JPH0230668B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing freeze-dried sensitized blood cells. In recent years, insulin, thyroid hormone, α-fetoprotein (hereinafter abbreviated as AFP), human placental gonadotropin (hereinafter abbreviated as hCG), HB
BACKGROUND OF THE INVENTION It is widely practiced to measure trace amounts of biological components secreted in urine and body fluids, such as antigens and antibodies, and to use the results for clinical diagnosis or disease prognosis. However, since all of these biological components are in trace amounts, it is difficult to measure them using ordinary chemical analysis, and immunochemical methods are generally used, especially radioimmunoassay (hereinafter referred to as RIA method), which has extremely high measurement sensitivity. (hereinafter referred to as EIA method) and enzyme immunoassay (hereinafter referred to as EIA method). However, the RIA law,
In addition to the EIA method, which has complicated operations and lacks simplicity and speed, the RIA method requires expensive equipment to obtain, manage, and measure isotopes. In this respect, passive hemagglutination (hereinafter abbreviated as PHA), which uses blood cells as carriers, does not require any special measurement equipment and is also superior in terms of operational simplicity, speed, and economy. Moreover, in recent years, improvements in blood cell processing methods, sensitization methods, and antigen and antibody purification methods have significantly improved the sensitivity of the PHA method.
The sensitivity has become comparable to the RIA method. Generally, the sensitized blood cells used in the PHA method are blood cells collected from mammals or birds in their natural state, or after being treated with an appropriate fixative such as glutaraldehyde, pyruvaldehyde, or formaldehyde, and then further treated with tannic acid, bis-diazobenzidine, etc. After treatment with a binding agent, the corresponding antigen or antibody can be sensitized and prepared. However, liquid sensitized blood cells have the disadvantage of poor storage stability even when stored in a cold place. Freeze-drying is used to preserve sensitized blood cells. However, even when lyophilized sensitized blood cells were used, a decrease in sensitivity and disturbance of the hemagglutination image were observed, and furthermore, there were problems with the appearance and solubility of the lyophilized sensitized blood cells. Based on this technical background, the present inventors conducted intensive studies and found that by coexisting disaccharides, serum albumin, amino acids, and sugar alcohols during freeze-drying, it is possible to achieve long-term stability, clear aggregation images, and improved appearance. It was discovered that freeze-dried sensitized blood cells with excellent dispersibility during use can be obtained, and as a result of further research based on this finding, the present invention was completed. The present invention is a method for producing freeze-dried sensitized blood cells, which comprises freeze-drying a sensitized blood cell suspension in which disaccharides, serum albumin, amino acids, and sugar alcohols are dissolved. Examples of disaccharides used in the method of the present invention include sucrose, lactose, trehalose, gentiobiose, cellobiose, and maltose. Among these, sucrose and lactose are preferred. As the serum albumin used in the method of the present invention, for example, serum albumin derived from a mammal and obtained by conventional means can be used. Examples include human serum albumin, horse serum albumin, bovine serum albumin, sheep serum albumin, goat serum albumin, rabbit serum albumin, and the like. Among them, bovine serum albumin is preferred. The amino acids used in the present invention may be aliphatic, aromatic, or heterocyclic. Examples include, for example, aliphatic acidic amino acids (e.g., glutamic acid, aspartic acid),
Aliphatic neutral amino acids (e.g., glycine, alanine,
(valine, leucine, isoleucine, serine, threonine, glutamine, asparagine), aliphatic basic amino acids (e.g. lysine, arginine), aliphatic sulfur-containing amino acids (e.g. cysteine, cystine, methionine), aromatic amino acids (e.g. enylalanine, tyrosine), heterocyclic amino acids (proline,
histidine), etc. Among them, glutamic acid and glycine are preferred. These amino acids may also be in the form of salts. Examples of such salts include sodium salts and potassium salts. In particular, glutamic acid,
Acidic amino acids such as aspartic acid are preferably used as sodium or potassium salts. Moreover, it is preferable to use basic amino acids such as lysine after neutralizing them with an appropriate acid. In the method of the present invention, it is preferable to further dissolve a sugar alcohol in the sensitized blood cell suspension. Examples of the sugar alcohol include hexylate (eg, mannite, sorbitol), pentite (eg, arabitite, xylitol), tetrit (eg, erythritol), and the like. Among them, mannit is preferred. The amount of the dissolved compound of the present invention is such that the disaccharide concentration is approximately 1 to 15 w with respect to the total amount of the sensitized blood cell suspension subjected to freeze-drying (when the sensitized blood cell concentration is approximately 4 v/v%). /v%, more preferably about 2~
10w/v%, the concentration of serum albumin is approximately
0.1-3w/v%, more preferably about 0.3-2w/v%
v%, the amino acid concentration is approximately 0.1 to 5w/v
%, more preferably about 0.3 to 2 w/v%, and the sugar alcohol concentration is about 1 to 30 w/v%, more preferably about 3 to 15 w/v%. The above sensitized blood cell concentration is approximately 4v/v
%, the amount of the above compound to be dissolved can be proportional to the above concentration. For the freeze-drying process, methods and conditions suitable for freeze-drying sensitized blood cells are employed. In this treatment, the sensitized blood cell suspension is heated at a temperature of about -40 to +40, for example.
℃, the pressure is about 0.5 to 10×10 ⁻² mmHg, and the time is about 24 to 72 hours. The freeze-dried sensitized blood cells obtained in this way are extremely stable, and even when stored at room temperature or higher for long periods of time, there is virtually no decrease in the activity of the antigen-antibody reaction, and specificity is maintained, with a clear image of the agglutination reaction. Furthermore, it is a beautiful powder with an excellent appearance, and has excellent dispersibility in distilled water, physiological saline, and various buffer solutions, making it easy to prepare a sensitized blood cell suspension before use. You can. As described above, the freeze-dried sensitized blood cells obtained by the method of the present invention meet the conditions required for sensitized blood cells, namely
It is an excellent product that satisfies all of the following: (1) storage stability, (2) clarity of aggregation images (negative images, positive images), (3) appearance of freeze-dried products, and (4) dispersibility. The sensitized blood cell suspension used in the method of the present invention is usually prepared by sensitizing immobilized mammalian red blood cells or avian red blood cells with a serologically active substance and then suspending them in an appropriate buffer. As the red blood cells, for example, mammalian red blood cells such as human, horse, cow, sheep, goat, and rabbit, and avian red blood cells such as chicken, geese, and turkey can be used. In the fixation treatment, any fixative commonly used for fixing red blood cells may be used, such as formalin, pyruvaldehyde, glutaraldehyde, and the like. The red blood cells fixed as described above may be further treated with tannic acid to sensitize them. This tannic acid treatment can be carried out according to a method known per se. For example, a fixed red blood cell suspension may be treated with a tannic acid-containing solution (usually about 0.001
~0.04 w/v% (preferably about 0.04 w/v%) is added in small amounts under shaking, and then left at room temperature for about 0.5 to 3 hours or shaken to produce red blood cells with enhanced adsorption ability for serologically active substances. can be obtained. The serologically active substance that sensitizes the red blood cells thus obtained may be any of antigens, haptens, antibodies, and the like. For example, immunoglobulin, albumin, fibrinogen (fibrin and its degradation products), AFP, C-reactive protein, β ₂ -microglobulin, myoglobin, carcinoembryonic antigen, hepatitis virus antigen,
Examples include proteins such as hCG, human placental lactogen, and insulin, hormones, administered drugs, and antibodies thereof. When sensitizing the above-mentioned red blood cells with such a serologically active substance, a known sensitization treatment can be used, and the red blood cells and the serologically active substance are mixed in an aqueous solvent (for example, water,
The contact is preferably carried out in a solution (physiological saline, various buffer solutions, etc.), and is generally carried out by mixing a solution containing a serologically active substance and an aqueous suspension of red blood cells and allowing it to stand, but if desired, it may be carried out by stirring or Shaking may be used to reduce contact time. Examples of the serologically active substance-containing liquid include serum, plasma, or purified active substance-containing liquid containing the active substance. In particular, it is preferable to carry out the sensitization treatment using a solution of a purified serologically active substance.
In this case usually about 0.001 to 10 mg/ml, especially about 0.01
A solution containing about 2 mg/ml of active substance at about 1-20% (V/V), especially 3-10% (V/V)
It is preferable to add an equal volume to the above-mentioned red blood cell suspension. This sensitization treatment generally has a pH of about 5 to 8 and a pH of about 20 to 60.
Preferably it is carried out at a temperature of .degree. After this sensitization treatment,
If desired, the sensitized red blood cells may be washed with an aqueous solvent. The sensitized red blood cells thus obtained are suspended in an aqueous solvent such as a phosphate saline buffer with a pH of 7.4 or a Hepes saline buffer with a pH of 7 at a concentration of about 2 to 20% (v/v). The present invention will be explained in more detail with reference to Examples below. Example 1 (1) Preparation of anti-hCG immunoglobulin Purified hCG (unit: 10,000 IU/mg) was mixed with complete Freund's adjuvant, and the anti-hCG serum (rabbit) obtained by immunizing a rabbit was added with saturated ammonium sulfate and salted. conducted an analysis. After centrifugation at 3000 rpm for 30 minutes, the resulting precipitate was dissolved in physiological saline and dialyzed against physiological saline overnight to obtain an anti-hCG immunoglobulin solution. (2) Preparation of anti-hCG sensitized blood cell suspension Sheep blood cells were fixed with pyruvaldehyde according to a conventional method, and then fixed using a known method {Journal of Experimental Medicine, No. 93
(1951), Section 107}, tannic acid-treated immobilized sheep blood cells obtained by tannic acid treatment were suspended in a phosphate buffer. This and the above hCG immunoglobulin solution diluted and dissolved in phosphate buffer to a concentration of about 0.13 mg/ml were mixed in equal amounts and reacted at 56°C for 2 hours to sensitize blood cells to anti-hCG. Next, the obtained anti-hCG sensitized blood cells were washed with physiological saline, left overnight in Hepes buffered saline containing 0.5% normal rabbit serum, washed with physiological saline, and added to Hepes containing the additives listed in Table 1. A 4% (v/v) anti-hCG sensitized blood cell suspension was prepared using buffered saline. (3) Preparation of freeze-dried anti-hCG sensitized blood cells Dispense 0.25 ml of the above anti-hCG sensitized blood cell suspension into vials (2 ml capacity), pre-freeze at approximately -30 to -40°C, and then vacuum freeze-dry. The material was placed in a machine, evacuated, vacuumed, and dried for approximately 40 hours while gradually raising the temperature to 30°C. Then, after filling with nitrogen gas, the vial was sealed and placed in a freeze-drying oven.
hCG sensitized blood cells were obtained. (4) Test of freeze-dried sensitized blood cells 37 for each freeze-dried product prepared in Example 1 (3)
The appearance at ℃, dispersibility in Hepes buffered saline, stability of sensitivity, and sharpness of hemagglutination images were investigated. The results are shown in Table 1. In addition,
The method for measuring sensitivity is as follows. That is,
1 ml of Hepes buffered saline was added to one vial of the freeze-dried anti-hCG sensitized blood cells to prepare a 1% anti-hCG sensitized blood cell suspension. Each sensitized blood cell suspension
0.2ml each with a hemispherical bottom and an inner diameter of approximately 10mm
into a small test tube, add 0.1 ml of hCG standard solution to it, mix by shaking, and leave it for 2 hours. If a sedimentation ring is formed at the bottom of the test tube, it is considered negative.
The result was determined as positive if no such occurrence occurred. The results are shown in Table 1.

【table】

【table】

[Table] As is clear from the above results, it is better to dissolve and lyophilize the disaccharide, serum albumin, amino acid, and sugar alcohol of the present invention than to dissolve and lyophilize only one of the individual components. two of the following: (a) disaccharides and serum albumin; (b) disaccharides and amino acids; (c)
(serum albumin and amino acids) dissolved and lyophilized, and sugar alcohols and one or two other components (i.e. (d) sugar alcohols and disaccharides; (e) sugar alcohols, disaccharides and serum albumin; (f) The appearance, solubility, sensitivity, and
It shows excellent stability effect in terms of negative image. Example 2 Table 2 shows the results of a stability test conducted by adding the following stabilizers to the anti-hCG sensitized blood cell suspension obtained in the same manner as in Example 1. Note that the test method was the same as in Example 1. The sensitivity of each sensitized blood cell is
In the test method shown in Example 1, it was expressed as the minimum hCG concentration that showed a positive reaction. As is clear from Table 2, the severe stability test (37
When stored at 10°C for 4 weeks) and 12 months at 10°C, when mannite, sucrose, bovine serum albumin, and monosodium glutamate (or glycine) were added as stabilizers, good results were obtained in terms of sensitivity, appearance, solubility, and negative image. It was hot.

[Table] Example 3 (1) Preparation of anti-AFP immunoglobulin Obtained from umbilical cord blood by a conventional method
AFP16mg 0.5M NaCl in 0.1M NaHCO ₃ 8
3 g of bromocyanide Cephalose 4B [manufactured by Pharmacia Huain Chemicals (Sweden)] dissolved in
and stirred overnight at 5°C. After the reaction
Wash thoroughly with 0.1M _NaHCO3 containing 0.5M NaCl, then add 10ml of 0.5M ethanolamine adjusted to pH 8 with HCl and react for 1 hour at room temperature, and then (1) 0.1M acetate buffer containing 1M NaCl. Wash the solution (PH4.0) sequentially with (2) 0.1M borate buffer (PH8.0) containing 1M NaCl and (3) 0.02M borate buffer (PH8.0) containing 0.15M NaCl. Filled (2.2〓×2.5cm). 35 ml of goat anti-AFP serum was salted out and precipitated by the saturated ammonium sulfate method, and the resulting γ-globulin fraction was applied to the AFP-bound Sepharose 4B column described above. 0.15M NaCl
The anti-AFP antibody was obtained by washing the column with 0.02M borate buffer (PH 8.0) containing 0.02M and then eluting with 0.17M glycine-hydrochloric acid buffer (PH 2.2). (2) Preparation of anti-AFP sensitized blood cell suspension Goat red blood cells were fixed with glutaraldehyde according to a conventional method and further treated with tannic acid.
V), and an equal volume of the anti-AFP antibody solution (0.7 mg/ml) prepared in Example 3 (1) in phosphate buffer was mixed therewith.
Goat blood cells were sensitized to AFP by reacting at ℃ for 3 hours. The obtained sensitized blood cells were then washed with physiological saline, left in a phosphate saline buffer containing 0.5% normal rabbit serum, washed with physiological saline, and treated with 5% mannitol, 2.5% sucrose, and 0.5% sucrose. 1.8% (V _/
Anti-AFP sensitized blood cells were suspended as shown in V). (3) Preparation of freeze-dried anti-AFP sensitized blood cells Dispense 0.5 ml of the anti-AFP sensitized blood cell suspension prepared in Example 3 (2) into vials,
Freeze-dried anti-AFP sensitized blood cells were prepared in the same manner as in (3). The anti-AFP sensitized blood cells thus obtained were subjected to the same test as in Example 1 (4).
When the standard solution was tested, as shown in Table 3, good results were obtained in terms of sensitivity, appearance, solubility, and negative image.

[Table] Example 4 (1) Preparation of anti-human IgE sensitized blood cell suspension Using sheep blood cells, fixation with glutaraldehyde according to a conventional method, followed by further treatment with tannic acid. 10% sheep blood cells (V/
V), suspended in phosphate buffer to a concentration of 0.1 mg/ml, mixed with an equal volume of anti-human IgE goat serum gamma globulin in phosphate buffer, and reacted at 56°C for 2 hours. Sheep blood cells were sensitized to anti-human IgE gamma globulin. Next, the obtained sensitized blood cells were washed with physiological saline, left in a phosphate saline buffer containing 0.5% normal rabbit serum, washed with physiological saline, and injected with 10% mannitol, 5%
Anti-human IgE sensitized blood cells were suspended at a concentration of 4% (V/V) in an aqueous solution containing % sucrose, 1% bovine serum albumin, 1% sodium glutamate, and 0.1% _NaN3 . (2) Freeze-dried anti-human IgE-sensitized blood cells The anti-human IgE-sensitized blood cell suspension prepared in Example 4 (1) was dispensed into vials (capacity 2 ml) in 0.5 ml portions, and the same was prepared as in Example 1 (3). Freeze-dried anti-human IgE-sensitized blood cells were prepared in a similar manner. (3) Sensitivity measurement Human IgE was measured for the freeze-dried anti-human IgE-sensitized blood cells thus obtained using a microtiter method using a V plate. That is, 1 ml of Hepes buffered saline was added to 1 vial of freeze-dried sensitized blood cells to prepare a sensitized blood cell suspension. On the other hand, aliquot 0.05ml of the human IgE standard dilution into a V-plate well, make a 2-fold serial dilution series using phosphate buffer saline, add 0.05ml of the sensitized blood cell suspension, and mix. After shaking and standing at room temperature for 1 hour, the agglutination image at the bottom of each well was judged to be positive if agglutination occurred, and negative if not. The sensitivity of sensitized blood cells is the lowest in humans with a positive reaction.
Expressed as IgE concentration. As shown in Table 4, the results showed good results in terms of appearance, solubility, sensitivity, and negative image.

【table】

Claims (1)

[Claims] 1. A method for producing freeze-dried sensitized blood cells, which comprises freeze-drying a sensitized blood cell suspension in which disaccharides, serum albumin, amino acids, and sugar alcohols are dissolved.