JPS60201259A - Bacillus type specific tuberculin diagnosing agent - Google Patents

Abstract

PURPOSE:To identify effectively a BCG vaccine-sensitized living body and a human type tubercle bacillus-sensitized living body by using the D material derived from BCG bacilli as a tuberculin antigen. CONSTITUTION:A D material which is the novel and powerful tuberculin antigen derived from tubercle bacilli is isolated in early time and is verified later that the D material obtd. from the BCG bacilli in particular is the tuberculin antigen extremely powerful as compared with refined tuberculin (PPD) in a BCG vaccine-sensitized living body. On the other hand, it is found that said material exhibits extremly weak activity only as compared to PPD in a human type tubercle bacillus-sensitized living body, unlike the BCG vaccine-sensitized living body. Such characteristic is utilized for the diagnozing agent of this invention in which the D material derived from the BCG bacilli is used as an effective component to induce the specific reaction with the BCG vaccine-sensitized living body. The effective identification of the BCG vaccine-sensitized living body and the human type tubercle bacillus-sensitized living body is thus made possible.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a tuberculin diagnostic agent which contains substance D derived from BCG bacteria as an active ingredient and which induces a specific reaction in living organisms sensitized to a BCG vaccine. It is.

Conventionally, as an indicator of Mycobacterium tuberculosis sensitization, a sarcastic reaction test using purified tuberculin (hereinafter abbreviated as PPD), the so-called tuberculin test, has been generally performed. however,
Although the PPD used in the tuberculin reaction is called purified tuberculin, it has been found to be a complex of various antigens, and cross-reactivity is observed in organisms sensitized to Mycobacterium tuberculosis and BCG bacteria. There is.

Therefore, when seroconversion is observed for the first time in a tuberculin skin test, it is extremely difficult to determine whether it is due to infection with Mycobacterium tuberculosis, which is dangerous to the human body, or whether it is due to BCG vaccination to provide anti-tuberculosis immunity. This is difficult, and in this case, sufficient consideration must be given to subsequent health management.

For these reasons, the emergence of highly purified tuberculin antigens, especially bacterial type-specific antigens, to replace PPD has been strongly anticipated, and much effort has been made. In recent years, with advances in substance purification methods, some highly purified tuberculin antigens have been reported, but those with clear properties as bacterial type-specific antigens are extremely rare and have not been put to practical use. For example, Nagai et al.
nd Irrvnity r31, 1152 (
MPB70, which was reported in 1981')), is a highly purified tuberculin antigen produced by BCG bacteria, and is said to be a type-specific antigen that reacts only in living organisms sensitized with BCG bacteria. It is said that the test becomes negative after 20 weeks after harvesting, so it is considered to be of little practical use in this field.

The present inventor previously isolated Substance D, which is a novel and potent tuberculin antigen derived from Mycobacterium tuberculosis, and has already filed a patent application (Japanese Patent Application No. 186,850/1983).

Subsequently, as a result of further detailed studies on the properties of Substance D, it was found that Substance D obtained from BCG bacteria was
It was confirmed that the tuberculin antigen is extremely strong compared to PPD in organisms sensitized with G vaccine, whereas it has extremely weak activity compared to PPD in organisms sensitized with Mycobacterium tuberculosis, which is different from organisms sensitized with BCG vaccine. I found that it only shows that Furthermore, when an amount of antigen sufficient to induce a sufficient skin reaction in a BCG vaccine-sensitized organism was subtly inoculated into a Mycobacterium tuberculosis-sensitized organism, it was completely impossible to induce a skin reaction. The present inventor has focused on these findings and has completed the present invention.

That is, the purpose of the present invention is to use Substance D derived from BCG bacteria as a tuberculin antigen, and to take advantage of the fact that BCG vaccine-sensitized living organisms react specifically to the BCG vaccine-sensitized living organisms. The object of the present invention is to provide a tuberculin diagnostic agent for identifying organisms sensitized to Mycobacterium tuberculosis.

Next, a production example of Substance D, which is a tuberculin antigen used in the present invention, and the physical and chemical properties of Substance D will be shown.

Mycobacterium + n bovis BCG (
ATCC19015) was statically cultured at 67° C. for 6 weeks using a meat extract-glycerin medium.

5 to 2 kg of wet bacterial cells obtained by filtering the culture through cheesecloth
After adding and suspending 0mM phosphate buffer (pi(7,0) 10A!), the bacterial cells were crushed in a Dyno-Mill while cooling on ice.

The obtained microbial cell fragments were centrifuged under cooling to remove solid matter, and a cell-free extract 8.21 was obtained. Streptomycin sulfate (24.61 mm) was added as a nucleic acid precipitant, and the mixture was thoroughly stirred, left to stand overnight at 4° C. to form a precipitate, and centrifuged to obtain 7.8 mm of supernatant.

6 kg of solid ammonium sulfate was added to the above nucleic acid-removed supernatant, and after stirring to saturation, the mixture was incubated overnight to form a precipitate, and centrifuged to obtain a precipitate. This precipitate was suspended in a small amount of distilled water, packed into a dialysis tube, and dialyzed against distilled water 101 for 2 days. After that, the external dialysis solution was added to 10mM acetate buffer (pH 4,
2) and further dialyzed at 4°C for 6 days. The dialysis fluid was replaced with fresh fluid twice daily. After completion of dialysis, centrifugation was performed to obtain 2.2 mm of supernatant.

This supernatant was added with cold ethanol 1. Ol was added little by little with stirring, and after standing at 4°C overnight, centrifugation was performed to obtain a supernatant, which was concentrated under reduced pressure to about 100 d.

A column was filled with CM cellulose (0M52, Whatman Co., Ltd.!8) equilibrated with 70mM acetate buffer (pH 4,1) to prepare a 15x200mm bed.The supernatant was dialyzed against the same buffer. was passed through this column at a flow rate of 50 tnl/h, and then eluted with the same buffer.The part of the eluate that showed an absorbance of 0.05 (280 nm) or more was fractionated. Da was included.

Both fractions were then concentrated under reduced pressure and then dialyzed against 5mM Tris-HCl buffer (PH8,0). This dialysate was transferred to a QAE Sephadex A-25 (manufactured by Pharmacia) column (15 x 160 m) equilibrated with the same buffer at 30 d/min.
After passing the liquid through the tube at a flow rate of 700 d, the tube was washed with the same buffer solution for 700 d.

The intermediate N product was then eluted using the same buffer supplemented with 0.15M NaCl. Absorbance in eluate o, oi (280n
m) The parts showing the above were collected and used as an intermediate purified product. The protein content of the obtained intermediate purified product was 106 Da. The intermediate purified product was lyophilized after dialysis against distilled water.

Intermediate purified product of Production Example 1 using Sephadex 0100 Canim (26,4x960xm) equilibrated with 10mM J acid buffer to which 0.15M salt was added and dissolved in 5d of the same buffer. was developed with the same buffer solution at a rate of h11rd/h according to a conventional method. Effluent volume 33
A portion of 0 to 375 go was separated and concentrated under reduced pressure to about 2 go.

Next, a Sephadex G75 Super Fine column (16x148
The above concentrate was developed with the same buffer at a rate of 6 g/h. Effluent volume: 160~160WLl! The portion up to this point was separated. This fraction contained a protein amount of 11 Da.

Furthermore, both of these aliquots were added to 5 mM) lithium-hydrochloric acid buffer (pi (
A QAE Sephadex A-25 column (10
After passing the solution through a X450 tube at a rate of 1 pm/h, linear concentration gradient elution was performed using the same buffer and 0.12 M saline-added buffer for 150 d each.

The protein content and carbohydrate content of the eluate were measured, and the glycoprotein peak eluted at a salt concentration of approximately 0.07 to 0.09 M was fractionated. Both volumes were packed into a dialysis tube, dialyzed against 1 volume of the above buffer solution, and again under the same conditions.
Chromatography was performed using an AE Sephadex A-25 column.

The obtained fraction containing the target product was dialyzed against distilled water and then lyophilized to obtain 5.4 m9 of Substance D (weight of lyophilized product). The protein content of the obtained substance D was 0.749.

In addition, the protein amount and sugar amount were measured by the following method.

(1) Lowry method using protein content bovine serum albumin as standard (8 tauffer, C, B+, Analytica
lBiochemistry+ Volume 69, 646 Sada, 1
975).

1i) Phenol-sulfuric acid method using 1 mm Conigel course standard (Dubois, M., etal, Analyti
calBiochemistry, vol. 28, p. 650, 1956).

Next, the physical and chemical properties of substance D will be shown.

(1) It is a glycoprotein and has a white powder appearance.

(2) Solubility in solvents; soluble in water, insoluble in methanol, ethanol, ether and acetone.

13) Molecule t: 26,000 (4) ultraviolet absorption according to the gel filtration method using Sephadex G75 Superfine; the combination shown in FIG.

(5) Infrared absorption spectrum; as shown in the second factor.

(6) Amino acid composition C1/D substance 1001, asparagine fi15, threonine 91, serine 1.4. Glutamic acid 11.4, proline 6&4, glycine 3.1, alanine 4.0, valine Z4, isoleucine 2,4, leucine 1.5 (7) Sugar composition C&/D substance 100. ! 9) Mannose 190. Glucose 1.2 (8) By the double diffusion method using a large green plate, a single sedimentation line is formed between Shiko and Canapa 177A.

(9) Delayed allergic reaction activity in BCG vaccine-sensitized guinea pigs is more than 8 times that of purified tuberculin.

As shown in the test example below, the substance D obtained in this way showed approximately 60 times the activity on a dry weight basis compared to PPD in a sarcastic reaction test in BCG vaccine-sensitized guinea pigs, but In guinea pigs sensitized with Mycobacterium tuberculosis, the PPD is about one-fifteenth of the PPD, and depending on the amount of inoculation of the antigen used, the -j7' is sufficient (also when measuring the durability of the tuberculin reaction after sensitization with the BCG vaccine, It shows almost the same trend as PPD used as a control system, and its practicality is considered to be high.

In addition, Pailin, which uses the manure of guinea pigs sensitized to Mycobacterium tuberculosis,
In an in vitro J-bulb rejuvenation test, a sufficient response was observed in the case of BCG vaccine sensitization even at an antigen addition dose of 0.2 μi/Id, but a similar level of response was observed in the case of M. tuberculosis sensitization. To obtain this, antigen addition of 25 μg/d or more is required. This result indicates that there is a good correlation between the skin reaction of this diagnostic agent in living organisms and the in vitro immune response represented by the lymphocyte blastogenesis reaction. This can be said to further clarify its character as a specific antigen.

The toxicity of Substance D used in the present invention is extremely weak, and when subcutaneously injected into mice, the acute toxicity is LD, o value of 100.
m9/ is 9 or more, and is sufficiently safe compared to the inoculated amount.

There are several methods for using large blood vessels, such as direct inoculation during half a day to examine the reaction, or in vitro immunoassay using immunocompetent cells, but the commonly used method is the tuberculin test. A sarcastic response test is easy to perform.

When using this diagnostic agent for a sarcastic reaction test, preferably use a phosphate buffered sodium chloride solution, with a concentration of 50 to 500.
1 ml of the preparation prepared to a volume of 97 ml is inoculated into the skin of an animal or human, and local redness and induration are measured 24 or 48 hours later.

If the reaction value (average value of vertical diameter and horizontal diameter) is 1Qmrn or more, the reaction with this diagnostic agent is positive and the organism is determined to be BCG vaccine sensitized.

For example, if the initial positive result after vaccination with PPD (7) is determined to be negative, infection with Mycobacterium tuberculosis is suspected, and sufficient observation is deemed necessary thereafter.

Examples of the present invention and its usefulness will be illustrated below using test examples.

Substance D 11n9 derived from BCG cells obtained in the production example was dissolved in 21 distilled water for injection, and then 10 g of lactose was added and dissolved, and then filtered using a Nuclebore filter (α2 μm; manufactured by Nuclehore Corporation). ) was used to sterilize and filter. The obtained filtrate was aseptically dispensed into vials in 1 d portions and lyophilized to produce a diagnostic drug preparation.

BCG vaccine (Nippon BCG Manufacturing Co., Ltd.) 9 weeks before the test
subcutaneous injection of 500 μg of physiological saline suspension, liquid paraffin suspension of 200 μg of heat-killed Mycobacterium tuberculosis H37 strain A, or 200 μg of heat-killed Mycobacterium tuberculosis H37 strain A. Substance D obtained in the production example and PPD (purified tuberculin, Nippon BCG Manufacturing Co., Ltd.' was inoculated intradermally into the flank.Table 1 shows the results of measuring the local redness diameter (arithmetic mean value of longitudinal diameter and transverse diameter) after 24 hours.

Test Example 2 Sustainability test of delayed allergic reaction in BCG vaccine sensitized guinea pigs Hartley female guinea pigs, in which delayed sensitization was achieved by subcutaneous injection of 500 μg of BCG vaccine suspension in physiological saline, were divided into 3 groups. As a group of 6 animals, after sensitization 6,
Substance D obtained in the production example and PPD in each week of 12.24
(Purified tuberculin, manufactured by Nippon BCG Manufacturing Co., Ltd.)
was inoculated intradermally into the flank. Table 2 shows the results of measuring the diameter of local redness after 24 hours.

Test Example 3 Lymphocyte rejuvenation test A test was prepared in the same manner as in Test Example 1, and 9 BCG vaccine sensitized guinea pigs and 2) W Mycobacterium tuberculosis blue and white 8 peripheral sensitized guinea pigs were used. The lungs of each guinea pig were removed aseptically using the method of Onozaki et al.
(1937, 1977) prepared a tile cell suspension. This was suspended in RPM11640 medium containing 10% tallow serum at a concentration of 2 x 10'/mA.
Dispense into well culture plates and apply to each well. ! 1) 3X10'
Individual cells were used. Add to this substance D or PP obtained in the reference example.
After adding D (P-TB reagent, manufactured by Mitsui Pharmaceutical Industries, Ltd.) as an antigen, 5 qA Co! Cultured at 67°C for 4 days in an incubator, and 0.5 hours before the end of culture.
μC1 sH-TdR was added. Hereafter, Ohno et al.'s method (
Immunology Experimental Procedures V, edited by the Japanese Society of Immunology, 1525.19
(1976), the radioactivity taken up into multicellular cells was measured.

The results are shown in Table 6.

The reaction value was calculated using the following formula.

Table 6 Lymphocyte rejuvenation test substances 0.2 9.0 0.9 1 18.6 1.9 5 21.0 2.7 25 26.5 4.4 1) PD 10 80.0 29.2 50 915 27.4 D obtained in the production example was given to 6 week old female ddY mice (10 mice per group).
Dissolve the substance in physiological saline and calculate the amount per kg of body weight, Ii o
Oda was administered subcutaneously. This substance did not show any inhibition of body weight gain during the observation period of 1 s after administration, and caused death.
．． LD, which can be ridden with one+-.

is 100 to 9 or more.

[Brief explanation of drawings]

Fig. 1 is an ultraviolet absorption spectrum diagram of substance D (11v/d distilled water), and Fig. 2 is a Kutle diagram of infrared absorption spectrum of substance UD. Agent Patent Attorney 1) Parent Male 1st Diagram Wavelength (nm)

Claims (1)

[Claims] A tuberculin diagnostic agent characterized in that it contains substance D derived from BCG bacteria as an active ingredient and induces a specific reaction in a BCG vaccine-sensitized organism.