the binding substance is bound to a chromogen or fluorometrically active binding partner, and transfer ves material, which then ii) forms a free phase of the labeling conjugate in a conventional manner, is determined quantitatively. 25 in which the specific binding proportion is not determined by a specific Further heterogeneous specific binding tests, which binding partners see bound to it, use different types of labeling substances, b) determination of the characteristics mentioned either in are described in the following literature: Report No. PB-224,875 of the bound phase or in the free phase as a measure of that of the National Technical Information Service (NTIS) of the US mentioned ligands in the liquid medium mentioned, Ministry of Commerce (1973) describes an unsuccessful method and the method according to the invention is characterized by the use of hemin chloride as the marking sub-character that the mentioned characteristic of the marker dance is found in a heterogeneous test system which is characterized by a substance is followed in the conjugate mentioned in the subsequent chemiluminescent reaction. In Nature 219: 186 reaction systems proved to be active: (1968) describe in detail certain radioimmunoassay methods 1) as a substrate in an enzyme-catalyzed reaction, while at the same time a general reference 35 2) as a reactant in a cyclic reaction to the possible use of coenzymes and virus systems, instead of radioisotopes as labeling substances 3) as reactants in a chemiluminescent is made. However, the author does not develop a reaction system concept, or on the implementation of such an alternative labeling 4) as a coenzyme in an enzyme-catalyzed reaction. Tests using substances. In Principles of Competitive 40 The reagent mentioned for performing in the Bestim Protein Binding Assays, Ed. Odell and Daughaday (JB mung a ligand in a liquid medium containing Lippincott Co., Philadelphia, 1972) will discuss various a conjugate of a labeling substance, which discusses a known test scheme and the different materials, determined characteristics and a specific binding sub-broad so far contains for labeling in a specific distance and the reagent and the ligand were used in a binding test. 45 form reaction system that produces a bound phase of the Of Interest is the labeled conjugate described in U.S. Patent No. 3,817,837, wherein the spe-immun test contained therein, which is followed by an enzyme. The specific binding substance there to a specific binding part method is of the “homogeneous” type such that it is not bound to the ner and forms a free phase, in which the use therein of a divided (i.e. insoluble fraction / specific binding substance contained does not relate to a specific) -Liquid fraction) specific binding reaction system and 50 see binding partner is bound and the said requires the corresponding separation process, since the ligand traced by means of characteristics either in the bound phase or in the enzyme is equipped in such a way that after the reactive phase a function of the amount of the ligand in which the binding partner of the ligand is an enzymatic liquid medium is characterized by activity being inhibited. The ratio of bound mate - that the mentioned characteristics of the labeling subrial to material can be determined in free form - 55 punch in the conjugate mentioned in the subsequent reaction - by proving the changes in the enzymatic activity systems to be active: tracked. However, this process has the difficulty of 1) as a substrate in an enzyme-catalyzed reaction, that well-characterized, enzyme-bound conjugates 2) have to be prepared as reaction participants in a cyclic reaction and that enzymes have to be found which are integrated in the basic arrangement system, system. 60 3) as a reactant in a chemiluminescent Although various new types of specific binding reaction systems, or tests have been proposed and studied, the radio 4) remain as a coenzyme in an enzyme-catalyzed reaction, immunoassays and the various enzyme-linked immune systems. The monitor system is preferably enzyme-catalyzed. tests the most widely used and best worked out- Usually you choose a monitor system that is compared to the tests you are testing. However, both systems have obvious post-reagent in the conjugate that is highly sensitive. Luminescent parts, namely the radioimmunoassay in the use of or fluorescent reaction systems are radioactive material in this regard, which is risky and a very careful careful one. Cyclic handling is particularly preferred, and the enzyme-linked immunoassay in reaction systems, especially those in which the reagent is required 635438 is the material in circulation. Among the preferred cyclic reaction systems, the enzyme-catalyzed ones are particularly advantageous. The reagent in the conjugate is usually an enzymatic reagent, for example an enzyme substrate or particularly preferably a coenzyme, and preferably has a molecular weight of less than 9000. The following designations are used in the following description: The ligand is the substance or group of substances whose presence or amount in the liquid medium is to be determined. The specific binding partner of the ligand is any substance or group of substances which has a specific binding affinity for the ligand to the exclusion of other substances. The specific binding analogue of the ligand is any substance or group of substances that behaves essentially like the ligand in terms of the binding affinity of the specific binding partner for the ligand. The new homogeneous test procedure is based in part on the fact that the reaction between a ligand and a specific binding partner, with the reagent coupled to one of these two partners, alters the activity of the reagent in the intended reaction, so that this reaction as Used to track the specific binding reaction. Various operations with different test mixtures and devices can be used to carry out the method according to the invention. The direct binding method and the competing binding method are preferred. In the direct binding method, a liquid medium in which the ligand to be determined is suspected is usually brought into contact with a conjugate which contains the reagent bound to a specific binding partner of the ligand, after which any change in the action of the reagent is determined. In the competing binding process, the liquid medium is typically contacted with a specific binding partner of the ligand and with a conjugate that contains the reagent coupled to the ligand and / or a specific binding analogue, followed by any change in the activity of the reagent determined. In both cases, the activity of the reagent can be determined by contacting the liquid medium with at least one agent which enters into the intended reaction suitable for tracking with the reagent. The qualitative determination of the ligand in the liquid medium generally compares one property, usually the extent of the resulting reaction, compared to the reaction used for tracking in a liquid medium without ligands, with differences indicating a change in the activity of the reagent. In the quantitative determination, a characteristic of the resulting reaction is preferably compared with the intended reaction in a liquid medium which contains known amounts of the ligand. In the homogeneous test scheme, the components of the specific binding reaction, i.e. the liquid medium in which the ligand is expected, the conjugate and / or a specific binding partner of the ligand can generally be combined with one another in any amount, in any way and in any order, provided that the activity of the reagent in the conjugate is measurably changed if the liquid medium contains the ligand in an amount or concentration that is significant for the purposes of the test. All components of the specific binding reaction are preferably soluble in the liquid medium. Do you work e.g. using homogeneous direct binding technique, the components of the specific binding reaction are the liquid medium in which the ligand is suspected and an amount of conjugate containing the reagent coupled to a specific binding partner of the ligand. The activity of the conjugate / reagent on contact with the liquid medium changes inversely to the extent of the bond between the ligand in the liquid medium and the specific binding partner in the conjugate. The activity of the reagent bound in the conjugate thus decreases with increasing amount of the ligand in the liquid medium. When competing binding in the homogeneous process, the components of the specific binding reaction consist of the liquid medium in which the ligand is suspected, an amount of a conjugate containing the reagent coupled to the ligand or a specific binding analog of the ligand, and an amount of one specific binding partner for the ligand. The specific binding partner is brought into contact with the conjugate and the liquid medium essentially simultaneously. Since each ligand in the liquid medium competes with the ligand or its specific binding analogue in the conjugate for binding with the specific binding partner, the activity of the conjugated reagent changes upon contact with the liquid medium in a direct manner according to the extent of the binding between ligands in the liquid medium and the specific binding partner. With increasing amount of ligand in the liquid medium, the activity of the conjugated reagent also increases. A preferred modification of the homogeneous process with competing binding is the homogeneous process with displacement binding, in which the conjugate is first brought into contact with the specific binding partner of the ligand and then with the liquid medium. There is then competition for the specific binding partner. In this method, the amount of conjugate brought into contact with the specific binding partner is usually such that it contains the ligand or its analogue in excess of the amount capable of binding with the amount of the specific binding partner which during the time in which the conjugate and the specific binding partner are in contact with each other before contact with the liquid medium. There are two ways to do this. In one case e.g. before contact with the liquid medium in which the ligand is expected, the conjugate is brought into contact with the specific binding partner in a liquid medium. In the second case, the liquid medium in which the ligand is expected can be brought into contact with a complex which contains the conjugate and the specific binding partner, the specific binding substance in the conjugate and the specific binding partner being reversibly bound to one another. The amount of conjugate that is bound to the specific partner, which in the second case is in complex form, is usually an excess over the amount that can be displaced by all ligands in the liquid medium in the period in which the specific binding partner or The complex and the medium are in contact before the determination of any change in activity of the conjugated reagent is complete. Another preferred modification of the homogeneous competing binding method is the homogeneous consecutive saturation technique in which the components of the specific binding reaction are the same as the competing binding but the order of addition or combination of the components and their relative amounts are different are. In sequential saturation, the specific binding partner of the ligand can be mixed with the liquid medium in which the ligand is 4th 5 10th 15 20th 25th 30th 35 40 45 50 55 60 6? 635 438 suspected to be brought into contact for a certain time before the liquid medium comes into contact with the conjugate. The amount of specific binding partner which is brought into contact with the liquid medium is usually in excess of the amount which is capable of binding the entire ligand in the liquid medium during the time in which the specific binding partner and liquid medium come into contact with one another stand before the liquid medium comes into contact with the conjugate. The amount of ligand or ligand analogue in conjugated form is usually an excess over the amount capable of binding the remaining unbound amount of the specific binding partner during the time that the liquid medium and conjugate are before determining the change in activity of the conjugated reagent are in contact with each other. The determination of changes in activity of the conjugated reagent as a component of the predetermined monitor reaction is expediently carried out by bringing the reaction mixture into contact with at least one substance which enters into the monitor reaction with the conjugated reagent, the effect of the specific binding reaction on one Characteristic of this reaction determined. The use of a reagent as a labeling substance can also be applied to the conventional heterogeneous test schemes in which bound and free phases of the labeled component are separated from one another and the amount of labeling substance is determined in one or the other phase. The means for performing such a heterogeneous test can take various forms. They generally comprise 3 basic components, namely (1) the ligand to be determined, (2) a specific binding partner of the ligand and (3) a labeled component, which is usually a labeled form (a) of the ligand, (b) a specific binding Analogue of the ligand or (c) the specific binding partner. The components of the binding reaction are combined simultaneously or in succession, and with a suitable incubation time or times, the labeled component is bound to the corresponding competing binding partner in such a way that the extent of the binding, that is to say the ratio between the amount of the labeled component, to a binding partner is bound to the unbound amount, a function of the amount of ligand present. The following is a brief description of some binding reaction schemes that can be used to carry out the method of the invention. In conventional heterogeneous test methods with specific binding, such as radioimmunoassays and heterogeneous enzyme immunoassays, the labeling property in the labeled conjugate, for example radioactivity or enzymatic activity, is essentially the same in bound and free form of the conjugate. According to the invention, however, the activity of the reagent, as a labeling substance, is influenced in certain cases by the binding of the labeled conjugate. In these cases, the monitor reaction shows a relatively constant character if the ligand is missing in the liquid medium or is present in an insignificantly small amount. If the ligand is present in the liquid medium, a property of the monitor reaction is changed, just as in the homogeneous method. The following symbols apply to the following diagrams: symbol definition L ligand to be determined 5 (l) ligand or specific binding analog B binding partner for the ligand * labeling substance, i.e. Reagent t insoluble phase - Incubation period followed by a suitable separation (lim) limited; is less than that Amount that could be bound to all available binding sites under the chosen reaction conditions at the chosen incubation time; i.e. the The component with which the other components compete for binding, (exc) excess, is present in a larger amount than can be bound by all available binding sites 20 under the chosen reaction conditions during the chosen incubation period. 1. Competing binding in a heterogeneous system 25 a) L + © * + B (lim) - ■ + for B or © * insolubilizing agent This is the classic competing binding process. Examples of insolubilizing agents are special precipitating antibodies, special insolubilized antibodies and, if 3o B or (t) + is a proteinaceous material, protein precipitators such as ammonium sulfate, or if B or (l) + consist of a small adsorbable molecule, Coal coated with dextran. The description of parallel systems can be found in Bio-chem. J. 88: 137 (1963) and U.S. Patent 3,839,153. 35 b) L + © * + l-B (lim) - » This process is commonly referred to as the solid phase technique. The description of parallel radioimmunoassay and enzyme immunoassay procedures can be found in US-PSS 3 505 019.3 555 143.3 646 346 and 3 654 090. 40 c) L + B * + I— (JXlim) Reference: U.S. Patent 3,654,090 d) L + l- (l) + B * (lim) - Reference: U.S. Patent No. 3,850,752. 45 2. Successive saturation in the heterogeneous system a) L + B (exc) - + (l) * (exc) - * + insolubilizing agent for B or (Ç) + In the saturation technique, some or all of the binding sites on B that remain after the first incubation period are bound by the labeled component. b) L + l- B (exc) - * + (l) * (exc) - * Descriptions of parallel radioimmunoassay and enzyme immunoassay procedures can be found in US Pat. No. 3,720,760 and in J. Immunol. 209: 129 (1972). 55 c) L + W * (exc) - + H (ÇXexc) - 3. Heterogeneous “sandwich” process L + i- B (exc) - B * (exc) - * ■ In the sandwich process, some or all of the ligand molecules bound to the insoluble binding partner are bound by the labeled component. Reference: US Pat. No. 3,720,760. 4. Solid phase dilution method with heterogeneous system L + (l) * + l- (not specific) - »+ B (lim) - In this process, the ligand and labeled component are bound to a non-specific binder and then pro- 10th 15 20th 635438 proportional amounts are dissociated therefrom by binding to a binding partner that has greater affinity for the ligand and the labeled component. The most convenient form of this method uses a column made of the non-specific binder according to US Pat. No. 3,659,104. This method is useful where the ligand is bound to endogenous binding substances in the sample, which, if not removed, the competing binding reaction would disturb. After binding to the non-specific binder, the endogenous binding substances can be removed by suitable washes. With regard to a discussion of the parameters that occur in conventional heterogeneous test systems, test forms and separation methods, reference is made to Principles of Competitive Protein Binding Assays, Ed. Odell and Daughaday (J.B. Lippincott Co, Philadelphia, 1972). Of course, other addition sequences and other binding reactions can be used for both homogeneous and heterogeneous tests without departing from the inventive concept. The appropriate reactants, which together with the reagent in the conjugate effect the monitor reaction, can (1) be brought into contact with the specific binding reaction mixture in the homogeneous process, or (2) in the heterogeneous process either with the separated or bound 25 Phase, individually or in any combination before, simultaneously with or after the onset of the specific binding reaction. After the onset of the specific binding reaction, the reaction mixture containing some or all of the components required for the monitor reaction is usually incubated for a predetermined period of time before changes in the activity of the reagent in the conjugate (homogeneous technique) or the amount of activity in the separate ones Phases (heterogeneous technology) can be determined. After the incubation period, any components which are required for the monitor reaction and are not already present in sufficient quantity are added to the reaction mixture, and the reaction is regarded as an indication of the presence or the amount of the ligand in the liquid medium. A preferred form of the monitor reaction is a luminescent reaction system, which is preferably enzyme-catalyzed, for example a reaction that produces bioluminescence or chemiluminescence. The reagent in the conjugate can be a reactant of the light-producing reaction or a reaction that is the precursor of an enzymatic or non-enzymatic luminescent reaction. Any change in the activity of the conjugated reagent resulting from the specific binding reaction causes a change in the light production or the total amount, peak intensity or character of the light produced. Examples of luminescent reaction systems are shown in Table A, which uses the following abbreviations: ATP AMP NAD NADH FMN FMNH2 hu 30th 35 40 <D H r- | <D £> CO Adenosine triphosphate adenosine monophosphate nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide flavin mononucleotide reduced flavin mononucleotide electromagnetic radiation, usually in the IR, visible or UV range 50 55 60 d •H U a) "H / N •H 01 O eu 3rd U + = 1-3 <D bû (0 CQ •H •H a> -P î) 0 + » + »Rö 3 days a> >> • r-s Ci a) M si d Q) •H bO a » O M N d tj M CÖ C0 rH w (D <CJ rH <$ m P4 <u CQ ÉH a> <U u d U TJ a> <D 0) O 'O 'Ö O d 0 CM M W £ 3 P4 n <D . E-l § <î bO ' ßH ^ 5 S a> + » ra> » m 03 d 0 • H + » M eö a> P3 03 CU T3 Ö 03 U dì • ri ta 01 a> ö • ri 10th (1) Ü + »-H U ^ <D <L> • H <H 'Ö -H ■ H O M 3 o nq F4 .d © CS u <D <H • H O P 1-3 0) <H : cö M + => si o 13 <u i-q 65 Ci • H Q) "H • H O PS 1-3 m 0) <D • H ta 3 Q> ßi EH «! 0) ai cö U Q> •H O £ 3 -I ■ H U <D X5 ra H CH Ph C \ l O TJ> » Sì <0 <0 iH <5 M o bû •H +> V> (U .M bO ci cO rH CM « O CM 0) U ë K> <D dew •H + » 0) M bÛ a ci r-J Ê4 + 7 " si CM M Ü4 s 0) CQ cd et 0 btj o u • a H si (SI R B ■ = 4 p % WHERE <4 •H a> 0 to Jh 0) «H • H O 3rd 1-3 h a> si 03 • H <H PH CM O O <M do 0 m s 33 0) •H + » +3 a> M tm d as rH + Si O Sulfate- D.1) 3f, Ç'-adenosine dipliosplate + reduced luciferinsulfate t ^ ran5fe? A.P.?> 3 ', 5'-adenosine diphosphate or reduced luciferin Adenosine-S'-phosphate-V-phosphosulfate + reduced luciferin 2) reduced luciferin + 02 hv + oxidized luciferin reduced luminol + H202 feroxidase ^ hv + oxidized luminol + H20 reduced pyrogallol + Hp02 ^ e ^ ° xi ^ ase) hV + oxidized Pyrogallol + H20 reduced luminol + 02> * V + oxidized luminol reduced pyrogallol + 02 Qxyg? nase> hV + oxidized pyrogallol Isoluminol + H202 I-aci; operoxida3e> hv + ^ inoplitlialat + N2 E. P. G. H. I. reduced luminol reduced pyrogal = loi reduced luminol reduced pyrogallol Isoluminol J. Isoluminol + KO, -> hV + aminophthalate + N, Isoluminol or catalase p \ CA 6 00 635 438 8th Umbellifero !! 3-indole Further details on luminescent reaction systems which can be used according to the invention can be found in the following literature: J. Biol. Chem. 236: 48 (1961); J. Amer. Chem. Soc. 89: 3944 (1967); Cornier et al. Bioluminescence in Progress, Ed. Johnson et al, Princeton University Press (New Jersey, 1966) pp. 363-384; Kries, P. Purification and Properties of Renilla Luciferase, PhD at the University of Georgia (1967); At the. J. Physiol. 41: 454 (1916); Biol. Bull. 51:89 (1926); J. Biol. Chem. 243: 4714 (1968). Another preferred, sensitive monitor reaction works with fluorescence and is enzyme-catalyzed. In this reaction system, the reagent in the conjugate is a substrate in an enzymatic reaction that produces a product with fluorescent properties different from those of the conjugate substrate. Any change in the activity of the conjugated enzymatic reagent resulting from the specific binding reaction causes a change in the fluorescent properties of the reaction mixture. A general reaction scheme for such enzyme catalyzed reaction systems is as follows: 20 enzymatic Reagent -X-Z (enzyme ^ product (Substrate) 25th In the above equation, X represents an enzyme-cleavable bond or binding group such as an ester or amide group, while Z is a specific binding substance which, depending on the technique used, consists of the ligand, a specific binding analogue of the ligand or a specific binding partner of the Ligan-3-pyridol. Specific conjugates that can be used in these reaction systems are various enzyme-cleavable derivatives of fluorescein, umbelliferone, 3-indole, β-naphthol, 3-pyridol, resorufin and the like. Examples of possible formulas for these derivatives are shown in the following β-naphthol Compilation: Derivatives Resorufin Worms 40 Fluorescein In the above formulas, R1 means -OH or -X-Z, R2 X-Z and R3-H or -CHs. A particularly preferred reaction system for tracking the new specific binding reaction according to the present invention is a cyclic reaction system. In such a reaction system, a product of a first reaction 50 is a reagent of a second reaction, in which a substance is formed as one of the products, which also represents a reagent or starting material of the first reaction. The following diagram illustrates a model of a cyclic reaction system: Products A ^ ^ in circulation .- / Reagents B Material (form 1) REACTION A l REACTION B Reagents jJ ^ in circulation. 1 ^ ^ products B Material (form 2) 9 635 438 In the above model cyclic reaction system, the amount of circulating material, if one uses a small amount of circulating material when supplied from the circulating material, is often a great invention as a reagent in the conjugate according to sufficient amounts of the starting materials A and B. Examples of Cyclic Reaction Systems Amounts of Products A and B. Because speed and for use with the new specific binding reaction amount of product formed by the reactions that make up the cyclic reaction system according to the present invention, the table stem reactions are highly dependent len B and C: Table B Product A ÌTAD ^ 5 Enzyme enzyme A WA UH +% K Reagent k '^ NADH Reagent B Product B Reagent A or Reaction product 3 1 3rd 4th 5 7 8th Lactaldehyde enzyme Alcohol dehydrogenase Reagent B or product A Propanediol o ^ -Ketoglutar- GELu camo in acid glutamate at + NH-oxaloacetate acetaldehyde Dehydrogenase Malic acid de-malate hydrogenase Alcohol de-hydrogenase C £ ~ Ketoglutar ~ Isocitrone at + CO acid dehydro = genesis Dihydroxyace- ot-glycerin = clay phosphate phosphate- Dehydrogenase Pyruvate 1,3-diphos = phoglycerate Lactic acid de = hydrogenase Ethanol isocitrate I -C £ glycerin = phosphate Lactate Glyceraldehyde-Glyceraldehyde-3-phosphate-Dehy = -3-phosphate + drogenase phosphate Nicotinamide adenine dinucleotide ++ reduced NAD 635 438 10th Table C. Product A Reagent A NADP -y enzyme enzyme NADPH ++ «" ^ Reagent A reaction or Product B 1 6-phospho = gluconate oxidized glutathione enzyme Glueose-6-phosphate dehydrogenase Glutathione reductase o-benzoquinone quinone reductase Reagent B Product B Reagent B or product A Glucose-6-phosphate reduced glutathione Hydroquinone 4th nitrate Nitrate reductase Nitrite o £ "-ketoglu = glutamic acid-glutamate tarat + NH, dehydrogenase Nicotinamide adenine dinucleotide phosphate 45 50 ++ reduced NADP The reaction systems in Tables B and C also include the combination of all the reactions listed there with one another. For example, reaction 1 of Table B can be paired with any of reactions 2 through 8 to form a useful cyclic reaction system. Tables B and C thus represent 56 and 20 possible cyclic reaction systems for use in accordance with the present invention. In addition to the cyclic reaction systems in Tables B and C, one of the reactions in the cyclic system includes the enzymatic or non-enzymatic conversion of a spectrophotometric inducer. In this system, any change in the reaction or circular speed is reflected in a change in the spectrophotometric properties of the indicator. Using the preferred colorimetric indicators, this change is a color change. An example of a cyclic reaction system with conversion of an indicator is the system which is obtained by combining a reaction reagent B - product B according to table B with a reaction between an oxidation / reduction indicator and an electron carrier. Phenazine methosulfate can be used as the electron carrier. Usable indicators are the oxidized forms of nitrotetrazole, thiazoyl blue and dichlorophenolindophenol. An exponentially cyclic reaction system can also be included in the monitor reaction system. An example of an exponential cyclic reaction system is as follows: 60 65 AMP + ATP myokinase ^ 2 ADP ADP + PEP pyruvate kinase .. ATP + pyruvate This cyclic reaction is autocatalytic in the sense that the amount of circulating material is doubled during each cycle. The amount of circulation therefore increases exponentially with time and leads to a high sensitivity. J. Biol. Chem. 247: 3558-3570 (1972) provides further details on such cyclic reactions.