JPS60209179A - Latex reagent sensitized with pneumonitic mycoplasma lipid antigen-lecithin composite and detection of pneumonitic mycoplasma antibody using said reagent - Google Patents

Abstract

PURPOSE:To diagnose the pneumonitic mycoplasma with a simple operation by conjugating pneumonitic mycoplasma lipid antigen-lecithin composite with latex particles. CONSTITUTION:The lipid antigen composite is easily formed by culturing pneumonitic mycoplasma, gathering bacteria, extracting the same with an org. solvent such as chloroform and adding lecithin prepd. into an ethanol soln. to such antigen. The latex used for producing the sensitized latex is latex consisting of a synthetic polymer such as polystyrene or the like and has preferably about 0.2+ or -0.005mu grain size. The lipid antigen-lecithin complex is deposited on the latex particles by bringing about 0.05-5% concn. of latex particles and the lipid antigen-lecithin composite into contact with each other for 30min-24hr at about 4-40 deg.C in a buffer soln. kept at about 6.5-8pH. One drop of the serum or the like of a person to be inspected is dropped on slide glass and one drop of the sensitized latex is dropped thereto, then the slide glass is slowly moved for 2-3min. Distinct agglutination is observed if the pneumonitic mycoplasma antigen exists.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel Mycoplasma pneumoniae lipid antigen lecithin complex sensitized latex and the ability and method for detecting Mycoplasma pneumoniae antibodies using the same. The present invention relates to a sensitized latex for passive latex agglutination reaction of Mycoplasma pneumoniae bound to substances, and a method for detecting the above-mentioned antibodies using the same.

Mycoplasma pneumoniae is the pathogen that causes mycoplasma pneumonia, which is the most common type of atypical pneumonia in adults, but there are no specific findings in acute respiratory infections such as atypical pneumonia caused by this mycoplasma, and therefore viruses and It cannot be clinically distinguished from other respiratory diseases caused by fungi.

Therefore, serological diagnostic methods such as cold agglutination reaction, metabolic inhibition reaction, and complement fixation reaction are currently used for this diagnosis. However, among these, although the metabolic inhibition reaction is superior in specificity, it has the disadvantage of requiring a long period of 5 to 7 days because it involves culturing operations, and the cold agglutination reaction, although easy to operate, The specificity is poor, and the complement fixation reaction requires complicated operations and time, both of which are unsatisfactory.

In view of the current situation, the present inventor conducted extensive research in order to overcome the above-mentioned drawbacks of conventional serological diagnostic methods, and as a result, developed a method using latex in which a complex of the lipid antigen of the bacterial cell and lecithin was combined. The present invention was completed based on the discovery that mycoplasma pneumonia can be specifically diagnosed in a very simple and easy reaction in just a few minutes by performing a slide agglutination reaction on a slide glass.

Therefore, an object of the present invention is to provide a sensitized latex useful for diagnosing various Mycoplasma pneumoniae infections, including Mycoplasma pneumoniae, in which a Mycoplasma pneumoniae lipid antigen lecithin complex is bound to latex particles.

Another object of the present invention is to provide a method for detecting antibodies to Mycoplasma pneumoniae using the sensitized latex.

The lipid antigen used in the present invention is obtained by extraction from Mycoplasma cells with an organic solvent. That is, as shown in the reference example below, Mycoplasma pneumoniae (Mycoplasma pneumoniae)
coplasma pneumoniae) (e.g. M
ac strain) and collect the bacterial cells, which were mixed with chloroform,
Extract with an organic solvent such as methanol, ethanol, or ether, particularly preferably a chloroform-methanol mixture, and prepare an ethanol solution. This lipid antigen can be stored stably for a long period of time if kept at a low temperature of 4°C or less. When lecithin in an ethanol solution is added to this antigen, a complex is quickly and easily formed at room temperature. Egg yolk lecithin is particularly preferred as the lecithin used here.

The latex used to produce the sensitized latex of the present invention is polystyrene, carboxylated polystyrene, carboxylated polystyrene having an amino group, polyvinyltoluene, styrene-butadiene copolymer, carboxylated styrene-butadiene copolymer, styrene-divinyl Benzene copolymer, vinyltoluene-tert-butylstyrene copolymer, polyester, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, acrylonitrile-butadiene-styrene copolymer, polyvinyl acetate acrylate, polyvinylpyrrolidone, vinyl chloride-acrylate The latex is made of synthetic polymer latex particles such as copolymers, and the surface of these synthetic polymer latex particles may be treated with a nonionic surfactant or the like. Among the synthetic polymer latexes mentioned above, polystyrene latex is particularly preferred. The particle size of the latex particles is usually 0.1-10g, preferably 0.1-1. Regarding OIL, in order to improve the reproducibility of analytical test results, it is desirable to have a narrow particle size distribution, for example, 0.2±0.005 Ji.

In order to support the lipid antigen lecithin complex on the latex particles, the latex particles and the lipid antigen lecithin complex at a concentration of 0.05 to 5% are mixed in a buffer solution at pH 5 to 1O1, preferably at pH [i, 5 to 8]. This is carried out by contacting at 40°C for 30 minutes to 24 hours with gentle stirring. As the buffer solution, for example, phosphate buffered saline or glycine buffered saline is used. After the reaction is completed, the latex particles are washed several times by centrifugation in a near-neutral salt solution such as glycine buffered saline or phosphate buffered saline (hereinafter referred to as rPBsJ), and finally, the latex particles are suspended in a diluted solution and stored. . The diluent used is glycine buffered saline, PBS, etc. with approximately 0.1% bovine serum albumin (hereinafter referred to as rBSAJ), and 0.01-0.5% sodium azide (N
Add aN3).

To diagnose mycoplasma pneumonia using the sensitized latex of the present invention obtained as described above, one drop of serum, pleural effusion, etc. from the patient to be examined is placed on a slide glass, and the latex described above is added to the sensitized latex of the present invention. Add one drop of the solution, shake slowly for 2 to 3 minutes, and visually judge whether or not there is aggregation of the latex. Clear agglutination is observed if Mycoplasma pneumoniae antibodies are present. If you want to determine the quantitative value of this antibody, first dilute the sample serum with physiological saline in a test tube, perform an agglutination reaction on each diluted serum, and calculate the reciprocal of the maximum dilution factor of the serum that shows agglutination. value.

As described above, antibodies can be specifically detected by extremely simple means with a reaction time of only 2 to 3 minutes. Furthermore, since the carrier is latex, antibodies against the carrier are not present in human blood, so the raw serum can be used as is for diagnosis without pre-treatment of the test serum with the carrier.

As described above, the present invention is a slide agglutination reaction using a lipid antigen lecithin complex sensitized latex, which has never been used in the diagnosis of mycoplasma pneumonia. They were able to create a sensitized latex that could be used in the reaction.

The composite prepared by adding lecithin to mycoplasma lipid antigen is unprecedented and completely new, and the slide agglutination reaction for detecting antibodies to Mycoplasma pneumoniae is also completely new.

That is, with this latex, it is possible to specifically diagnose mycoplasma pneumonia with a reaction of just a few minutes using a groundbreaking simple operation, which has extremely great significance from a public health and clinical perspective.

Next, reference examples and examples will be given and explained.

Reference example Preparation of lipid antigen: Mycoplasrrra pneumoniae M
The ac strain was cultured with shaking in mycoplasma medium at 36°C for 2 weeks. This culture solution was centrifuged at 30.00 rpm/80 minutes to collect bacterial cells, which were thoroughly washed with PBS and extracted by vigorous shaking with chloroform-methanol (3:1). The bacterial cells were removed by high-speed centrifugation, and the solvent was removed from the extract under reduced pressure to obtain mycoplasma lipid antigen. This lipid antigen was dissolved in 1/100 volume of ethanol of the culture medium to obtain a lipid antigen solution. This product can be stored stably at temperatures below 4°C.

Example I To 10 volumes of PH, a mixture of 1 volume of the above lipid antigen (complement fixing antigen titer 320 units) and 1 volume of a 10 mg/m egg yolk lecithin solution was added. Add an equal amount of 2 to this complex solution (PH7,2)
% latex (Japan Synthetic Rubber Co., Ltd., GOIOI; particle size 0.72±o, oo5 JL) and sensitized at room temperature for 2 hours. During this time, slow mixing was performed approximately every 10 minutes. This sensitized latex was washed three times with PBS (PH7,2) and diluted to 0.5% (BSAo, 1% in PBS).
% and 0.1% sodium azide).

Example 2 Using the latex obtained in Example 1, a slide agglutination reaction was performed on patient serum.

Patient serum was diluted multiple times with physiological saline in a small test tube.
Then, one drop at a time was applied onto a slide glass. Add latex one drop at a time and shake slowly for 3 minutes.
Aggregation was determined visually. The results are shown in the table, and the reciprocal of the maximum dilution factor of serum showing agglutination was taken as the antibody titer. As shown in the table, high antibody titers were observed only in patients with mycoplasma pneumonia, and the method also showed higher sensitivity than the complement fixation method. Furthermore, no cases of agglutination were observed in normal subjects.

In other words, Shinmachi believes that this method is more sensitive than the complement fixation reaction method, has an extremely short reaction time of 3 minutes, is extremely easy to operate, and is extremely suitable for detecting and diagnosing antibodies in patients. .

Wood complement fixation reaction method *Thursday Passive hemagglutination method

Claims (2)

[Claims] (1) A latex sensitized with a Mycoplasma pneumoniae lipid antigen lecithin complex, characterized in that it contains latex particles carrying a Mycoplasma pneumoniae lipid antigen lecithin complex on its surface. (2) Contact the test sample with the Mycoplasma pneumoniae lipid antigen lecithin complex sensitized latex containing latex particles carrying the Mycoplasma pneumoniae lipid antigen lecithin complex on the surface on a slide glass, and observe the agglutination image. Characteristic method for detecting antibodies against Mycoplasma pneumoniae.