JPS6097273A - Measurement of syphilis antibody - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] It is about the method.

It is known that IgM antibodies appear in the early stages of infection, and IgG antibodies increase in the later stages of infection. Various methods are known for measuring specific antibodies such as IgM antibodies and IgG antibodies for syphilis, and one of them is the solid phase hemoadsorption method (SPHA method, Schmidt B.L., Sex.
Transf-Dis-, Vol. 7, pp. 53-5.8 (
(1980)). This method has the advantage of being easy to operate, but has significant measurement errors, such as the one reported by Muller et al. (Muller F, Lindensch
midt B-G XBr. J.

Vener. Dis. 5 Volume 8, 1 2-1-7
(1982), 55% of patients with early syphilis
thing fal senan - reactive is h
It's called Tsuda.

The present inventors conducted various studies in order to develop a method to easily and accurately measure these specific antibodies of syphilis.
If the above-mentioned SPHA method is performed using
The present invention has been completed based on the discovery that alse non-reactive can be almost eliminated and the above object can be achieved.

That is, the present invention involves contacting a solution containing a Treponema ballidum-specific antibody to be measured with the wall of a reactor to which anti-human immunoglobulin animal immunoglobulin obtained when an animal is immunized with human immunoglobulin is reacted. In the method of measuring Treponema vallidum-specific antibodies by contacting the wall surface with particles sensitized with an antigen derived from Treponema vallidum bacteria and causing an antigen-antibody reaction after removing the solution after the reaction, The present invention relates to a method for measuring Treponema noglydum-specific antibodies, which is characterized by using a culture of Treponema noglydum from which both components with a specific gravity of 1.01 or less have been removed.

The material of the reactor is one capable of adsorbing immunoglobulin, such as reed, such as polystyrene, glass, acrylic resin, and polyvinyl chloride. The shape of the reaction container may be a microplate, a test tube, a flat plate, etc.

The immunoglobulin adsorbed to the reactor is an anti-human immunoglobulin animal immunoglobulin obtained by immunization with zeta immunoglobulin. The animal may be any conventional animal used to obtain immunoglobulins, such as rabbits, goats, etc.
These include horses, chickens, and mice. In principle, one type of human immunoglobulin is used, but the type is not critical and may be any of rgMllgG, IgA, IgE, and IgD. This anti-human immunoglobulin animal immunoglobulin may be used as is from immune serum collected from animals, but it is usually used after purification.

Adsorption may be carried out by natural adsorption, such as by placing a solution containing anti-human immunoglobulin and animal immunoglobulin in a well of a microplate, bringing the anti-human immunoglobulin and animal immunoglobulin into contact with the wall of the reactor, and then heating at about 0 to 20°C. Just leave it for the required time. After adsorption, the reaction wall surface should be washed thoroughly if necessary.

This reaction wall usually still has a portion that has protein adsorption activity, and since this portion may cause a non-specific reaction during measurement, it is preferable to adsorb proteins such as BSA and NR8. In the conventional SPI (A method), the solution containing the Treponema varidum-specific antibody to be measured was diluted with a non-specific reaction absorption solution after blocking with a protein, but the present inventors It was discovered that by coexisting a blocking protein, si-blocking and absorption of non-specific reactions can be performed simultaneously, and as a result, the reaction time can be cut in half.

The wall surface of this reactor is brought into contact with a solution containing a Treponema ereadum-specific antibody to be measured and allowed to react. The Treponema no.4reedum-specific antibody to be measured is an antibody against an antigen derived from Treponema ballidum,
In the method of the present invention, specific antibodies are selectively measured depending on the type of animal-derived immunoglobulin adsorbed to the reactor.

That is, when it is desired to measure only IgM antibodies against Treponema ballidum, anti-human IgM animal immunoglobulin obtained when an animal is immunized with human 11gM may be used. Although the type of solution containing the antibody to be measured does not matter, it is usually a test serum and a series of dilution solutions obtained by diluting the serum to a ratio of 1:1. It goes without saying that in the case of other materials, pre-treatments such as pH adjustment, concentration adjustment, centrifugation, etc. are performed as necessary.

Normally, the contact is carried out at 37°C for 2 hours, but in the above-mentioned method in which the blocking protein is allowed to coexist with the non-specific reaction absorption solution, the contact is completed after about 1 hour at 37°C.

After the reaction, this solution is removed from the reactor, and the reaction wall is washed with physiological saline solution or the like.

On the wall of this reactor, Treponema glidum (hereinafter referred to as
It's called TP. ) Particles sensitized with bacterial antigens are brought into contact to cause an antigen-antibody reaction. The method of the present invention is characterized in that a TP bacterial culture from which both parts with a specific gravity of 101 or less are removed is used as the antigen.

Any method may be used to obtain the TP antigen, but for example, the method commonly used in the TPHA test may be used as is. As the TP inoculum, for example, the WHO pathogenic standard Nichols strain or the TP bacteria used by various testing institutions for syphilis testing may be used as is.

In addition, the wHO pathogenicity standard Nichols strain is, for example, CDC (C
enter for Disease Control
)Public HealthSe)vices U9
．． S-, Department of ) (ealt
h,gducationandWelfareXAt
Lanta, Georgia). As a method for culturing and collecting TP bacteria, for example, the method of Miller et al. (Miller et al.
of Immunology Vol, 96, p 4
50 (1966)). In other words, T. aeruginosa was grown from minced pieces of Kosomaru after culturing in a solvent containing inactivated rabbit serum and 1/7M saline in equidistant conditions.
Extract P bacteria. The extract is centrifuged at 200 x 9 for 10 minutes to precipitate and remove large particles of the rosacea tissue. then 1
Centrifuge at 9000Xp for 90 minutes to precipitate TP bacteria.

The resulting precipitate is washed three times with cold 0.075 M sodium oxalate. The method for culturing TP bacteria and the method for collecting bacteria from the culture in the present invention are not limited to these examples, and any known methods can be adopted.

Conventionally, the washed precipitate is suspended in an appropriate buffer solution,
Immediately remove T by homogenizer treatment, ultrasonication, etc.
The P cells were crushed and used as a stock solution for carrier sensitization. In the method of the present invention, a step is added to separate and remove impurities other than TP bacteria in the antigen sensitizing solution with a specific gravity of 1.01 or less by some method before or after crushing the bacterial cells. The unique feature of this method is that the TP antigen was used as the sensitizing stock solution.

The method for separating and removing impurities may be appropriately selected from among the methods known for general molecular weight fractionation, such as density gradient method, gel filtration method, centrifugal sedimentation method, ultrafiltration method, ammonium sulfate fractionation method, The fraction may be removed by electrophoresis or the like.

Among these, density gradient methods and centrifugal sedimentation methods are particularly suitable for this purpose. This separation method is not limited to the molecular weight fractionation method, but may also be, for example, a method in which TP bacteria are selectively adsorbed to a solid-phase TP antibody through an antigen-antibody reaction. In the present invention, the components with a specific gravity of 1.01 or less need only be removed to the extent that they do not inhibit the antigen-antibody reaction between the antigen-sensitized carrier and the syphilis I and p-M antibodies, and are not necessarily completely removed. Does not need to be removed. In addition, although the purpose of the present invention can be achieved whether this removal u is performed before or after crushing the TP cells, industrially it is most preferable to fractionate by a density gradient method before crushing the cells. .

In the present invention, impurities with a specific gravity of 1.01 or less are removed from the TP bacterial culture, but from the viewpoint of ensuring sufficient sensitivity of the antigen-sensitized carrier, impurities with a specific gravity of 1.05 or less are removed. It is more desirable to remove the fraction having a specific gravity of 1.20 or more. In the conventional TPHA manufacturing method, the TP axillary solution obtained by extracting and washing the whole rabbit carcass, that is, the TP antigen stock solution, contains 109 TP bacteria and more than 90.6 m9 of protein, but the protein obtained by the method of the present invention Since a large amount of impure protein has been removed from the purified antigen stock solution, the protein content per 109 TP bacteria is 0.3 to less than 0.1 m9, and is usually less than 0.1 m9. In addition, the protein concentration in this case was determined by the Lowry method (Lowry et al.
J, Biol, chem, %Vo1.193, pp
265-275 (1951)), and the number of TP bacteria was measured using a bacterial counter (C, A, Hauser & Son).
The sample solution was taken on a plate (manufactured by MITSUMI CORPORATION) and directly counted using a dark field microscope.

The TP cells may be disrupted according to conventional methods for preparing TPHA reagents, such as homogenizer treatment, ultrasonic treatment, surfactant treatment, enzyme treatment, autolytic method, freeze-thaw method, and the like. When the antigen is purified before the bacterial cells are disrupted, the TP bacterial fraction from which impurities have been removed is suspended in the necessary buffer solution and centrifuged to wash the bacterial cells, and then the cells are disrupted.

The disrupted bacterial cells are diluted with a buffer or the like if necessary, and then sensitized to carrier particles such as Hisso red blood cells by a conventional method. In addition, carrier particles include red blood cells of various animals such as chicken red blood cells, and gelatin particles that have recently been newly developed
153658, etc.) or latex, or any other material that can be used for indirect agglutination reactions based on antigen-antibody reactions.

The antigen-antibody reaction may be determined in the same manner as the known 5PHA method, and is usually determined visually.

The method of the present invention is false non-reactive.
This method dramatically improves measurement accuracy by eliminating most of the problems, and also improves sensitivity and reduces the amount of antigen used.

Examples are shown below.

Example: WHO pathogenicity standard Nichols strain Treponema e varitum 6, OX 1, provided by the National Institute of Preventive Health.
0'7m13 of suspension per rabbit, 1 m
1 was inoculated. After culturing for 12 days, 10 rabbits and Shigomaru were collected, minced, and mixed with inactivated rabbit serum and 1/7M.
The grown TP bacteria were extracted using an extraction solvent of 10100 O mixed with saline. The extract was centrifuged for 200410 minutes, the precipitate was removed, and the supernatant was further filtered for 1. 9000xg
The mixture was centrifuged for 90 minutes to precipitate the TP bacteria. The resulting precipitate was washed three times with 0.075M cold sodium oxalate/10mA of 1/15M phosphate buffer, pH 6.4! After calculating the number of bacteria, the cell number was adjusted to 2 x 109/ml, and this was used as a crude TP stock solution.

Sodium Diatrizoate was placed in a 15 ml cellulose cup.
) 60.0%, 37.5%, 250%, 19.0%
A density gradient was created in the order of (both W/V%), and finally 1 ml of the crude TP stock solution was layered. This layered product was centrifuged at 20° C. for 2 hours at 25.00 Or, p, m (7,6000×g) using a Meese ultracentrifuge (5uper 5peed 65).

After centrifugation, 1 ml of fractions were added from the top of the layer to 15 fractions.

Tl) bacterial count of crude TP stock solution, protein concentration, and bacterial count of each of the above fractions (triangle mark), density (white circle mark), protein concentration (×
), TPvC original activity (black circle) was measured, and the results shown in the figure were obtained. The measurement method used is abbreviated below.

Bacterial count: The test liquid was placed on a bacteria S1 counter manufactured by CA-Hausser & son, and counted using a dark field microscope.

The test solution was weighed into a pipette with a density of 100 μt.

Protein concentration was measured by the Lowry method. That is, potassium sodium tartrate and a sodium carbonate solution containing copper sulfate were added to the sample, and the mixture was allowed to react at room temperature for 10 minutes or more, then Folin's reagent was added, and the color was compared at 750 nm after 30 minutes or more had elapsed.

Antigenic activity: The test solution was subjected to ultrasonic treatment to destroy TP bacterial cells, and then the treatment was carried out according to the procedure.

(1) Take 25 μt of each antigen fraction onto the left end of the microtray.

(2) Syphilis antibody diluted to a certain concentration is used as a diluent (
Dilute the antigen obtained in step 1) on the tray.

(3) Incubate at 37°C for 30 minutes. At this time, the antibody in the well on the left side of the tray will bind to the antigen and be consumed, but the antibody in the well on the right side, where the antigen concentration is low,
remains unconsumed.

(4) Sensitized blood cells are added to the well, and the antigen activity is determined by the boiling in the well that causes an agglutination reaction with the remaining unconsumed antibodies.

In the figure, the fractions with high TP bacteria concentration and low protein concentration, ie, fractions 4 to 7, were combined to obtain a purified antigen stock solution], Ome. The number of TP bacteria in this stock solution was 1.91 x 10', and the protein content was 0.116. The number of TP bacteria in the crude antigen stock solution was 2.0 x 109, and the protein content was 1,213~.

Therefore, the protein content of 109 TPs is 0.607 for the unpurified antigen stock solution and 0.058 m for the purified antigen stock solution.
It was 9.

Using this purified antigen stock solution, sheep red blood cells were sensitized by the tannic acid method to obtain TP antigen-sensitized blood cells.

Using the thus obtained TP antigen-sensitized blood cells, SPHA
Measurements were carried out using the method.

if, 1 gM of anti-Human in each well of a U-shaped microplate.
Rabbit r-globulin (μ chain specific, Dak.

50 µt of a 400-fold diluted solution (manufactured by Co., Ltd.) with a physiological saline solution was added to each well, and the gamma globulin was allowed to adsorb onto the well walls by standing overnight at 4°C. The microplate was washed three times with tap water, and 25 μL of a non-specific reaction absorption solution for TPHA containing 1% BSA was added to each well. 25 μt of untreated early stage syphilis patient serum was placed in the left end of the well, and diluted 2n using an automatic dilator. 1 at 37℃
After incubating for an hour, the microplate was washed three times with tap water.

The TP antigen-sensitized blood cells and TPHA non-specific reaction absorption solution were suspended at 0.18%, and 50 μt of this suspension was added to each well, left overnight at room temperature, and the agglutination image was visually determined the next day.

On the other hand, for comparison, measurements were carried out in the same manner using commercially available TP antigen-sensitized blood cells.

The results obtained are shown in the table below.

Method of the present invention 54 1 Conventional method 3520

[Brief explanation of drawings]

The figure shows the results of measuring the number of bacteria (triangle mark), density (white circle mark), protein concentration (x mark), and TP antigen activity (black circle mark) for each fraction obtained by fractionating the crude TP stock solution using the density gradient method. There is. Patent applicant Fujirebio Co., Ltd. Agent Patent attorney 1) Masahiro Naka

Claims (1)

[Claims] Anti-human immunoglobulin obtained when an animal is immunized with human immunoglobulin A solution containing a Treponema flJ-dam specific antibody to be measured is brought into contact with the wall of a reactor to which animal immunoglobulin has been adsorbed, and reacted. After removing the solution after the reaction, in the method of measuring Treponema no. 4 leadum-specific antibodies by contacting the wall surface with particles sensitized with an antigen derived from the Treponema golium bacterium and causing an antigen-antibody reaction, Treponema i
A method for measuring Treponema bulidum-specific antibodies, characterized by using a culture of R. bulidum from which both parts with a specific gravity of 1.01 or less are removed.