JPS60209181A - Anticandida polysaccharide antibody sensitized blood cell - Google Patents

Abstract

PURPOSE:To obtain PS antibody sensitized blood cells by conjugating anticandida polysaccharide antibody (PS antibody) via a coupling agent with red blood cells. CONSTITUTION:The red blood cells to be used for sensitized blood cells are preferably fixed red blood cells in terms of preservation and are formed by treating the raw red blood cells suspended by an alsever liquid with gaseous carbon monoxide and adding formalin thereto to fix the cells. A tannic acid, etc. are used for a coupling agent. The bacteria for candida are obtd. by inoculating the same in the Sabouraud's broth, etc. and culturing the same at about 37 deg.C. The anticandida bacterium antibody is obtd. of the bacteria killed by a heating treatment, etc. and are immunized with a rabbit, etc. A polysaccharide antigen is then mixed with the anticandida antibody and the precipitate of only the immune complex is made. The precipitate is centrifugally dispensed and an acetic acid buffer soln. is added thereto, to which a satd. ammonium sulfate soln. is added to settle the antibody. The PS antibody is refined by centrifugal sepn., dialysis, etc. The red blood cells are treated by using the coupling agent and the liquid contg. the PS antibody is brought into contact therewith, by which the PS antibody sensitized red blood cells are obtd.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to anti-Candida polysaccharide antibody-sensitized blood cells. More specifically, the present invention relates to sensitized blood cells sensitized with specifically purified anti-Candida polysaccharide antibodies.

Candida, especially Candida albicans
A albicans) is a type of yeast-like fungus that is resident on human skin, oral cavity, etc., and it is well known that it sometimes causes opportunistic infections in humans. In particular, candidiasis secondary to leukemia or chronic diseases can become serious, and has become one of the infectious diseases that has received attention in recent years. However, candidiasis has no clinically specific symptoms, making it difficult to make a definitive diagnosis. Furthermore, even if this bacterium is isolated, it is difficult to differentiate between a commensal bacterium and an infectious disease, and a high proportion of normal people have antibodies derived from subclinical infections, so unless a particularly significant value is shown, Measuring antibody titers also cannot aid in diagnosis.

However, when Candida infection is established in humans, bacterial components are first liberated and released into the blood, causing antigenemia. It becomes possible to diagnose infectious diseases at an extremely early stage.

In this case, the antigen to be detected must be a stable substance and unique to Candida, which is present in all strains. In addition, antigens released into the blood due to infection combine with antibodies that were already present in the patient's blood before infection, forming antigen-antibody combinations, that is, immune complexes, and exist as free antigens. Therefore, it is necessary to create a system that can easily dissociate the antigen from this immune complex and use it for measurement. From this point of view, the present inventor used polysaccharides contained in all Candida, especially Annan, as an antigen. Candida contains mannan polysaccharides, which are heat-resistant and extremely stable substances that are solubilized and released into the blood after infection. Since this polysaccharide specifically binds to anti-glycoside antibodies, anti-Candida polysaccharide antibodies (hereinafter referred to as rPs antibodies) are specifically separated and purified from anti-Candida bacterial antibodies prepared by immunization. We have established a method for detecting polysaccharide antigens by sensitizing them to blood cells via a coupling agent and performing a reverse passive hemagglutination reaction, leading to the completion of the present invention. Using this method, Candida infection can be reliably diagnosed in the early stages of infection with extremely simple operations. Until now, there was no known easy practical method for detecting small amounts of antigens present in the blood, either alone or in immune complexes.
Although early diagnosis by antigen detection was almost impossible,
The invention of this diagnostic method for Candida antigenemia has made it possible to diagnose Candida infection at an early stage and to treat it based on this diagnosis.

The purpose of the present invention is to provide PS to red blood cells via a coupling agent.
The object of the present invention is to provide Ps antibody-sensitized blood cells which are bound to antibodies.

The red blood cells used for the sensitized blood cells of the present invention are bovine blood cells or fixed red blood cells, but it is preferable to use fixed red blood cells from the viewpoint of preservation of the sensitized blood cells. Bovine blood cells are obtained from sheep, goats, cows, horses, guinea pigs, chickens, turkeys, humans, etc. according to conventional methods. Fixed red blood cells are prepared as follows. That is, Alsepar
ver) Bovine blood cells suspended in a solution are treated with carbon monoxide gas, and formalin is added to the red blood cells for fixation.

As the coupling agent, tannic acid, glutaraldehyde, chromium chloride, etc. can be used, and among them, tannic acid is preferred.

If Candida is inoculated into a Sabouraud medium or the like and cultured at about 37°C, a large number of cells can be obtained in one day.

Anti-Candida antibodies can be easily obtained by killing the bacteria by heat treatment or formalin treatment, and immunizing rabbits, goats, etc. with the bacteria in a conventional manner. These antibodies also include PS antibodies.

The strain used in this case does not need to be particularly specified as long as it belongs to the desired species.

Candida polysaccharide antigens can be extracted from both live and dead bacteria. Several extraction and purification methods are known, including Gorin's method (P, A.

Gorin and J.F.Pencer; Ca
n, J, Chem,, 4f(.

2299-2304.19 [18].

That is, after heating the microbial cells, the crude polysaccharide is obtained by heating and extracting with potassium hydroxide and precipitating with methanol, and Feilinda's solution is added to the crude polysaccharide to separate it as a copper complex.

Next, the following method is followed to separate and purify the PS antibody from the anti-Candida cell antibody. First, a bacterial antibody is mixed with a polysaccharide antigen to form a precipitate of immune complexes, and the precipitate is separated by centrifugation. This is thoroughly washed with physiological saline etc. to obtain a pure polysaccharide immune 1 complex, and when an acetate buffer of pH 2.8 is added to this, the antigen and antibody dissociate and become soluble. p to this
When a saturated ammonium sulfate solution of H2,8 is added, the polysaccharide does not precipitate and only the antibody precipitates. This is collected by centrifugation, washed with 1/2 saturated ammonium sulfate, dissolved in water, and then added to physiological saline etc. The Ps antibody is purified by dialysis to remove ammonium sulfate.

The sensitized blood cells of the present invention are produced as follows. That is, red blood cells are treated with a coupling agent to obtain coupling agent-treated blood cells (red blood cells with a coupling agent bound to the surface of the red blood cells), which are then brought into contact with a solution containing a PS antibody to produce PS antibody-sensitized blood cells. obtain.

The diluent used is glycine buffered saline, phosphate buffered saline, etc. with approximately 0.1% bovine serum albumin (hereinafter abbreviated as rBSAJ), and diluted with 0.05 to 0.5%.
of sodium azide (NaN3) is added.

The sensitized blood cells obtained in this way may be suspended in a diluent to a concentration of about 2.5% by weight and stored in an ice chamber.
(You can also freeze-dry it.) For freeze-drying, add 0% each of various amino acids, especially glycine or sodium glutamate, and dextran as stabilizers to the diluted solution.
．． Add 2 to 2% by weight and 0.3 to 3% by weight and quickly freeze in liquid nitrogen or the like, and then freeze-dry. Freeze-drying further extends the shelf life and is usually stable for more than two years.

Since the PS antibody-sensitized blood cells of the present invention are agglutinated by the Kandig polysaccharide antigen, first dilute body fluids such as human serum 4 times with distilled water, and heat this at 100°C for about 10 to 20 minutes. When the antibody portion constituting the immune complex is inactivated and the polysaccharide antigen is made into a free state, when PS antibody-sensitized blood cells are brought into contact with this solution or its diluted solution, the inside of the heated body fluid or its diluted solution is In the presence of antigen, sensitized blood cells undergo an agglutination reaction. When this reaction is carried out using the microtiter method, it is also possible to observe the aggregated image on the bottom of the tube on a microplate. That is, a certain amount of the diluent is dispensed dropwise into the plate, then a certain amount of heated body fluid is added to the first hole, and the diluent is sequentially diluted with a gilutor. Sensitized blood cells are dripped and dispensed into this, and the image of aggregation at the bottom of the tube is determined after a certain period of time. In this case, if a standard polysaccharide with an accurately known concentration is used together, it is easy to calculate the concentration in the unknown body fluid by proportional calculation from the agglutination value for this standard substance, and It can reduce body concentration. That is, quantifying polysaccharide antigens is extremely easy and simple, and does not require any special techniques. Further, since the antibody has been specifically purified, it is specific, and has sufficient sensitivity for measurement in human body fluids, and can perform qualitative and/or quantitative determination of a large number of analytes at the same time.

By using the sensitized blood cells of the present invention, the Candida polysaccharide antigen concentration in body fluids, for which there was no conventional method for detection except by radioimmunoassay, can be easily and conveniently quantified in a short time, thereby preventing Candida infection. This will enable accurate early diagnosis and appropriate treatment from an early stage.

Currently, there is no known practical method for diagnosing Candida antigenemia, and there are no documents describing PS antibody-sensitized blood cells, and the Candida antigen agglutination reaction using PS antibody-sensitized blood cells is completely new.

In addition, these sensitized blood cells react only to Candida polysaccharides and do not react at all to other antigens or plasma proteins, and unsensitized blood cells do not react to Candida polysaccharides at all.
It can be said that the PS antibody is bound to these sensitized blood cells.

Next, the present invention will be explained in more detail with reference to Preparation Examples and Examples, but the present invention is not limited thereto unless it exceeds the gist thereof.

Preparation Example 1 Preparation of Candida cells Candida φ albicans was inoculated into Sabouraud liquid medium,
Shaking culture was performed at 37°C for 24 hours. This culture solution is 3.0
Centrifuge at 0 rpm for 30 minutes to sediment and collect the bacterial cells.
After washing three times with physiological saline, the cells were suspended in physiological saline and heated in a 100°C water bath for 30 minutes to kill the bacteria. The cells were collected by centrifugation, washed twice with distilled water, and dehydrated with acetone to obtain dry cells.

The cells of Preparation Example 1 were suspended in physiological saline to a concentration of about 108/ml, and this bacterial solution was injected once into the ear vein of a rabbit weighing about 2.5 kg. Immunization was carried out by injecting each OmJL 10 times every two days. One week after the last immunization, whole blood was collected from the carotid artery. Serum was separated according to the standard method and heated at 56°C for 30 minutes to prepare anti-Candida bacteria antibodies. I got it.

Inoculate Candida albicans into Sabouraud liquid medium,
After culturing at 37°C for 48 hours, the cells were collected by centrifugation, washed three times with pure water, made into a 10% suspension with pure water, and heated at 100°C for 60 minutes. Centrifuge this to collect the bacterial cells, and
Add 0 times the volume of 2% potassium hydrate solution and bring to 100°
After heating at C for 2 hours, the pH was made neutral with acetic acid, and the supernatant fraction was collected by centrifugation. This supernatant was concentrated under reduced pressure, then 4 times the volume of methyl alcohol was added, left to stand overnight, centrifuged to collect the precipitate, washed 3 times with methyl alcohol and then 3 times with acetone, dried, and the crude A polysaccharide was obtained.

This crude polysaccharide was made into a 2% aqueous solution and heated at 100'C.
It? After heating for a while, add an equal amount of Fehling's solution, leave it in an ice chamber overnight, centrifuge to collect the precipitate, and wash it three times with 2% potassium hydroxide solution and three times with methyl alcohol.
1R120 was added, stirred well to mix, and the mixture was centrifuged to collect the supernatant. After concentrating this under reduced pressure, 4 volumes of methyl alcohol were added to precipitate the resulting precipitate, which was washed three times with methyl alcohol and once with acetone, dried, and made into an aqueous solution to obtain mannan.

The polysaccharide prepared in Preparation Example 3 was added to 40 mJL of the anti-Candida bacterial antibody prepared in Preparation Example 2 until a precipitate was formed. The resulting immune complexes were collected by centrifugation at 3.00 rpm for 30 minutes and washed 3 times with physiological saline. pH 2.8
, 0, LM acetate buffer, dissolved in 10 ml, pH 2,
10+J1 of a saturated ammonium sulfate solution adjusted to 8 was added. After mixing with a stirrer for 1 hour at room temperature, 1
Only the antibody was precipitated by centrifugation at 0.00 rpm for 80 minutes, and this precipitate was washed three times with 1/2 saturated ammonium sulfate solution. This was dissolved in 5 mfL of leather distilled water and dialyzed against physiological saline for 18 hours to remove ammonium sulfate. During this time, the external solution was replaced twice. After dialysis, 10. ．． 0
Centrifuge at 00 rpm for 60 minutes to remove insoluble matter, and transfer the supernatant to PS
It was used as an antibody. This antibody produced a single sedimentation line with candida mannan polysaccharide using the Octarlony method, proving that it was a single antibody.

Alsepar was added to fresh blood obtained from chickens.
ver) solution, and after blowing CO gas in the flask for 30 minutes, immediately add the fixative solution [physiological saline with 5% sodium citrate + 37% formalin (volume ratio 29
:1)] was added in an equal amount, and the mixture was left in an incubator at 37°C for 24 hours, with occasional shaking during this time. Thereafter, it was washed five times with pure water and five times with physiological saline. Sodium azide 0.1%
The fixed red blood cells were suspended in 7.2 p) of phosphate buffered saline at a concentration of 10% and stored in an ice chamber. This fixed red blood cell was stable for over 1 year.

Proboscis - tongue 2.5% in phosphate buffered saline (pH 7,2) (hereinafter abbreviated as "rPBS") containing the fixed red blood cells obtained in Preparation Example 5 1: too, ooo tannic acid/PBS
After adding an equal amount of the following, tannic acid treatment was performed at 37° C. for 15 minutes, the cells were washed once with PBS, and suspended in an equal amount of PBS to prepare a tannic blood cell solution. An equal amount of the t:teo PS antibody obtained in Preparation Example 4 was added to this tannin blood cell solution, treated at 37°C for 30 minutes, and then incubated once with FBS.
BSAo, 1%, NaN30. i%
The sample was washed once with a diluted solution to which 100% of the sample was added, and then suspended in the diluted solution.

These sensitized blood cells aggregated with Candida polysaccharide on a microplate, but did not aggregate even when Cryptococcus polysaccharide was added. -Also, tannin blood cells that are not sensitized to PS antibodies did not aggregate even when Candida polysaccharide was added under the same conditions. That is, it was confirmed that these sensitized blood cells specifically reacted to Candida polysaccharide and aggregated.

Example 2 ＾ Sphere - The reaction was carried out by the microtiter method, and first, the diluted solution was dispensed into a plate in 0.025 ml portions using a dropper.

0.025m of 1:4 heated serum for the first dose! l added. Diluted 21 with Guiluta. Standard polysaccharides were also diluted in the same way. After adding 0.025 al of sensitized blood cell fluid dropwise and leaving it in a mixer at room temperature for 1 hour or more, evaluation was performed. The polysaccharide concentration was calculated by comparing the aggregation end point of the standard polysaccharide and the agglutination end point of the sample.

The results of measuring the polysaccharide concentration in human serum using this method are shown in the table below.

As is clear from the above results, blood polysaccharides were detected only in patients suspected of having Candida infection.

In other words, measuring the blood polysaccharide concentration is extremely useful for early diagnosis of Candida infection. It can also be applied to determining the extent of lesions.

By using the sensitized blood cells of the present invention, polysaccharide antigens can be extremely easily quantified using only a trace amount of a sample of about 0.025m, and moreover, by simply heating the sample, so it can be said that it is particularly suitable for clinical diagnosis.

Claims (4)

[Claims] (1) Anti-Candida polysaccharide antibody-sensitized blood cells comprising an anti-Candida polysaccharide antibody bound to red blood cells via a coupling agent. (2) The sensitized blood cells according to claim 1, wherein the red blood cells are fixed red blood cells. (3) The sensitized blood cell according to claim 1 or 2, wherein the Candida polysaccharide is mannan. (4) The sensitized blood cell according to any one of claims 1 to 3, wherein the coupling agent is tannic acid.