JPS604423B2 - Method for quantifying peptide hormones - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for quantifying peptide hormones.

Methods for measuring blood and urine concentrations of physiologically active substances, drugs administered to living organisms, or their substitute products include immunochemical measurement methods that measure the immunological activity of these substances, and biological measurement methods. Although there are biological measurement methods for measuring activity, it is most rational to measure the latter biological activity as an indicator, and conventionally, biological measurement methods (Bioas
say) was used.

However, since this method has low measurement sensitivity and requires complicated procedures, the former immunochemical method has been increasingly used in recent years.
Among them, radioimmunoassay (hereinafter abbreviated as RIA) or enzyme immunoassay (8nzym
el-mmunoassay: Hereinafter abbreviated as E-butsu. ) is widely used because of its high measurement sensitivity and excellent quantitative performance. The measurement principles of RIA and EIA are common, and both utilize a specific and sensitive reaction between an antigen and an antibody, and both utilize a substance to be measured (hereinafter referred to as a measurement substance).

) is measured using its immunological activity as an index. Immunological activity often shows a good correlation with biological activity, but for certain substances, such as corticotropic hormone and growth-stimulating hormone, the activities of the two dissociate, resulting in RI.
In some cases, the values measured by A or EIA do not necessarily indicate the biological activity of the substance.

For this reason, recently, radioreceptor analysis has been developed as a method that has extremely high measurement sensitivity and whose measured values are considered to indicate the biological activity of the substance.
orassay: Hereinafter abbreviated as RRA.

) is now used. The above-mentioned RRA utilizes the binding activity of receptors present in the cell membranes or cells of target organs for physiologically active substances and drugs, and RRA cannot be said to strictly measure biological activity. However, it is expected to be a method that can measure biological activity values more accurately than RIA or EIA.

The measurement method of RRA is almost the same as that of RIA, and involves a binding reaction between a measuring substance and a fixed amount of a labeling substance, which is a labeling agent bound to the measuring substance, against a fixed amount of receptors for those substances. When the amount of the analyte and the labeled substance is increased, they bind to the receptor in proportion to their abundance, so as the amount of the analyte increases, the amount of the labeled substance bound to the receptor decreases in inverse proportion to it.Next Next, separate the labeling substance that bound to the receptor and the labeling substance that did not bind to the receptor using an appropriate method, measure the activity of the labeling agent in either fraction, and at the same time perform the same procedure using a labeling substance with a known concentration. The amount of the unknown amount of the analyte can be measured using the standard curve created by the manipulation.The above measurement method corresponds to the competitive method in RIA, and in RRA it corresponds to the sandwich method in RIA. No method has been used to do so.

In addition, as a labeling agent, a method using an enzyme or a fluorescent substance in addition to a radioactive isotope [EmMmerceptor
assay: (hereinafter abbreviated as ERA).

) or rass of Fluoresencerecep
ay] is theoretically conceivable, but conventionally only radioactive isotopes have been used. The measurement sensitivity of RRA is
Although it is very expensive compared to bioassay,
It has the disadvantage that it is inferior (compared to RIA) and that the fluctuations in measured values are large.

One reason for this is that the receptor being measured is usually not the receptor itself, but rather various receptor-containing fractions (hereinafter referred to as (referred to as receptor distribution), in addition to specific binding between the substance to be measured and the receptor, non-specific binding occurs, and the degree of this is thought to vary depending on various conditions. To eliminate such fluctuations, it is necessary to sufficiently purify the receptor fraction to remove substances that cause nonspecific binding.
For this purpose, very complicated operations such as repeated density gradient ultracentrifugation are not necessary. Furthermore, ``The binding between the receptor and the cell membrane in the receptor fraction remains a natural bond, so the receptor may be naturally solubilized and eluted during the preparation process of the receptor fraction or the measurement process. The loss of the receptor fraction itself due to the centrifugation operation during measurement is also thought to be one of the causes of fluctuations in the measured values.The present inventors
The present invention was completed as a result of repeated research into the development of a simple measurement method that does not require complicated operations to highly purify the receptor and has excellent measurement sensitivity and accuracy.

In the present invention, after solubilizing and separating the receptor from the cell membrane,
By using an insolubilized receptor obtained by binding this to an appropriate insoluble carrier, the present invention provides a method for measuring biological activity with high accuracy and sensitivity, which has not been possible with conventional RIA and RRA.

The implementation of the present invention requires three things: an immobilized receptor, a labeled antibody prepared by labeling an antibody that specifically reacts with the antigen to be measured, and the antigen to be measured.

The first stage reaction produces a biological bond between the receptor and the substance to be measured, and the second stage reaction produces an immunological bond between the substance to be measured and the labeled antibody. By performing both reactions simultaneously or sequentially, it became possible to accurately measure biological activity.
The present invention is generally carried out as follows.

First, the receptor for the substance to be measured (for example, hormone) is solubilized, and receptor fractions such as organ slices, free cells, tissue homogenates, and purified cell membranes, which have been conventionally used as measurement receptors in RRA, are solubilized. As it is, or after homogenizing and removing large tissue lumps etc. by filtration or centrifugation, it is further heated to 20,000 to 3,000 m. p.
m. Centrifuge and collect the supernatant. Add a suitable buffer solution (e.g. phosphate buffer (pH 7.2)) or surfactant solution (e.g. Triton X-100 (Trip
nX-100, trade name, manufactured by Koso Chemical Co., Ltd.).
1 to 2.0 phosphate buffer solution] to make a suspension, and leave on ice or at 370℃ for 30 minutes to 2 hours. The receptor solubilized by the buffer as described above has a strong binding ability with the substance to be measured, and the unmelted receptor obtained by binding it to an immovable carrier has good measurement sensitivity and accuracy. Therefore, it is an excellent receptor for measurement, but it has the disadvantage that the amount eluted from the cell membrane is small. On the other hand, when solubilized with a surfactant solution, a larger amount of solubilized receptor can be obtained, although the binding capacity is inferior to that obtained by solubilization with a buffer solution.

Next, the solubilized receptor is bound to a suitable insoluble carrier. Insoluble carriers include blood cells, polystyrene latex, plastic tubes, Sepharose, Sephadex, etc. Conventionally, in the field of immunochemistry, antigens or antibodies are used. Those used as carriers can be used.

For example, when using blood cells, blood cells from various animals such as mammals such as cattle, horses, sheep, rabbits, and humans, and birds such as chickens and turkeys are used, and the blood cells are treated with formalin, pyruvaldehyde, etc. After treatment with a fixative such as hydrogen peroxide, tannic acid, bis-diazobenzidine, 1.
Treat with a binder such as 3-difluoro-4,6-dinitrobenzene, glutaraldehyde. For example, when using sheep blood cells, the sheep blood is centrifuged to separate the blood cells, which are washed with physiological saline and then concentrated to approximately 8.
% suspension. An equal volume of a formalin solution with a concentration of about 3% is added to this, mixed, reacted at 370 for one hour, washed, and made into a suspension with physiological saline at a concentration of 10%. To treat this suspension with a binder, e.g. tannic acid,
The formalin-treated blood cells are mixed with phosphate saline (hereinafter abbreviated as PBS) to a concentration of 1.0 to 6.0%, preferably 3.3%.
, and an equal volume of a tannic acid solution with a concentration of 0.0025 to 2.0% is mixed therein, or 10 to 5% of a tannic acid solution with the above concentration is placed on top of the formalin-treated blood cells 1.
Add and mix at a ratio of 0 to 25 to 56 to 0 to 30 to 9.
Let it react for 0 minutes. With this treatment, 0 per M blood cells
．． 3 to 500 9 tannic acids are bound. Note that the amount of blood cells is expressed by the apparent volume of blood cells obtained by centrifuging a blood cell suspension.

In order to use the blood cells treated in this way as a carrier and bind the solubilized receptor to them, the deceased combination-treated blood cells are suspended in PBS at a concentration of 1.6 to 6.0%, and this and the previously Mix with an equal volume of the prepared solubilized receptor solution and react at 25 to 56 circles for 20 to 60 minutes.

In addition, in a measurement method that combines an immobilized receptor using blood cells as a carrier and a method using an enzyme as a labeling agent, the presence of enzyme activity in the blood cells themselves may interfere with the measurement of enzyme activity as a labeling agent. Therefore, it is necessary to remove the enzymatic activity of the child's blood cells themselves. In order to remove the enzyme activity of blood cells themselves, the enzyme activity varies depending on the type of enzyme to be removed, but in general, pH, temperature, etc. are introduced into the unstable region of the enzyme to inactivate it. For example, when using horseradish peroxidase as a labeling agent, an enzyme that exhibits the same type of activity is present in blood cells, so the formalin-treated blood cell suspension should be stored under acidic conditions with a pH of 3 or less. Deactivates enzyme activity. Furthermore, when polystyrene latex or plastic tubes are used as carriers, treatment with fixatives and binders is not necessary. The receptor can be bound to the carrier by conjugation.

When using cellulose or Cephadex as a carrier,
Activate these with bromsyanide and bind to the receptors. To label the antibody of the analyte, use an enzyme (
For example, horseradish peroxidase, alkaline phosphatase, glucose oxidase, etc.) are used, and known methods such as the glutaraldebide method, the maleid method, and the periodic acid method are used.

Measurements using the thus obtained insolubilized receptor are carried out as follows. First, a specimen and an insolubilized receptor are placed in a test tube and incubated. After adding a certain amount of labeled antibody to this and incubating it, it is centrifuged to collect the pillow sand. Next, an appropriate substrate solution is added to the precipitate and incubated, and the absorbance of the supernatant is measured. At the same time, the amount of the analyte is measured using a standard curve prepared using a standard solution of known concentration instead of the analyte.

The measurement method of the present invention is applied to a method called a sandwich method in RIA. Generally, the sandwich method is known to have advantages over competitive methods, such as better measurement sensitivity and accuracy, and a wider measurable range. Therefore, the measurement method of the present invention, which uses an unmelted receptor as a partner of the analyte and combines it with a labeled antibody and applies the principle of the sandwich method, can achieve good measurement sensitivity and Can be measured with precision. Substances that can be measured by the measurement method of the present invention include, but are not limited to, hCG, growth hormone tinsulin, etc., and substances for which the presence of receptors is known can be measured. It can be applied.

The insolubilized receptors of the present invention have various characteristics.

Conventionally, the receptor fraction of a tissue homogenate has been mainly used as the receptor for measurement, but in order to prepare this fraction, the tissue homogenate is sedimented by applying a large centrifugal force. Therefore, in the measurement operation, separation of this receptor fraction from the reaction solution requires a centrifugal force comparable to that used during preparation, so the operation is not easy. For this reason, some measures have been taken, such as adding reagents such as CaH and Mg to the reaction solution to cause the receptor fractions to associate or aggregate to facilitate centrifugation. Since the receptor is solubilized and separated from the cell membrane and then bound to a faceless carrier such as blood cells or polymer latex, it can be easily sedimented using a small centrifugal force. The insolubilized receptor of the present invention does not require any special treatment, and the measurement operation is easy.Also, since the insolubilized receptor of the present invention is tightly bound to an easy-to-handle inert carrier, it can be easily handled using conventional receptor molecules. As shown in the picture, the measurement accuracy does not change significantly due to the receptors gradually dissolving from the cell membrane during the measurement operation or the receptors themselves leaking out, and the measurement accuracy can be greatly improved. Further, the measurement method of the present invention combines the advantages of a measurement method using a receptor with the advantages of the antigen-antibody reaction (particularly the sandwich method), thereby making it possible to measure the substance to be measured with good measurement sensitivity and accuracy.

The present invention will be explained in more detail with reference to Examples.

Example 1 Production of an insolubilized gonadotropin receptor a Production of a solubilized gonadotropin receptor A mineball was extracted from a rad killed by decapitation, and after peeling off its albuginea, it was mixed with about 3 quarts of silica sand using a Virtjs homogenizer. Homogenized for a while.

This is 100 go-p. m. After centrifuging for 10 minutes, the supernatant was further centrifuged for 2,500 centimeters. p. m. Centrifuged for 30 minutes. The supernatant was removed and the resulting diluted solution was added with 0.5% TribnX-1.
After floating in 20% of phosphate buffer (pH 7.2) containing 0.00% and left at 4°C for 30 to 60 minutes, it was heated to 2700%. p
．． m. Centrifugation was performed for IQ minutes at Equal amounts were mixed and incubated for 20 minutes at 5 to bind the gonadotropin receptor to the polystyrene latex.

After the reaction is completed, the position is 800. p. m. After centrifuging for 5 minutes at
0' floating and immobilized gonadotopin receptors were obtained.
Example 2 Production of Insolubilized Growth Hormone Receptor a Production of Solubilized Growth Hormone Receptor The liver of a domestic rabbit from which the gallbladder, large blood vessels and connective tissue had been removed was cut into small pieces with scissors and washed twice in a 0.3 M glucose solution. ,
A 0.3M glucose solution 5 times the amount of the liver tissue was added and homogenized.

After heating this with gauze, apply the furnace liquid at 60℃ for 20 minutes (
Centrifuge the supernatant at 1500 °C for 20 minutes (400 °C).
00) far '0 separated. This condition was further centrifuged for 90 minutes (400 °C) at 10,000 °C, and the obtained
．． The cells were suspended in PBS containing 5% TrinX-100 and incubated at room temperature for 1 hour. Do this for 30 minutes at 10,000 targets (
400) Centrifugation was performed to remove insoluble portions to produce solubilized growth hormone receptor. b Preparation of blood cells for carrier 50% of commercially available sheep blood for complement fixation reaction (manufactured by Toshiba Chemical)
005. p. m. Centrifuge for 10 minutes to separate blood cell components,
After washing three times with physiological saline, the suspension was made into a suspension at a concentration of 8% with physiological saline.

Add an equal volume of 3% formalin solution to this, and at 370
After incubation for glue time, wash 4 times with purified water,
It was stored at 4°C as a 10% suspension until use. c. Production of insolubilized receptor Blood cells were separated by centrifugation of the blood cell suspension 2' in b above and washed twice with P plaques.

This is mixed with 6 P of P already floating and the same volume of tannic acid solution (
(concentration 0.25%) and then incubated at 5600 for 30 minutes. After cooling, it was washed twice with P-hydrochloride, suspended in P-acid 6 secretion, mixed with the receptor solution 6 of the above a, and incubated at 56oo for 20 minutes to inject growth hormone into the blood cells. bound the receptor. Next, this was rapidly cooled, washed twice with PBS, and then floated on two P plaques containing 0.1% bovine serum albumin (hereinafter referred to as Mother A) to obtain an insolubilized growth hormone receptor. Example 3 Production of Insolubilized Gonadotropin Receptor a Production of Solubilized Gonadotropin Receptor From rats killed by decapitation, the tunica albuginea of the brush circles was peeled off, and the seminiferous tubules were collected in P spots to obtain interstitial cell fragments.

After treating this with gauze, pour 100 ml of furnace liquid. p. m.
The supernatant was further centrifuged for 2,500 centimeters. p. m
．． Centrifuged for 30 minutes. The supernatant was removed and the resulting precipitate was suspended in 2' P containing 1% TripnX-100 per mineball. This was stirred at room temperature for 3 minutes to obtain a solubilized gonadotropin receptor. b. Production of insolubilized gonadotropin receptors Place the receptor solution 1' produced in step a above in a polystyrene test tube with an inner diameter of 1.5 and a length of 10 cm, and react at 56q○ for 20 minutes. Combined.

After the reaction, wash with PBS and add PBS containing 0.5% BSA.
After adding BS solution 2 and leaving it for 3 hours at 4°C, the mixture was washed with P mother to produce an insolubilized gonadotropin receptor. Example 4 Production of insolubilized gonadotropin receptor (a) Production of solubilized receptor was carried out according to the method of 31a to obtain 2700 ml of gonadotropin receptor. It was used as a solubilized receptor fluid.

b Production of blood cells for carrier Sheep blood cells were fixed according to the method of Example 2-b, and 10%
It was made into a floating liquid.

0.8M glycine-hydrochloric acid buffer (p
After adding H2.0) 5 and mixing and leaving it for 5 minutes,
300 enemies. p. m. The mixture was centrifuged for 5 minutes. The supernatant was removed, and the resulting precipitate was suspended in the buffer solution 1ow' and allowed to stand for 5 minutes, and then centrifuged again to remove the supernatant. The resulting night clear liquid was washed twice with P plaques and then made into a 10% blood cell suspension with LPBS. c. Production of immobilized gonadotropin receptors Using the receptor solution 2 of a above and the blood cell suspension 2 of b above, an insolubilized gonadotropin receptor was produced according to the method of Example 2-c. .

Example 5 Production of insolubilized gonadotropin receptor a Production of solubilized insulin receptor After removing large blood vessels and connective tissue, rat liver was cut into small pieces with scissors and washed twice with ice-cold 0.28 glucose solution. did.

To this was added an ice-cold 0.29M glucose solution in an amount 5 times the amount of the liver tissue, homogenized, and centrifuged at 60 eaves for 1 minute. This supernatant was centrifuged at 1200 eaves for 30 minutes, and sodium chloride and magnesium sulfate were added to the resulting supernatant at 0% concentration, respectively.
．． They were added at 1M and 0.2M. This is 4
After centrifugation for 40 minutes at 0.000, the resulting sediment was
．． 09M Tris-HCl buffer (pH 7.4),
Homogenized for 20 seconds. Centrifuge again for 40 minutes at 4,000 °C, suspend the obtained pillow clear in 0.0M Tris-HCl buffer (pH 7.4) containing 1% Trion The mixture was centrifuged (400) for 30 minutes in a boat, and the supernatant was collected to obtain undocked insulin receptors.

b Production of immobilized insulin receptor 0.5% glutaraldebide solution dissolved in 0.1M carbonate buffer (pH 9.0) 20 polyethylene rods with 3 legs in diameter and 8 ribs in length were placed in 2 bars.
Books were added and incubated for 2 hours at room temperature.

After thoroughly washing the polyethylene rod with purified water, this comb was added to the receptor solution 2' of a above and left at 4° C. to bind the Iwasthrin receptor to the polyethylene rod. After the reaction was completed, this was washed with purified water and then placed in IM ethanolamine P pre-existing solution 2. After incubation at room temperature for 30 minutes, it was washed with purified water to produce an insolubilized insulin receptor. Example 6 Measurement of placental gonadotropin (a) Placental gonadotropin (hereinafter abbreviated as hCG).

) Preparation of standard solution The hCG standard product listed in the Japanese Pharmacopoeia was mixed with 0.
P containing 1% mother A has already dissolved, 30,000,3000?
hCG standard solutions of 300, 30, and 370 iU' were prepared.

b. Preparation of anti-hCG antibody hCO was dissolved in physiological saline to a concentration of 2:2 and 2:1, and the complete adjuvant of Front was mixed and thoroughly emulsified.

Apply this subcutaneously to the rabbit's back once a week (see figure 1).
was administered five or more times, and blood was collected i weeks after the final administration to obtain antiserum.This antiserum was salt-folded twice with sodium sulfate to produce anti-hCG antibody.c Anti-hCG antibody/enzyme conjugation Product Production 5-9 Horseradish peroxidase (hereinafter abbreviated as HRP) was dissolved in 0.9% sodium bicarbonate solution 1% 2.4-dinitrofluorobenzene 0.

I added 1 fence and lit the lamp for 1 hour at room temperature.

To this was added one volume of 0.08 M sodium periodate solution and mixed for 3 minutes at room temperature, and then 1.0 volume of 0.18 M ethylene glycol solution was added and mixed for 1 hour at room temperature. Next, after dialysis overnight against 0.01M carbonate buffer (pH 9.5), a solution of 1.0 μl of the anti-hCG antibody in b above dissolved in the buffer at a concentration of 5 μl was added to this. After adding and reacting at room temperature for 3 hours, sodium borate hydride from 5 to 9 was added and the reaction was further carried out at 4°C for 3 hours. After the reaction was completed, the anti-hCG
An antibody/HRP conjugate was obtained. d Measurement of hCG: 0.1% of the standard hCG solution from a above, 0.1% of the insolubilized gonadotropin receptor suspension from Example 4 [and 0.1%]
PBSO containing BSA. Put 4' into a test tube and heat to 37℃.
and incubated for 40 minutes.

Next, the anti-hCG antibody/HRP conjugate solution 041 described in c above was added to this, and the mixture was incubated at 370℃ for 6 minutes. 300 enemies. p. m. After centrifuging for 5 minutes at
Substrate solution 3 containing ' was added and allowed to react at room temperature for 60 minutes.

After adding one drop of 1.6% sodium azide solution to stop the reaction, the mixture was briefly centrifuged and the absorbance (46 m) of the supernatant was measured.

An example of the results is shown in FIG. Comparative Example 1 Measurement of hCO using a gonadotropin receptor fraction a Production of a gonadotropin receptor fraction A rat was killed by decapitation. , homogenized for a while.

Pass this through a gauze, centrifuge the solution at 150 °C for 10 minutes, and add 10 volumes of PBS per fist to the resulting precipitate.
A gonadotropin receptor fraction was prepared by floating in
Measuring hCG hCG standard solution prepared in Example 6-a 0.1', gonadotropin receptor fraction in a above 0.1
PBSO. 4' was placed in a test tube and incubated at 370 for 40 minutes.

Next, 0.1 volume of the anti-hCG antibody/HRP conjugate solution of Example 61c was added and further incubated for 60 minutes. 300 enemies. p. m. After centrifuging for 10 minutes and removing the supernatant, the mixture was washed three times with P.sub., and the substrate solution 3' used in Example 61d was added thereto, and the mixture was allowed to react at room temperature for 6 hours.

After adding one drop of 1.6% sodium azide solution to stop the reaction, the mixture was separated by reflux and the absorbance of the upper layer was measured.

An example of the results is shown in Figure 2.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the results of Example 6, and FIG. 2 is a graph showing the results of Comparative Example 1. Figure 1 Figure 2

Claims (1)

[Scope of Claims] 1. A peptide hormone receptor is extracted from a mammalian tissue or cell in a solubilized state with a buffer containing a surfactant, 2. The extracted receptor is transferred to an insoluble carrier with tannic acid, tannic acid, The insolubilized receptor is bound and insolubilized using a binding agent such as glutaraldebide, and 3. i. a test sample of blood components or urine, and ii. an enzyme as a labeling agent to the antibody that specifically binds to the peptide hormone. react simultaneously or separately with a labeled antibody bound to an insoluble receptor-peptide hormone-labeled antibody to form an insolubilized receptor-peptide hormone-labeled antibody conjugate, 4. separating the conjugate from the reaction solution, 5. A method for quantifying a peptide hormone, which comprises measuring the activity of a labeling agent in either one of the reaction solutions.