JPS647341B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to human myoglobin (hereinafter, in the present specification, "myoglobin" refers to "human myoglobin").
This is related to radioimmunoassay (RIA).

Myoglobin is a heme protein that exists in muscle tissue, and is extracted from muscle using the salting-out method (Singer et al.
Blood
2240 (1961)) and gel filtration method (Awad et al.
Nature, 198, 1201-1202 (1963)) and (Kagan et al., Amer. J. Physiol, 211,
656−660 (1966)) or with (Stone et al., J.
Clin. Invest. 56, 1334-1339 (1975)) is known as a purification method.

Recently, there has been interest in changes in blood myoglobin concentration related to myocardial infarction, etc., and research has been conducted on microquantitative methods using RIA, but there are not many reports and sufficient results have not been achieved. Not yet. That is, the myoglobin purification method described above yields myoglobin that gives a single band electrophoretically.
It was still immunologically impure and unsatisfactory for antibody production and labeled antigen (tracer) production. In addition to these causes, RIA for myoglobin has low sensitivity due to the inability to obtain good anti-myoglobin antibodies, which has caused problems in clinical application. As a result of repeated research, the present inventors established a method for producing myoglobin with high purity, and also made it possible to conduct RIA with high sensitivity using an excellent anti-myoglobin antibody and tracer, thereby completing the present invention.

The present invention uses purified myoglobin obtained by treating crude myoglobin by a combination of ammonium sulfate fractionation method, gel filtration method, and ion exchange chromatography method as a standard material, and uses this as a standard material to obtain a tracer labeled with radioactive iodine using the chloramine T method. This paper relates to RIA of myoglobin using antibodies purified by affinity chromatography and gel filtration.

To obtain purified myoglobin, crude myoglobin obtained by centrifugation or the like from a homogenate of myoglobin urine, myoglobin serum, or skeletal muscle is used as a raw material, and ammonium sulfate is added to a buffer solution of this crude myoglobin to salt out. For example, salting out by adding 40% (saturated concentration) ammonium sulfate, then salting out with 50% saturated ammonium sulfate, centrifugation, and then sequentially converting the supernatant to 65%, 79%, 80%,
It is appropriate to carry out the fractionation in stages using ammonium sulfate at a concentration of 100%. Next, the obtained precipitate was dissolved in a small amount of water or buffer solution and added to Sephadex G-
Perform gel filtration using 75, G-50, etc. moreover,
When this product is subjected to ion exchange chromatography using diethylaminoethyl (DEAE)-cellulose, trace amounts of albumin etc. can be completely removed and purified myoglobin can be obtained. This product shows a single band in SDS electrophoresis, and is immunologically pure as it does not react with various serum proteins. When an animal is immunized with this purified myoglobin, a highly specific resistor can be obtained, and it can also be directly labeled with radioactive iodine. These are the original points of the present invention. That is, Reichlin et al. reported that direct labeling of human myoglobin was not successful (Clinical Research, 24, 421A (1976)), and
The above-mentioned literature reporting on RIA of myoglobin
Stone et al. and Rosano et al. (Clin.Chem.23, 69-75
(1977)) both use an ^indirect labeling method (Bolton & Hunter,
Biochem. J. 133, 529-539 (1973)). This method requires complicated operations and expensive reagents, making it impractical.

Direct RI labeling of myoglobin is an originality of the present invention, but this is only possible using purified myoglobin that has been uniquely purified as described above. Specifically, a method known as the Hunter-Greenwood method (chloramine T method) is applied to this myoglobin.
A suitable method is to add and react for about 1 minute, and then add sodium metabisulfite to stop the reaction. The labeled myoglobin obtained by this method is very stable and can be used even after 50 days. On the other hand, it is said that the tracer obtained by the above-mentioned indirect labeling method can be used for up to 30 days.

To produce antibodies for RIA, in the same way as the commonly used method, for example, an antigen (purified myoglobin) emulsified in Freund's complete adjuvant is injected subcutaneously into the toe tip of a domestic rabbit. Thereafter, booster immunization is performed every week, and after 5 to 6 weeks, blood is collected when the antibody titer reaches the maximum to obtain anti-myoglobin antiserum. This antiserum is then treated with myoglobin-conjugated agarose to obtain purified antibodies.
Myoglobin-bound agarose is prepared by reacting myoglobin with agarose activated with cyanogen bromide. Anti-myoglobin antiserum is added to this, reacted at room temperature, and excess protein is removed to obtain an agarose-myoglobin-anti-myoglobin antibody conjugate. When this column is developed with, for example, an acidic buffer, an anti-myoglobin antibody is obtained. However, since it was found that this antibody contained a small amount of myoglobin, a purified anti-myoglobin antibody was obtained as a colorless and transparent solution by further gel filtration using a Sephadex or the like. This thing is
It can also be stored by dialysis with 0.01M phosphate buffer or freeze-dried. The anti-myoglobin antibody obtained in this way is immunologically equivalent to domestic rabbit IgG.
It reacts with antiserum, reacts only with human myoglobin in the Auctelony method, does not react with human hemoglobin or human albumin, and forms only one sedimentation line with human skeletal muscle homogenate supernatant, making it a highly pure antibody. This purified anti-myoglobin antibody has approximately 23 times the activity of the original anti-myoglobin antiserum.

By performing RIA using the labeled myoglobin (tracer) obtained as described above and the purified anti-myoglobin antibody, blood myoglobin can be easily quantified with high sensitivity. The RIA can be operated in the same way as in a normal method, but an example is as follows. Add 0.1 ml of antibody, 0.1 ml of standard myoglobin (or sample) and 0.1 ml of labeled myoglobin to 0.5 ml of diluent such as buffer, incubate at 37°C for several hours, then add 1.0 ml of polyethylene glycol and 0.2 ml of bovine γ-globulin. In addition, incubate (polyethylene glycol method), normal rabbit serum, anti-rabbit γ-globulin goat serum (double antibody method), or also tyakol 5g/dl 0.4ml, dextran 250 10.5g/
By dl0.4ml (charcoal dextran method)
Perform BF separation. Measure the radioactivity of the precipitate or supernatant.

The RIA of the present invention has a sensitivity 10 times higher than that of conventional methods and can detect up to 0.3 ng. An example of a standard curve is shown in Figure 1, and the curve of the present invention, represented by d, has a steeper slope than other curves using antiserum, and B/T is low even at concentrations of 10 ng or less. It is over 30% and has high sensitivity and reliability. In addition, there is little influence of sample serum, and there is no need to correct using horse serum or the like even in samples with low myoglobin concentrations of 100 ng/ml or less. This is clear from the standard curves shown in FIGS. 2 and 3. In other words, when RIA is performed using antiserum as is commonly done, nonspecific binding between the tracer and serum components occurs as shown in Figure 2, and human There is a dissociation between the curves containing serum or horse serum. On the other hand, RIA using the method of the present invention using purified antibodies does not have this effect, and the three types of curves are expressed as almost one line, as shown in Figure 3, so there is no need for correction. is understood.

Next, an example will be given and explained.

Example 1: Production of purified human myoglobin Fat tissue and connective tissue were removed from 0.5 g of human skeletal muscle.
Add an equal amount of distilled water, homogenize, and heat to 4-5℃.
Leave to stand overnight. Then, centrifuge at 10,000 rpm for 60 minutes.
Add 0.5M basic lead acetate to the supernatant and adjust the pH.
Adjust to 7.0 and centrifuge again at 10,000 rpm for 60 minutes. Next, ₄ mg of Na ₂ HPO and 4 mg of Na ₃ PO ₄ are added to this supernatant, the pH is adjusted to 7.0, and the mixture is centrifuged at 10,000 rpm for 60 minutes. The obtained supernatant was salted out by adding 40% saturated ammonium sulfate, and further salted out with 50% saturated ammonium sulfate. After centrifugation, the supernatant was sequentially added with 65%, 79%, 80%, and 100% ammonium sulfate. Salt out. The final precipitate was dissolved in a small amount of water or buffer (0.01M phosphate buffer PH7.2).
Pass through a column of Sephadex G-75 (2 x 100 cm). This effluent was then treated with DEAE-cellulose (2
A solution of purified myoglobin was obtained by elution with the same buffer solution. This solution is
When added with 0.05% NaN ₃ and stored at 4°C, it is stable for 6 months.

This purified myoglobin shows a single band in SDS electrophoresis, and is separated into four subcomponents by acrylamide gel and cellulose acetate membrane electrophoresis.

Visible absorption spectrum (PH5.4) Absorption maximum (mμ): 409, 500, 630 Shows a pattern characteristic of met myoglobin at 580-610mμ.

Ultraviolet absorption spectrum (PH8.6) Absorption maximum (mμ): 281 As for immunological properties, this purified myoglobin reacts with anti-human muscle creatine kinase (serum), anti-hemoglobin serum, or anti-myosin serum using the Auctelony method. No sedimentation line was observed for any of them. Furthermore, when human myoglobin was identified using an antiserum obtained by sensitizing a rabbit with this myoglobin, only a single sedimentation line was produced. This anti-myoglobin antiserum forms a single sedimentation line with human skeletal muscle homogenate supernatant, but does not react with human hemoglobin, human creatine kinase, or human serum.

Example 2: Labeling of myoglobin with radioactive iodine To a mixture of 1 mCi of Na ¹²⁵ I, 2.5 μg/5 μ of myoglobin and 25 μ of 0.4 M phosphate buffer (PH7.5) was added chloramine T (25 mg/10 ml of 0.04 M phosphate buffer (PH7.5)). PH7.5)) Add 10μ and react for 60 seconds. Add to this sodium metabisulfite (25mg/10ml)
Add 25μ of 0.04M phosphate buffer (PH7.5) to stop the reaction, and then add 10μ of 10% KI. This reaction solution was pre-coated with 2 ml of 2% BSA using Sephadex G-75 Super Fine (1 ml).
Using a phosphate buffered saline (hereinafter abbreviated as PBS: 0.15M phosphate buffer (PH7.4)) column (×20cm)
+0.85% physiological saline) and fractionation of 1 ml yields ¹²⁵ I-labeled myoglobin in the 8th and 9th tubes. The specific radioactivity of this ¹²⁵ I-labeled myoglobin is 60 μCi/μg (yield: 33 μCi). This product is stable and usable even after 50 days when stored at -20°C.

Example 3: Production of anti-myoglobin antibody (1) 2 mg of purified human myoglobin is diluted with physiological saline to make 2 ml, and an equal volume of Freund's Complete Adjuvant is added to make an emulsion. This is injected subcutaneously into the tip of an adult rabbit (male, approximately 3 kg) to immunize it. Booster immunizations are then given weekly by injecting 0.5 mg/ml human myoglobin diluted in 1 ml of physiological saline into the ear vein. Usually 5~
At 6 weeks, the antibody titer reaches its maximum value, so blood is collected to obtain antiserum.

(2) Purified human myoglobin 2 mg/ml in 2 g of CNBr-activated Sepharose 4B (manufactured by Pharmacia)
Add 5 ml, then add 0.5 M common salt-0.1 M borate buffer (PH8.3), and react at room temperature for 2 hours. Next, wash with the same buffer, add 1M ethanolamine (PH9.0), and react at room temperature for 2 hours to block excess active groups. This gel is washed three times each with 0.5M salt-0.1M acetate buffer (PH4.0) and 0.5M salt-0.1M borate buffer (PH8.5) to remove excess protein. The myoglobin-bound cephalose thus obtained was mixed with the anti-human myoglobin rabbit serum obtained in (1).
By adding 20 ml and reacting at room temperature for 2 hours, a cephalose-human myoglobin-anti-human myoglobin antibody conjugate is obtained. This was washed with 0.5M salt-0.1M borate buffer (PH8.3) to remove excess protein, and then packed into a 0.8 x 15 cm column.
0.5M NaCl-0.17M Glycine-HCl buffer (PH2.5)
When developed, anti-myoglobin antibodies are eluted.
Since this antibody solution contains a trace amount of myoglobin, it is further applied to a Sephadex G-75 column (2 x 100 cm) and the antibody is eluted with 0.17M glycine-hydrochloride buffer (PH2.5). The purified antibody obtained is a colorless and transparent solution. This is rabbit IgG, which exhibits the mobility of γ-globulin in electrophoresis and reacts with anti-rabbit IgG antiserum. Furthermore, immunologically, it reacts only with human myoglobin using the Auchterrony method, and does not react with human hemoglobin or human albumin. Only one sedimentation line is formed compared to human skeletal muscle homogenate supernatant. In radioimmunoassay, if the cross-reactivity with human myoglobin is 100%, there is no cross-reactivity of 0.003% with human hemoglobin, 0.00001% with human albumin, and 0.0005% or more with human muscle-type creatine kinase. The antibody activity of this purified antibody is approximately 23 times that of the original anti-myoglobin antiserum. For storage of this purified antibody, 0.01M
It is appropriate to freeze-dry the product after dialysis with a phosphate buffer.

Example 4: RIA dilution of myoglobin (0.01M phosphate buffer PH7.4, 0.15M NaCl, 0.05M EDTA, 0.01% NaN ₃ , 1% BSA, 1
Add 0.1 ml of purified antibody, 0.1 ml of standard myoglobin (or sample), and 0.1 ml of ¹²⁵ I-labeled myoglobin to 0.5 ml of mM FOY (ethyl-p-(6-guanidinohexanoyloxy)benzoate methanesulfonate)) at 37°C. Incubate for 4 hours. Next, 1.0 ml of polyethylene glycol and 0.2 ml of bovine γ-globulin were added to this mixture.
The radioactivity of the precipitate obtained by incubation at ℃ for 15 minutes and centrifugation was measured using a γ-ray counter.
The obtained standard curve is shown in FIG. 1 together with the curve using antiserum. Furthermore, in a similar manner, the influence of human serum or horse serum when antiserum was used and when purified antibody was used was investigated using a standard curve, as shown in FIGS. 2 and 3. As is clear from these figures, the RIA of the present invention has good sensitivity and is an extremely excellent method even for low-concentration samples with little influence from serum. Furthermore, purified myoglobin has high immunological purity, and the labeled myoglobin obtained from it is easy to produce and stable for a long period of time, making it a useful substance.

[Brief explanation of drawings]

Figures 1 to 3 are standard curves for myoglobin RIA.

Claims (1)

[Claims] 1. Purified human myoglobin obtained by a combination of ammonium sulfate fractionation, gel filtration, and ion exchange chromatography using diethylaminoethyl cellulose is used as a standard material, and the standard material is directly purified with radioactive iodine by the chloramine T method. A radioimmunoassay method for human myoglobin, which is characterized in that it uses radioactive iodine-labeled human myoglobin as a tracer and an anti-human myoglobin antibody purified by affinity chromatography and gel filtration.