JPS5924386B2 - Immunological analysis method for polyiodothyronine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunological assays, and in particular to methods and enzyme combinations for determining physiological amounts of polyiodothyronine in physiological fluids, particularly serum.

Thyroxine concentrations in serum fall within a relatively narrow range that is essential for the proper functioning of the body.

This thyroxine concentration is extremely low and can only be detected with very sensitive methods. Triiodothyronine (T
-3) is also an important element for the healthy functioning of the body.

T-3&ζ Differs from thyroxine (T-4) in lacking the 5 or 5'' or 5''-iodine. T-3 is believed to have an important influence on the overall metabolic effects of thyroid hormones. One method used to measure thyroid hormones is radioimmunoassay.

There are many variations of this method, but all use a combination of antibodies to thyroid hormone and radioactive or two-hot (HOt) thyroid hormone in conjunction with serum to separate bound hormone from unbound hormone. Antibodies The amount of hot hormone bound to or present as a free hormone is a function of the amount of hormone in the serum. The amount can be calculated. Using radioimmunoassay requires a separation step that introduces errors and is time consuming.
Furthermore, one has to work with radioactive materials that decay and therefore have a short lifetime. Additionally, work using radioactive materials is generally undesirable due to health hazards. Therefore, there remains a need for a simple method that requires fewer steps and is highly sensitive. US Patent No. 381783
No. 7 describes an enzymatic assay that has been found to be generally useful for the detection of a wide range of ligands. Immunoassays using homogeneous enzymes have been used to measure thyroid hormones.

The enzyme reagent used in this immunoassay is enzyme-containing polyiodothyronine (EBP), which is inactivated by approximately 50% or more by binding to polyiodothyronine, and the receptor binds to this polyiodothyronine. We use enzymes that can be partially or completely reactivated by That is, the enzyme formulation used has a low conversion rate (TurnOverr).
ate), and this rate is substantially increased in the presence of antibodies against receptors, such as polyiodothyronine. The analytical method of the present invention is carried out by combining the serum sample to be measured, the appropriate receptor, EBP and enzyme substrate in a buffered aqueous sample and measuring the rate of the enzyme-catalyzed reaction.

If desired, one or more incubation periods can be provided between additions of reagents. known quantity T
-3. Create a calibration curve by preparing a standard containing Reverse T-3 or T-4 and relate the unknown sample to it. In the EPB composition, on average at least one of the polyiodothyronine are bonded.

According to the present invention, as a homogeneous enzyme immunoassay, 10-7 M
Methods and compositions are provided for determining concentrations of polyiodothyronine having 3 to 4 iodo groups (T-3 and T-4).

Compared to the invention described in U.S. Pat. This is an antibody against T-4. In particular, the enzyme is malate dehydrogenase (Malate) 1yd.
r0genase) or triose isomerase (Tr
In the homogeneous enzyme immunoassay of the present invention, enzyme-linked polyiodothyronine (particularly T-3 and T-4), polyiodothyronine receptor (particularly antibody, e.g. anti-T-4), 3 and anti-T-4), an aqueous buffer that is usually alkaline and usually contains a substrate for the enzyme and one or more additives to increase the stability of the enzyme, measure the enzyme reaction products, etc. use The order of addition of reagents to the aqueous sample to be analyzed is not critical. However, a detailed workbook is preferred. Additionally, it may be desirable to incubate after each reagent addition. After all reagents have been added, the rate of the enzymatic reaction, which increases depending on the amount of polyiodothyronine in the sample, is usually followed spectrophotometrically.

By comparing the results obtained with the unknown sample with the results obtained using a known amount of polyiodothyronine, the amount of polyiodothyronine in the unknown sample can be determined. Reagent Enzyme 1 Binding - In the production of polyiodothyronine, polyiodothyronine is used as it is, and the carboxyl group to be bound to the available groups (e.g. lysines, tyrosines) of the enzyme is activated, and Preferably, the chain is extended from an available functional group such as an amino group or a carboxy group, especially an amino group.

Phenolic hydroxyl groups are not used as attachment sites. The enzyme used is one that is substantially reversibly inactivated by the incorporation of polyiodothyronine, and therefore substantial activity is restored by binding of the antibody to polyiodothyronine.

i.e. malate dehydrogenase, particularly glomerular, preferably porcine heart glomerular malate dehydrogenase (MDH).
) and triose phosphate isomerase, especially Rabbit muscle triose isomerase (TIM
). Usually, polyiodothyronine is attached to the enzymes by means of a linking group attached to its amino group, and its carboxy group is esterified, especially with a lower alkyl group (usually having 1 to 3 carbon atoms). has been done. Conveniently, polyiodothyronine can be attached through two non-oxocarbonyl functional groups. (In this specification, a functional group having a non-oxocarbonyl functionality refers to an oxygen-containing carboxylic acid group: 1C-0H1, a nitrogen-containing imide group: 1C-0H
, sulfur-containing thionocarboxy: EC-0H and their derivatives, e.g. amides, esters and anhydrides) The number of polyiodothyronines attached to the enzyme is on average at least 1 and not more than about 10. Yes, usually 1~
8 pieces, more commonly 2-6 pieces.

Polyiodothyronine can be bonded to an available site, such as amino or hydroxy, to form amides and esters by an amino or carboxy group of polyiodothyronine, particularly a bonding group following the amino group. When the carboxy group becomes part of the linking chain, the amino group will have to be protected during linkage. This is because most binding groups attack the amino site. For convenience, esters of polyiodothyronine are used, and groups with non-oxocarbonyl functionality (see definition above) are used for attachment to the amino and hydroxyl groups of the enzyme. The polyiodothyronine can be attached to the enzyme by a bond or linking group. It has been discovered that the identity of the linking group is not critical to the invention, although certain classes of linking groups are preferred. The number of atoms other than hydrogen in the linking group is generally 20 or less, usually 16 or less, and usually 1 to 6 heteroatoms, especially chalcogens (0 and S) (usually alone). (bonded to carbon), and nitrogen (bonded to carbon and hydrogen alone, and is neutral (eg, amide) when bonded to hydrogen). The enzyme used is reversibly inactivated by polyiodothyronine,
Therefore, in the absence of a receptor for polyiodothyronine, the enzyme has less than about 50%, usually less than about 35%, more usually less than about 25% of the initial enzyme activity that existed before formulation; At least 0.0% of the initial enzyme activity present before formulation.
5%, usually at least 1%.

By introducing an excess of receptors for polyiodothyronine into the enzyme formulation, the enzyme activity is increased by at least 50%;
Furthermore, usually at least 100%, often 20%
It increases by 0% or more. The amount of increase in activity depends on the actual degree of decrease in activity upon combination of polyiodothyronine and enzyme. Other enzymes that have been found to be inhibited by thyroxine, particularly dehydrogenase, are
WOlfff)″BiOchimicaetBiOph
ysica ACtal 26, 387 (1957) I. These include glutamine dehydrogenase, lactate dehydrogenase, yeast alcohol dehydrogenase, yeast glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and the like. Generally speaking, EBP is expressed by the following formula. [In the formula, ENZ is polyiodothyronine having 3 to 4 iodo groups (especially T-3 and/or T-4)
x is an integer 0 or 1; x is an integer 0 or 1; ; z is (in the formula, G and G1 are H or I, and cannot be H at the same time.

); one of W and 5Wl is a bonding group, preferably an aliphatic group, has O to 1 ethylenically unsaturated site, and has 1 to 20 atoms other than H; , these atoms are carbon, chalcogen and nitrogen, where chalcogen is bonded solely to carbon as oxy or oxo (including thio analogues), especially forming a non-oxocarbonyl, and nitrogen is bonded solely to carbon and hydrogen. When bonded to hydrogen, for example to form an amide, it is neutral and the number of heteroatoms is usually 1 to 8, more usually 2 to 7, preferably 2. ~4, and the bonding group chain includes t-amino, oxy,
There are usually 5 or less, usually 4 or less, preferably 2 to 3 functional groups such as amide, but W1 may be a mere bonding chain especially when x is an integer 0; when it is other than a bonding group, W1 is present. is H,. Wl is hydroxyl or C,~3 alkoxyl, especially methyl; m is on average at least 1 and not more than 10, usually from 1 to 8, more usually from 2 to 6. In most cases, the EBP used in the invention is expressed by the following formula.

[In the formula, ENZ is a polyiodothyronine whose activity is reversibly inhibited by polyiodothyronine having 3 to 4 iodo groups (particularly T-3 and T-4), and therefore polyiodothyronine binds to its receptor.] is an enzyme that is activated when
averages at least 1 and below 10, usually between 1 and 8,
More usually 2 to 6: a is O or 1, usually 1
and; an alkyl group, usually from 1 to 3, preferably 1; Z is 3
・3', 5-, 3-5, 3'-triiodo or 3, 3ζ
5,5'-tetraiodo-4-(hydroxyphenoxy)-phenol]: A is a linking chain, or at least 1, usually at least 2, but not more than 12;
More usually it has 10 or less carbon atoms, 0 to 12, usually 1 to 10, more usually 2 to 9, preferably 3 to 7 (for cyclic groups, ring atoms chalcogen atoms in the chain between the non-oxocarbonyl groups, and 0 to 4, usually 0 or 1 to 3, more usually 1 to 2 heteroatoms [chalcogen ( O or S) (as oxo, thio or sulfonyl) or N (as t-amino or amido)]; the total number of heteroatoms is O
to 6, typically O to 5, usually O or 1 to 4, more commonly 1 to 3, and the heteroatom is a chalcogen (O or S) (non-oxocarbonyl, non-oxothiocarbonyl,
as oxy, thio or sulfonyl) or N (as oxy, thio or sulfonyl)
(as amino or amide): there are usually two carbon atoms between the heteroatoms in the chain; A is hydrocarpylene (aliphatic, cycloaliphatic, aromatic or a combination thereof);
or non-hydrocarpylene (substituted aliphatic, cycloaliphatic, aromatic, heterocyclic, or combinations thereof), and generally has O to 1 cyclic groups in the chain (0 to 2 ring atoms, Usually O
~1 heteroatom and the cyclic group usually has substituents separated by 2 to 4, usually 3 to 4 ring atoms. However, the hydrocarpylene has O to 1 ethylene groups as its only site of aliphatic unsaturation and is preferably saturated, particularly preferably O to 1 carboxamide, imino, or It is an alkylene having an oxy group.

When A is carbocyclic aromatic, it usually has from 6 to 10 carbon atoms, more usually from 6 to 8 carbon atoms. The total number of functional groups in the chain (oxy, amide, amide, and their sulfur, nitrogen analogues) is generally from 0 to 4, usually from 0 to 3, more usually from 0 or from 1 to 2.

ENZ monobond -T-3 or -T- with a dinonoxocarbonyl chain interrupted by about 1 to 2 heteroatoms (chalcogen, N) or with an uninterrupted aliphatic chain
4 applies.

Most of these compounds are represented by the following formula. [In the ceremony, ENZ.
D. Z is as defined above: n1 has the same definition as n, but on average is between 1 and 10, preferably between 1 and 8, more preferably between 2 and 6; X1 and Y1 are the same as X and Y, respectively; , where X1 is usually chalcogen and Y
1 is preferably oxygen: P. q and r are each 0 to 1
And P. q. The sum of r is preferably at least 1 (p+q+r〉1): s is O~2, usually 0~1; α and γ are 1~8, usually 1~6, more usually is 1
It is a hydrocarbon group having ~3 carbon atoms, and the total number of carbon atoms is 2 to 10, preferably 2 to 8, more preferably 2.
~6; when p or r is an integer 0, α or γ preferably has 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms; α and γ preferably have 1 to 8 carbon atoms; , more preferably 1 to 6 carbon atoms; α and γ may be aliphatic, cycloaliphatic, aromatic, or heterocyclic, and together with β and δ are within the definition of A above: Preferably α and γ are saturated aliphatic, especially linear, such as methylene or polymethylene, or aromatic, especially monocyclic, in which case only one of α, γ is an aromatic group; β Oxy (-0-), thio (-S-), sulfonyl (-SO2-) or amino (
-N-), where T is 1 to 6, usually 1 to 3
or H; when β is attached to a non-oxocarbonyl (p-0), β is usually amino or alkylamino; δ is -CH-N
In a preferred embodiment, the above polyiodothyronine is combined with a dibasic dicarboxylic acid to form an amino acid (monoamide of a dibasic acid). Can be formed.

The dibasic acids usually have 2 to 8, usually 2 to 6 carbon atoms, have O to 1 sites of ethylenically unsaturation in the chain, and have an atomic number of 0 to 1. 7-8 (N, 0) atoms (
N,. O is singly bonded to C, ie forming a t-amino or ester). Preferred dibasic acids form cyclic anhydrides with 5 to 7 ring atoms, preferably 5 to 6 ring atoms. The carboxyl group of the dibasic acid bonded to thyronine is E
In order to ensure the reaction with NZ, the carboxyl group of thyronine is usually esterified with an alkyl group having 1 to 3 carbon atoms, preferably 1 (methyl). The modified polyiodothyronine or its analogs used for conjugation with ENZ are most often represented by the following formula. [In the formula, X.
Y. Z. a is as defined above; D' is H1 or alkyl having 1 to 3 carbon atoms, preferably 1; R is A
but preferably has 0 to 1 ethylenically unsaturated sites and O to 1 heteroatoms with atomic number 7 to 8 (completely substituted by carbon atoms) is a divalent group and has 1 to 6 carbon atoms; E is hydroxyl or can be taken together with the terminal N of R to form a double bond, e.g. isocyanate or isothiocyanate. The compound has a carbocyclic or heterocyclic ring having 5 to 6 ring atoms in the chain, and the heterocyclic ring contains 1 to 2, usually 1, heteroatoms (0.S or N).
has.

Preferably, these compounds have an isocyanate or isothiocyanate bonded directly or indirectly to a ring atom, especially if the ring is aromatic. In most cases these compounds have the formula: [In the formula, D and Z are as defined above: X2 and Y2 are chalcogen (0 or S), preferably X2 is S; Ar is marylene or aralkylene, and the number of ring atoms is 5 to 6 a carbon ring and a heterocycle (having 1 to 2, preferably 1 heteroatom, 0.N.S.), containing 4 to 101 carbon atoms and usually 4 to 8 carbon atoms; When it is a carbon ring, it is preferably 4 to 6, and when it is a carbon ring, it is preferably 6 to 8.] The ENZ conjugate of the above-mentioned series of compounds is represented by the following formula.

(wherein all symbols are as defined above) The polyiodothyronine analogs contain available non-oxocarbonyl reactive groups of ENZ, such as amino and hydroxyl,
It is used in activated or activated form in order to be able to react with

As carboxy, mixed anhydrides, N-hydroxysuccinimide, p-nitrophenyl, phenylthio, etc., ester derivatives or carbodiimide coupling reagents are conveniently used, while imidate esters and isothiocyanates are used directly for coupling with ENZ.

The reaction is carried out in a buffered mixed aqueous medium (usually at a mildly alkaline PH value, generally at a PH value of about 7 to 10) at mild temperatures, generally from about -5 to 30<0>C.
Co-solvents such as hexamethylphosphoramide can be used. Approximately 10 to 40 V/ when using a cosolvent
V%, preferably in an amount of about 20-30 V/V%. The molar ratio of T-3 or T-4 analog to ENZ is generally from about 1:1 to 20:1, more usually from about 3:1 to 1.
The ratio is 0:1.

The T-3 or T-4 analog can be added in portions or all at once, and reaction times generally range from about 1 minute to about 48 minutes.
It's time.

After the reaction is complete, the reaction mixture is treated in a conventional manner. Preferably, the untreated analog is ENZ-conjugated to T-3 or T-4.
The mixture is chromatographed in order to separate it. A convenient chromatography material is Sephadex (Se
phadex). Fractions are isolated, their enzymatic activity is measured, and fractions with enzymatic activity are combined. Desamino-T-3 or T-4 can be coupled to ENZ, except for analogs formed by coupling polyiodothyronine with di-non-oxo compounds.

In the case of polyiodothyronine, the amino group can be protected upon formulation with ENZ, while in the case of desaminopolyiodothyronine, no protecting group is required and the carboxy group can be activated in the same manner as other carboxy groups. Desaminopolyiodothyronine and its monocyclic analogs are represented by the following formula. HO2
CCH2(CH2)TZ (in the formula, Z is as defined above, and t is O or 1)
On the other hand, the ENZ blend of these compounds is represented by the following formula.

ENZ-(COCH2(CH2)TZ)n1 (all symbols in the formula are defined as above) ENZ-1 bond-T-3 or T-4 is usually at least about 50%, usually at least about 50%, compared to the enzyme activity before combination. is at least about 65% and no more than 99.5% inactivated.

Almost completely inactivated and excess amount of anti-T-3 or -T
It is desirable that the addition of -4 substantially completely restores its activity. However, compounded enzymes exhibit approximately 5-60% of the original activity of uncombined enzymes due to receptor binding, and usually 10-50% of the original activity of uncombined enzymes.
% has been found to be activated. Of primary interest is the difference in units of measurement of ENZ-binding-T-3 or T-4 in the presence of excess amounts of antibody in the absence of antibody. 2
Excess amount 2 refers to the amount of antibody sufficient to bind substantially all of the available T-3 or T-4.

Conveniently, the enzyme is porcine heart phloemmal malate dehydrogenase or triose phosphate isomerase, which is present in rabbit muscle. Anti-T-3 or T-4 A T-3 or T-4 binding antigen is injected into a vertebrate (usually a poultry animal, such as a sheep, rabbit, goat) to produce the appropriate antibodies.

Since antigens are usually polypeptides or proteins and have a molecular weight usually between 5,000 and 10,000,000, antigenic complexes are formed by the combination of T-3 or T-4 analogs and antigenic polypeptides or proteins. be done. The same or different analogs used for conjugation with ENZ can be used. Analogs other than those used to make ENZ formulations can also be used. Additionally, thyroglobulin can be used in the production of antibodies. Depending on the particular analog used, various methods described in the literature can be used to produce antigen combinations.

The antigen combination contains at least one, and preferably 2.
000-50.000 with approximately 1 analog molecule per antigen of molecular weight. T-3 or T-4 for antigen molecular weight
The ratio increases as the antigen molecular weight decreases. Antigens can be injected into animals using conventional methods, but pressure injection (BOOsteri
It would be preferable to use complete Freund's supplement.

Antigens include a wide range of proteins, such as albumin, globulin, and keyhole limpets.
mpet) hemocyanin, etc. can be used.

Buffers The buffers used can vary widely and include phosphates, carbonates, glycinates, Tris, etc.

Phosphates must not be used in high concentrations (>0.1M). Glycinate or triethanolamine are preferred buffers. Due to the other additive reaction pathway, the substrates for MDH are malate and NA.
Dl or oxaloacetate and NADH.

In the case of triosephosphate isomerase (TIM), the enzymatic reaction of the enzyme is coupled with a second enzyme to enable spectrophotometric measurements. Therefore, the analytes include substrates for TIM, glyceraldehyde phosphate, NADH, and α-glycerophosphate dehydrogenase. The latter enzyme and NADH
is used in substantial excess so as not to limit speed. It is beneficial to include small amounts of ethylenediaminetetraacetic acid to reduce bacterial growth and also sequester heavy metals.

The stability of the enzyme can also be increased by including protein and glycerin in the sample to be analyzed. Other stabilizers such as dithioerythritol and other antioxidants may also be included. Furthermore,
Small amounts of sodium azide can be added as a preservative. The analytical method reagent solution buffer is about 0.001-0.5M, usually about 0.01-0.2M, preferably 0.05-0.
．． It is usually at a concentration of 15M.

The protein content (albumin such as rabbit serum albumin and/or gelatin) is generally about 0.005 to 0.
．． 5, and more commonly present in the final analyte mixture at about 0.01-0.2% by weight. Glycerol 0.1-5,
Usually it can be present in amounts of 0.4 to 4% by weight.
The pH of the solution is generally about 6.0 to 10.5, usually about 7.
~10.5, more usually about 8-10, and preferably 8.5-10.5 when using NAD and malate or NADH and oxaloacetate, and preferably 7-10 when using glyceraldehyde phosphate. It is 9. The concentration of ENZ in the analyte can vary widely, but is generally about 10-5 to 10-12M1, more usually about 10-7 to 10-11M.

Antibody concentration is based on the ratio of antibody binding sites to the concentration of polyiodothyronine bound to EVs. Generally, the T of the binding site
-3 or T-4 (as ENZ-bonded -T3 or -T-4) is at least 0.5 and less than 1000, more usually about 1 to 100, most usually about 1 ~25. Usually 25% of recoverable enzyme activity
Add sufficient antibody to recover ~75%, preferably 25-50%. Adding a large amount of antibody inevitably reduces the binding constant.

The amount of antibody used in each analysis is usually determined empirically. Other small amount additives that can be added are approximately 0.001 to 0.1
Ethylenediaminetetraacetic acid, which may be present at % by weight, sodium azide, which may be present at about 10-5 to 10-3 M, TritOn −
100 or the like.

When using MDH, malate and NA may be mixed depending on the direction of the reaction.
Either Dl oxaloacetate or NADH is used, with the former being preferred.

The concentrations of these substances directly affect analytical sensitivity and must be determined empirically to optimize the difference in reaction rates in the presence and absence of added antibodies. The concentration of malate or oxaloacetate is generally about 0.01 to 0.5
M1 is more commonly about 0.05-0.3M. N.A.D.
or the concentration of NADH is generally about 0.001-0.05M
．． More typically it is about 0.005-0.2M. usually,
The concentrations of enzyme, antibody, and substrate are selected to optimize analytical sensitivity. When using TIM, glyceraldehyde phosphate is generally present at a concentration of about 10-1 to 10-4 M, α-glycerophosphate dehydrogenase is generally present in an amount of 10-5 to 10-9 M, while N
The ADH concentration is generally about 10-2 to 10-4M. It is convenient to add various reagents as aqueous solutions.

Usually, a buffer solution is used when the amount of sample to be analyzed is large. Analytical Process The order in which the various reagents are combined is not critical, but some are preferred, and in some cases one order is preferred over another.

Incubation may occur after each reagent addition or after all reagents have been combined. Normally, 10
Do not incubate the analyte mixture in the presence of the enzyme substrate for more than a minute. All reagents can also be combined at the same time.

Alternatively, the sample (eg, serum), buffer, and antibody can be combined and incubated if desired. In most cases, the antibody and ENZ formulation will not be combined prior to sample addition. However, when using monovalent antibodies to avoid precipitin formation, it may sometimes be desirable to store a mixture of antibody and enzyme. The order of addition produces different results.

If the sample and antibody are combined, the polyiodothyronine will bind to the antibody, reducing the number of available sites for binding to ENZ-T-3 or -T-4. The concentration of effective binding sites is therefore a function of the amount of T-3 or T-4 in the sample. The amount of antibody that binds to the ENZ-conjugated T-3 or T4 formulation is a function of the amount of T3 or T-4 in the sample. In this method, T-3 or T-4 and ENZ-bond-T-3
or -T-4, the sensitivity to the difference in binding constant is low. Alternatively, the sample, appropriate antibody, and ENZ formulation can be combined to allow free T-3 or T-4 to compete for available binding sites with T-3 or T-4 formulated in ENZ. . In any event, the antibody (antiligand) must be present in an amount sufficient to substantially partially or completely restore enzyme activity in the absence of the ligand in the sample.

This is because the enzyme-ligand conjugate has no or only a slight enzymatic activity compared to the free enzyme, but the enzyme can be released by bringing the anti-ligand into contact with the conjugate to bind the anti-ligand and the ligand. This is to restore enzyme activity. If the ligand is present in the sample,
Less anti-ligand will bind to the ligand of the enzyme-ligand conjugate and the increase in enzyme activity will be less than when there is no ligand in the sample. This allows the ligand in the sample to be measured. If desired, after each addition of reagents,
The analyte mixture may also be incubated either before or after the addition of enzyme substrates and other reagents.

Incubation times range from 2 minutes to 1 hour, typically from about 15 to 40°C, usually from about 25 (ambient) to 37°C.
It will be held in After adding the analyte mixture to the substrate for the enzyme, it may be incubated for up to about 10 minutes prior to initial readings, which can be taken using either a flow cell or a QVET.

Rates are measured at temperatures in the range of about 20-40°C, more commonly about 25-37°C. Generally, readings last about 0.1 to 45 minutes when using a flow cell, usually 0.5 to 2 minutes, and 5 to 45 minutes when using a QVET. For specific automatic equipment, a measurement period of 5 to 20 minutes may be sufficient. By using a standard containing a known amount of T3 or T4 in serum, a calibration curve can be established that can be used to determine the amount of T3 or T4 present in an unknown sample. Although spectrophotometry is most convenient for tracking enzymatic reactions, ie absorption spectra, other techniques such as Fluorimetry and Titrimetry can also be used.

The following examples are illustrative of the invention.

Experiment 1 All temperatures are in °C unless otherwise specified.

All percentages are by weight unless otherwise specified. Example 1 N-Methyl, N-carboxymethylglycylthyroxine methyl ester (T4-MEMIDA) 25 equipped with a stirrer and a septum stopper.
1.0547 (1.27 x 10-3M) in m1 flask
of thyroxine hydrochloride methyl ester was added.

The ester was dissolved in 8 ml of dimethylformamide (DMF) under an argon atmosphere, 10 ml of dry tetrahydrofuran (THF) and 253 μl of dry triethylamine were added. After stirring for 15 minutes, dry T of 2.5 mj
0.279V (2.16 x 10-3M) N- in HF
Methylimino diacetic anhydride was added in one portion.

The reaction seemed immediate. The volatiles were removed in vacuo on a rotary evaporator leaving a foamy solid, which was evaporated into 25 TL of TH.
Dissolved in deionized water (30 mO/ethyl acetate (5
Extracted at 0m0. After layer separation, the aqueous layer was extracted three times with 25 Tn1 each of ethyl acetate. Next, combine the organic layer and
It was extracted once with 0 ml of saturated NaCl solution and then dried over anhydrous magnesium sulfate. After suction filtration, the solvent was removed in a rotary evaporator and the remaining white solid was dissolved in 30 ml (7) TH.
Dissolved in F. 35 mj of chloroform was added, heated to reflux, and n-heptane was slowly added.

The solution was concentrated until an obvious cloudiness persisted. After cooling at room temperature and then cooling in a freezer, the white flocculent product was
Wash with hexane and vacuum dry with phosphorus pentoxide to 1.267
(75%) of white flocculent product was obtained. Example 2 Binding of T4-MEMIDA to Bovine Serum Albumin (BSA) 0.107 (1.09 x 10-4M) of T4MEMID was added to a reaction vessel equipped with a stirrer and a septum glass stopper.
A and 0.013y (1.1 x 10-4M) of N-hydroxysuccinimide were added.

Add 1 ml of dry THF to this reaction vessel under an argon atmosphere.
Then 15 μl of dry triethylamine was added. After cooling the mixture in an ice bath at 0.0247 (1.25 x 1
0-4M) of 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (ECDI) hydrochloride was added as a powder all at once.

The mixture was stirred for 2 hours at 0 and then for 12 hours at 4 degrees.

3.0.1007 (1.55X10-6M) of BSA.
A solution (PH 9.4) was prepared by dissolving in 0 ml of sodium bicarbonate monocarbonate buffer and P by addition of 6N NaOH.
H was readjusted to 9.5.

This BSA solution was cooled to 0.00 and added to the T4-ME described above.
While vigorously stirring the MIDA ester solution, 50 It,
It was added dropwise at a rate of e/min. The mixture was stirred at 0 for 1.3 hours and then gently stirred at 4 speed for 2 days.

Then, a 3M solution of hydroxylamine hydrochloride neutralized to pH 8.9 with 6N NaOH was added dropwise.

After stirring for 10 hours with 4 salts, put in a dialysis bag and give 500 ml each.
ml Tris-HCl buffer (0.05M, pH 7.8
) was dialyzed twice for one day.

Next, Aquaside (CalbiO)
chem) and subjected to gel filtration chromatography twice (each time using fresh Seph).
Using adexGl5, first add Tris-HCl buffer (
0.1M; PH9. swelled at O)]. Column size was 2.6 x 22.2 (ml flow rate was 48 drops/min, 40 drops of each fraction were collected, and the eluate was diluted with Tris-HCl.
(0.1M; PH9.O).

Combine the protein fractions and add deionized water (5 x 2.00
Dialysis was performed for 3 days at 0 m. Then freeze-dried to 0.15f
A white flocculent solid of 7 was obtained which was vacuum dried over P2O5 for 3 days to yield a 0.140y conjugate. UV analysis showed that 22 haptens were bound to each mole of BSA. Example 3 Carboxymethoxyacetyl thyroxine methyl ester (DGMA) Dry THF (80 ml) of thyroxine methyl hydrochloride (1.657) in a light-tight flask
/Chloroform (300 m0 while stirring the solution)
Injected t1 of triethylamine.

Then, diglycolic anhydride (255 instant; 0.0022 mol) was added and stirred overnight. It was then washed three times with water, dried over sodium sulfate, and the volatiles were removed in vacuo. 3 residues
Purified on a 0y Sephadex LH-20 column (using 20% methanol solution in dichloromethane as eluent). The clear fractions were collected, the solvent removed and the residue precipitated from methanol with water to give 1.537 (84
%) was obtained. Example 4 DGMA blend of bovine serum albumin 100 watts of BSA was dissolved in 30 mj urea aqueous solution (300), 25 ml of DMF was slowly added, and then 5 mj
1 (7) 453Tf19 in DMF (.5.9×104
M) DGMA was added dropwise.

After completing the dropwise addition, adjust the pH of the reaction mixture to 4.5, maintain it at 0, and add 100m9 (0.0005M
) of hydrochloric acid ECDI was added in 10η portions at 30 minute intervals. After the addition was complete, the reaction mixture was adjusted to pH 9 and dialyzed against 21 10% DMF in 4M urea (PH 9). The precipitate was allowed to settle, and the supernatant liquid was transferred to a DowBeak dialyzer (DOwBeak).
0.05M in 25 gallons
Carbonate (PH9) and then dialyzed against 5 gallons of aqueous ammonia (PH9). Freeze-drying yielded 95% of the formulation (23 DGMA per mole of BSAl by UV analysis).
) was obtained. Example 5 Methyl methoxycarboimide methoxyacetyl tyroxinate blend of BSA A. Cyanomethoxyacetic acid (12
98 μl of oxalyl chloride (146 m9) in suspension in 1 m0 of dichloromethane (1.1 M)
;1.15M) was added.

The reaction was started by adding one drop of dimethylformamide and stirred for 30 minutes until bubbling stopped and the starting acid was dissolved.

The solvent was then removed with ROtavap and the remaining yellow oil was dissolved in methyl tyroxinate hydrochloride (827η; 1 mmM) in dry tetrahydrofuran (280 μl: 2 mmM).
of triethylamine) was added to the suspension. The reaction was stopped after 2 hours when analytical thin layer chromatography (TLC) (silica gel, 25% ethyl ether in chloroform) showed no remaining starting thyroxine ester. The reaction mixture was poured into water/ethyl acetate and the aqueous layer was extracted three times with 50 ml of ethyl acetate. The organic layers were combined, washed once with water and once with brine, dried over magnesium sulfate, and evaporated in vacuo to yield 888 m9 (quantitative).
A pale yellow foam was obtained. Sephad
ex) Chromatography on LH-20 (257, 10% methanol in ethyl acetate) to remove polar impurities. Yield 85% o{. A solution of methyl cyanomethoxyacetyl tyroxinate (89η: 0.1 mmol) in dry methanol (1 ml) was placed in a flask (dry and purged with nitrogen) equipped with a septum stopper.

A methanol solution of sodium methoxide (containing 1.1 equivalents of base) was added and stirred overnight in the dark. After 20 hours, Tlc (25% ethyl ether in dichloromethane, silica gel) indicated that the reaction was virtually complete with the formation of some colored impurities.

-G. A 2m0 solution of the above imidate (0.1 mmM) in basic methanol (untreated imidate-forming reaction mixture)
was added dropwise to a solution of bovine serum albumin (112 w; 0.1 mmol lysine) in 0.05 M Tris buffer (PH 8.5; 5 ml).

The pH of the reaction mixture was adjusted to 9.5 with 0.05 MHC1 and stirred for 24 hours while cooling.

Dialyzed against 101 parts of dilute sodium bicarbonate solution and 31 parts of deionized water.

The extinction coefficient of thyroxine measured in experiment 1 was 6.2 x 10 M-1c by UV spectroscopic analysis.
It was shown to be about 20 based on 7rL-1. Sephadex G-15 gel filtration (0.05M Tris, P
There was no difference in the number of formulations depending on the H9 buffer). Example 6 N-carboxymethyl 3-aza-3-methylglutaramic acid amide blend of methyl tyroxinate of MDH 0
．． 2017 (0.22 mm M) T4 Ichika MIDA, 1
A solution (00) of 1 m dry THF, 25 m9 (0.22 mmM) N-hydroxy succinimide, 30 μl dry triethylamine (00) was mixed with 0.0487 m
0.25 mmM) of ECDI was added.

The mixture was stirred at 00 for 35 minutes and then at 4 mm for 14 hours.

1N Na0H'('00 deionized water adjusted to pH 9 (1.5 m0/pyridine (0.033 in 0.5 m0)
The above solution was added dropwise to y (0.44 mm) of glycine with vigorous stirring.

Stirring was continued in the dark for 36 hours at 4 strokes, the solvent was removed in vacuo, the residue was dissolved in 5 ml of absolute ethanol, the ethanol was evaporated and this ethanol treatment was repeated three times to give a white foamy solid. The residue was dissolved in 107 TL1 methanol, poured into 15 ml deionized water and extracted three times with 25 ml each of ethyl acetate.

The ethyl acetate layers were combined and extracted with 25ml of saturated brine.
It was then dried with MgSO4. The volatiles were removed in vacuo and the residue was dissolved in 2 ml of methanol and purified with silica gel (Et3
N:CH3OH:CH2Cl2-2.1:10:90)
I performed chromatography. The product was converted into CH3OH/CH
Desorbed with 2Cl2 (1:1), filtered, volatiles were evaporated and the residue was dissolved in 5ml THF and filtered again.
After removal of volatiles, the residue was taken up in THF and THF/HCCl
3/recrystallized from cyclohexane to give 0.0467(2
1.6%). B. 39μ1f) Radioactive Cl4N- of methyl tyroxinate from 4.9 dissolved in DMF
Carboxymethyl 3-aza-3-methylglutamic acid was placed in a 1 ml container and cooled to 4 ml.

While stirring this DMF solution, add 0.1 in DMF at 4.
51 μl of 1 MECD solution and 0.5 MN in DMF of 4D
10 μl of hydroxysuccinimide was added and stirred overnight in the dark. C. Pig heart threadocular MDH was dialyzed against 0.05M carbonate buffer (PH9.O) to obtain 4 ml of MDH1.
A 3l x 10-5M solution was obtained.

445μ1f) DMF was added at 15μf/min.

The above-produced ester was added in 2 μl portions and the enzyme activity was analyzed at 5 minute intervals. In the first reaction 5 μl of the above ester was used and in the second reaction 9 μl was added (thyroxine vs. M
Both reaction mixtures (DH ratios were 4.1 and 7.4, respectively) were diluted with 300 ml of 1M K2HP04 (PH 9.8) three times and 300 ml (1) of 0.05M carbonate buffer (pH 9.8).
Dialysis was performed once against O) and then twice against phosphate buffer. Both reaction mixtures were then loaded on a Sephadex G-50M column (0.9 x 54 cT) equilibrated with the same phosphate buffer for 4 cycles.
The mixture was chromatographed with the same phosphate buffer at a flow rate of 4 drops/min and collected as a 20 drop fraction. Combine the active fractions, adjust the volume to 5 ml with the same phosphate buffer, and add 0.5 ml of the 500
Dialyzed against ml of deionized water. Radioactivity measurements estimated that there was no protein loss, and the number of haptens per protein molecule was 2.0 and 3.0. Example 7 T4-MEMIDA combination of malate dehydrogenase was added to 250 μl of dry DMF with T4-MEMIDAlOm9 (
11 μM) and N-hydroxysuccinimide 1.3m9
was dissolved.

Maintained at 00 while stirring under nitrogen atmosphere, ECD2.3
η was added and maintained at 00 until the ECDI was dissolved. 4η
I left it there overnight.

The number of haptens is varied depending on the amount of T4-MEMIDA hydroxysuccinimide ester relative to MDH. A general method for making a blend is provided. MDH (pig heart)
D while stirring 4 ml of a carbonate buffer (PH 9.2) solution of filamentum, Miles, 5.0 ml/ml).
Added to MFlml. T4-ME approximately every 60-90 minutes
MIDA ester was added continuously, and one portion was removed each time to analyze enzyme activity. The following table shows the order of addition, the amount added, the time of addition, the ratio of added T4 to MDH, and the observed inactivation rate (%). For measuring the number of compounds, 2 to 20μ
1 of the blend mixture was added to 0 constant 1M potassium dihydrogen phosphate
．． Added to 5ml.

For enzyme activity measurements, 2-20 μl of this diluted formulation
0.1% in glycine buffer (0.1M. pH 9.5)
Diluted to 0.8g with rabbit serum albumin and diluted to 0.108g.
100μl of MNAD and sodium malate (2M.pH
9.5) Added 100It1. 60-120 after introduction
GilfOrd type 30 at 300 per second
Velocity was measured with a 0-N spectrophotometer. Example 8 T4 of trione phosphate isomerase (TlM)
- Dry DMF5OOμ in MEMIDA formulation vial
T4MEMIDA6. Oη (6.8 μM) and N-hydroxysuccinimide 0.8η (7.3 μM) were introduced, purged with dry argon, stoppered, and cooled in an ice bath.

While stirring this, ECDIl. 5 wafers were added and the vial was purged with dry argon and stirred until everything was dissolved. Wipe the vial dry and add Drierite (D
The mixture was placed in a plastic cup with a lid containing rierite 8), wrapped in foil, and left overnight at 4°C with stirring. Triose phosphate isomerase (1 ml:2η) in 44 aqueous carbonate buffer (0.1 M. pH 9.2)
DMFO. 3 ml was slowly added using a syringe.

The ester solution was slowly added in portions using a syringe and the enzyme activity was monitored. When the enzyme was approximately 72% inactivated, DMF was added to bring the amount of DMF to 40 V/V%. The cold reaction mixture was passed through a Sephadex 8G-25-(medium) column equilibrated with 0.1M carbonate buffer (PH 9.2).

The column has a diameter of 1.1 (7fL1 bed volume (BedvOlu
me) It was a 50CC biuret of ~19m1. Elution was carried out using four solutions of 60 V/V parts of 0.1MC03 (PH9.2)/0.3M ammonium sulfate aqueous solution and DMF4O parts. Fractions were collected in varying amounts from about 1 to 4 mj. Combine fractions 5 and 6 (2.
4m0, first 20V/V% DMF/0.02M triethanolamine (TEA) (PH7.9) aqueous solution ~10
0ml, then 0.02M TEA aqueous solution (PH7.9
) Dialyzed 3 times with 250ml. The ratio of blended T-4 to enzyme was approximately 6. Example 9 Desaminothyroxine combination of MDH Desaminothyroxine (9.1×10−3y11.2×
10-5M), NHSl. 4 x 10-37 (1.2 x 1
0-5M), dry DMFO. 25 ml were added one after another to a 1 ml reaction vessel.

Cooled in an ice bath and diluted with ECDl2,5 x 10 under N2 atmosphere.
-37 (1.3 x 10-5 M) was added. 4, and stirred overnight.

Cooled in an ice bath and stirred, DMF (0.23 mO) was added to 0.05 MNa, cooled in an ice bath and stirred.
NC03-Na2CO3 (PH9.O) 0.83m11
It was slowly added to 1.9 x 10-37 (2.8 x 10-8 M) of 1MDH. Then, the ester solution (5.8μ
Add l) with stirring, and continue stirring while maintaining the temperature.
It lasted for hours. The blend mixture was gel-P filtered three times on a 0.9x13C7rL Sephadex 8G-50M column to produce the desaminothyroxine/MDH blend. This formulation is 91% inactivated and has a hapten count of 2.5.
(according to iodine analysis). The enzyme activity of this formulation was found to be 30% activated when treated with anti-T4 serum. Example 10 T4-MEMIDA Glycine 3ml Pierce Reacti Vial TM (Pierce
Reacti-VialTM) to T4-MEMIDAO
．． 2Ol7 (2.18 x 10-4M) and N-hydroxysuccinimide (NHS) 0.0257 (2.17 x 1
0-4M) was added.

2ml dry THF and 30μl dry triethylamine (2ml)
．． 15×10 −4 M) was added and cooled to 0.00 in an ice bath. ECDI (0.048y, 2.50×10-4M)
was added as a powder and stirred at 00 for 35 minutes. The mixture was then placed in a cold room (2e) and stirred for 15 hours. At the end of stirring, Tl
c is an analytical silica gel plate (10
% methanol) and the R1 value was 0.06 (T4-ME
MIDA), 0.60 (T4MEMIDANHS ester).

Glycine 0.033 in a 25ml flask with Stirringflea and septal stopper.
7 (4.39 x 10-4M), distilled water 1.50ml11 pyridine 0.50ml, 1.0N Na0H100p.
1 (1.00×10 −4 M). Cooled down to 0.00 in an ice bath. T4-ME produced above while stirring vigorously.
The MIDANHS ester solution was added dropwise, and after the addition, the reaction mixture was placed in a cold room (2 baths) and stirred for 36 hours. The solvent was removed on a rotary evaporator to give a pyridine-smelling oily solid. This solid was taken up in 10 mj Me0H, poured into 15 ml H2O and extracted with ethyl acetate (2 x 25 m0. The organic layers were combined and extracted once with 27 ml saturated brine, anhydrous MgSO
It was dried at 4. After 7f3 filtration, ethyl acetate was removed on a rotary evaporator to obtain a white crystalline solid.

This solid was placed in 2 ml of methanol and placed on 4 preparative silica gel plates TLC plates, and triethylamine rimethanol lydichloromethane (2.1:
10:90). The plate was run twice and then discarded and the product was washed with methanol lydichloromethane (1:
It was removed in step 1). Remove the silica gel and remove the silica gel by vacuum.
Concentrated to mj. The Tlc of the product in THF was determined by Rf on an analytical silica gel TLC plate [triethylamine rimethanol lydichloromethane (2.1:10:90)].
Only one spot with a value of 0.50 is shown. The product is TH
Recrystallized from F/chloroform/cyclohexane. Example 11T4-MEMIDA Glycylglycine 3ml Piercing Reacti Vial TMVCT4MEM
IDAO. 2O27 (2.20X103M), NllS
O. O257 (2.17 x 10-4M) was charged.

Dry THF (2m0 and dry triethylamine (31μf)
! ) was added and cooled to 0 dryness. ECDI (0.05
1f7, 2.66 x 10-4M) and put it in the cold room (2.)
The mixture was stirred for 8.25 hours. Tlc is T4-MEMIDA
Formation of NHS ester was shown [Rf value = 0.63, analytical silica gel plate, triethylamine rimethanol lydichloromethane (2.1:10:90)]. Glycylglycine 0.05 in a 25ml flask with Stirringflea and septal stopper.
8y (4.39 x 10-4M), H2Ol. 5Oml,
Pyridine 0.50ml 111.0NNa0H100μl (
1.0×10 −4 M). The mixture was cooled to 0.00C, and the T4-MEMIDANIIS ester solution was added dropwise under stirring.

After dropping, add 1.0ml of deionized water and 0.50ml of pyridine.
added. Stir in a cold room (2 m) for 93 hours, then use T4-ME.
Treated in the same manner as MIDA glycine to give 0,0187 (9%
) was obtained as a brownish-golden solid. Tlc [silica gel, triethylamine rimethanol lydichloromethane (
2.1:10:80) was obtained. The product was homogeneous according to Rf value = 0.81, plate developed twice].

Example 12 1 m of T4-MEMIDA glycine/MDH blend Stirringflea
1 pierced rare vial TM with T4 1 MEMIDA
O. O497 (5.0X10-6M) and dry DMF39
μl was added.

0. 5.0 x 10-1 M in DMF at 0D.
10 μl of NHS solution (5.2 x 10 M) and 00 D
51 μl of 1.2 x 10-1 MECDI solution in MF (6.
1×10 −6 M) was added thereto and stirred in a cold room (2μ) for 49 hours.

Tlc after stirring [analytical silica gel plate, triethylamine rimethanol lydichloromethane (2.1:1
0:90), the Rf value was 0.49 (T4-
MEMIDA glycine), 0.68 (T4-MEMID
Two spots of A glycine NHS ester) were shown.
MD in a 10m1 round bottom flask equipped with a stir bar and septal stopper.
H (4,0m111.3×10-5M10.05MNa
HC03-Na2cO3, pH 9.2), cooled to 0C, and enzyme activity was measured as described above. dry DM
Add F (445 μl) at a rate of 15 μl/min to 10%
A DMF reaction mixture was obtained. Enzyme activity was measured again. 4
,3×10-2MT4゛-MEMIDA glycine NHS
The ester solution was added to the reaction mixture in 1-2 μl portions and the enzyme activity was measured after each addition.

An 82% inactivated enzyme formulation was obtained from 9 μl of the active ester solution.

The enzyme reaction mixture was adjusted to 1.0M after the final addition of active ester.
K2HP04 (containing 1.0X10-3M NaN3, 2
2) was completely dialyzed. After dialysis, the enzyme combination was carefully removed from the dialysis bag and placed in two pieces of Cephadex RG-50.
M (1.0×10-3M(7) 1.0M containing NaN3
(pre-swollen with K2HP04) column. 2
The size of one column is 0.9 x 54.0CTL10.9
×51.0(:Tn), the flow rate was 4-5 drops/min, and fractions of 20 drops were collected.

The protein fraction was concentrated using a collodion bag apparatus in a cold room. The hapten number was determined to be 3.0.
Example 13T4-MEMIDA glycylglycine/M
DH blend ECDI (0.0157, 7.8×10-5
M) in dry DMFO. Dissolve in 50ml and 1.6 x 10
-1M solution was obtained.

NHS (0.75y, 6.5x10-4M) 1.0m
1 in dry DMF to obtain a 6.5 x 10-1M solution. Both solutions were cooled to 0.000 in an ice bath prior to use. 1m1 pierced earrings with sterling free 2.4 x 10-3g (71) T4-ME in Reacti Vial TM
Add MIDA glycylglycine, then 78 μl of ice-cold dry DMF, cool in an ice bath, then add 4 μl of DM
18 μl of 6,5 x 10 M NHS solution (00) in F
A 1.6 x 10-1 MECDI solution (00) in DMF was added. The reaction mixture was placed in a cold room (2-) and stirred for 20 hours.

At the end of stirring, the Tlc of the reaction mixture [analytical silica gel plate, triethylamine rimethanol lydichloromethane (2.1:10:90)] had an Rf value of 0.25 (T4
-MEMIDA glycylglycine), 0.59 (T4-
Two spots of MEMIDA (glycylglycine NHS ester) are shown. MDH (1.5×1 of 4.0mj
0-5M10.05MNaHC03-Na2CO3(P
H9.2)] was placed in a 10 ml round bottom flask equipped with a stir bar and septal stopper. Cool in an ice bath to 440μ
1 of dry DMF was added at a rate of 50 μl/min. DMF
Enzyme activity was measured before and after addition. 1.9×10-2MT
3-10 μl of 4-MEMIDA glycylglycine NHS ester solution was added to the reaction mixture and enzyme activity was measured after each addition.

Addition of 29 μl of activated ester solution resulted in an 82% inactivated enzyme formulation.

The reaction mixture was then diluted with 1.0MK2HP04 (1.0MK2HP04).
(containing 0x10-3M NaN3). After dialysis, the formulation was loaded onto three Sephadex RG-50M columns (1.0MK2H containing 1.0 x 10-3M NaN3).
(pre-swelled with P04) and eluted with the same buffer.

Column size is 0.9×55CT1110.9×56C
7n10.9×56CTf1, flow rate was 4-5 drops/min, and fractions of 20 drops each were collected. The protein fraction was concentrated using a collodion bag apparatus in a cold room. When the putene number was measured by the method described above, it was 3.9. The antigen combination was used to produce antibodies.

First, the 2W19 formulation was injected. 0.25η of the formulation was then injected at two week intervals. For the sheep, the total injection volume was 2 ml, of which 0.5 ml was the formulation dissolved in saline and 1.5 ml was Freund's complete supplement. Subcutaneously inject 0.25 ml each into 4 sites, each 0.5 m
1 was injected intramuscularly into the hind limb. For rabbits, the total injection dose is 0.7
5m1, of which 0.257r9 is 0.25m1
0.5 ml of the formulation dissolved in salt water was Freund's complete auxiliary solution.

Subcutaneously inject 0.09 ml each into 4 sites, and 0.19 ml each.
was injected into the hind leg. Laboratory animals typically receive approximately 5 to 7 hours after each injection.
Blood was collected on the following day, and antibodies were isolated in a conventional manner.

A number of analyzes were performed to demonstrate the utility of the thyroxine-MDH combination for analyzing T4. In the first series of analyses, we varied the amount of antibody and demonstrated that the activity of the enzyme combination increased with increasing amount of antibody. In a second series of analyses, the amount of T4 added to the antibody was varied to vary the effective concentration of antibody available for binding to MDH-binding-thyroxine.

These results demonstrate that the amount of free thyroxine in serum can be determined by establishing a standard curve based on samples containing known amounts of thyroxine and relating enzyme activity measurements to this standard curve. Ta. The analysis method is as follows.

PH is 9.5, contains 10-3 MEDTA, 0.
0.8ml of 1W/W% RSA (7) 0.1M glycinate buffer solution was prepared for the antibody solution, MDH-conjugated-T4.

After 45 minutes of incubation, the substrate (100tt1
2M malate and 100μ117) 0.108MNA
Add D), transfer to a spectrophotometer, and wait for 120 seconds and 60 seconds after transfer.
Values were read at 300 as the change in optical density over seconds.

The results when sheep antibodies were used for the combination prepared in Example 7 are shown in the following table. 0.0 of thyroxine (9.7W19)
A series of analyzes were performed using serial dilutions of 5N NaOH (25 mO solution with 0.05N NaOH).

These assays were carried out using 600μ1f) 100μ110-3Mf) of the antibody in 0.1% rabbit serum albumin, 0.1M glycine buffer (PH9.5) EDTA and T4 solution 1
This was carried out by combining 00 μl and incubating at room temperature for 15 minutes. The amount of MDH-conjugated-T-4 solution specified in the table below was then added and incubated for 10 minutes. Substrate was then added and readings taken on a spectrophotometer as described above (30e). The results are shown in the table below. Decks G-50
Chromatography was carried out using a column.

The flow rate was slow and 39 fractions of 20 drops each were collected.
Fractions of 99 drops were then collected. Second G-5
The above procedure was repeated using a 0 column, and the fractions collected each time were super concentrated using Sephadex G-200. Analysis shows that the number of T-4 groups per enzyme is between 4.3 and 3.
It was measured to be 5.3 with an average of 4.8. A series of analyzes were performed varying the antiserum source and thyroxine concentration.

Antibody and thyroxine were incubated together in 0.1% glycine buffered RSA solution for 15 minutes as described above, then the indicated amount of enzyme solution was added, incubated for an additional 10 minutes, substrate was added, and then introduced into the spectrophotometer. The reading for the change in optical density units was taken in 2 minutes. The amount of antibody added was 1 μl, and the amount of enzyme added was 1 μl. The results are shown in the table below. Another formulation was prepared by the method described in Example 7, containing Sepha TI equilibrated with 1 M potassium dihydrogen phosphate.
The following reagents were used in implementing the T-4 assay using the M-T-4 formulation.

TIM-T-4 analytical reagent buffer: 0.02M triethanolamine (TEA) H
Cl. ．． pH 7.9; 0.0054M ethylenediaminetetraacetic acid (EDTA) Rabbit serum albumin solution: 0.2W/W%R in buffer
SATIM-T-4 solution: ~7 x 10-3 μMf)T-
4 (as TIM-T-4) RSA solution (analysis rate will be ~2000 D/min) Anti-T-4 solution: ~4X10-
5M anti-T-4 (based on binding site) in RSA solution NAD
H solution: 2.5W NADH/1ml buffer (stock solution; made according to TEKIT instructions and diluted to 1:7 parts by weight with buffer) DL-Glyceraldehyde-3-phosphate solution (GAP): DL- Glyceraldehyde-3
- Phosphate diethyl acetal, Sigma Chemical Co., St. Louis;
It was made from Ba salt obtained from Missouri (1999) and processed with 1.57 salt to give a final volume of 10 ml.

α-glycerophosphate dehydrogenase (α-G
PDH) solution: 0.2η/1ml buffer (BOehri
mger Mannheim) Thyroxine (T-4 solution)
:2.57×10 −4 Mf) RSA solution analysis was performed as follows.

A number of dilutions of the T-4 solution were prepared. First 0.4m1
Mix TIM-T-4 solution, 0.4 ml (7) of TIM-T-4 solution and 0.4 ml of antibody solution, and add 12
I added a minute. Then 200tt1(7) NADH solution, 2
5 μl of α-GPD]I solution and 100 μl of GAP
solution was added. The analytical tube was covered with parafilm and a portion was blown into the thermocouple of a 300 N GilfOrd spectrophotometer with the machine temperature at 300 and the first reading taken after 30 seconds. Optical density readings were taken at 366 nm 1300. The remainder of the analyte solution was kept in the 300 bath and readings were taken after 13 minutes.
The results are shown in the table below. The results in Table 1-V show that extremely low concentrations,
This shows that extremely small amounts of thyroxine can be detected by the method of the present invention.

This method is very simple in that it does not involve complicated steps. By combining the reagents in a buffered sample, incubating if desired, and then adding the enzyme substrate, a spectrophotometer reading can be taken to determine T-4 in a short period of time. The device can be automated so that samples and reagents can be mixed automatically and readings can be taken automatically. Although the present invention has been described above in some detail for illustrative purposes and for ease of understanding, it is clear that changes and modifications can be made within the scope of the present invention.

Claims (1)

[Scope of Claims] 1. A method for measuring the amount of a ligand that is polyiodothyronine having 3 to 4 iodine groups in a sample, in which in an aqueous liquid zone: (1) the sample; ) a soluble ENZ-binding ligand; and (3) an anti-ligand, wherein the anti-ligand is a soluble ENZ-binding ligand;
NZ binding ligand (ENZ is malate dehydrogenase or triose phosphate isomerase) and has a site capable of binding to them, and the antiligand substantially partially inhibits the enzyme activity in the absence of the ligand. the ENZ is present in a concentration sufficient to restore or completely recover; and increases depending on the amount of ligand in the specimen.
A method comprising analyzing the enzymatic activity of bound ligand within said zone. 2 Claim 1 in which the ligand is thyroxine
Section method. 3 Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, ENZ is malate dehydrogenase or triose phosphate isomerase; a is an integer 0 or 1; n is at least 1 on average, and about 1
0 or less; X and Y may be the same or different;
chalcogen or imino; D is H or an alkyl group having 1 to 6 carbon atoms; Z is 3-iodo-4-
(3',5'-diiodo-4'-hydroxyphenoxy-1')phenyl-1, or 3,5-diiodo-4-
(3'-iodo or 3',5'-diiodo-4'-hydroxyphenoxy-1')phenyl-1; A is a bonding chain, or 1 to 12 carbon atoms and 0 to 6 carbon atoms; an organic divalent group having a heteroatom which is oxygen, sulfur or nitrogen; the total number of heterofunctional groups is from 0 to 4.