JPS58131913A - Preparation of lyophilized erythrocyte - Google Patents

Abstract

PURPOSE:To obtain lyophilized erythrocyte having stbility for a long-term storage with preventing the denaturation of erythrocyte during drying, by adding a specific amount of albumin to a water dispersion of erythrocyte, followed by lyophilization. CONSTITUTION:Albumin is added to a water dispersion of erythroctye (preferably sensitized erythrocyte or fixed erythrocyte) in such a way that the concentration of albumin is >=2%(w/v), preferably 3-6%, and the erythrocyte is lyophilized. A dispersion of distilled water, a dispersion of physiologic salt solution, an isotonic buffer solution of phosphoric acid may be cited as the water dispersion. In case of sensitized erythrocyte used as a reagent, etc. for detecting an antigen or an antibody, it shows no reduction in detection sensitivity during lyophilization, is stable for a long-term storage, and usable as various kinds of reagents for hemaggulutination.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention #3 relates to a method for efficiently producing a stable lyophilized red blood cell.

When a suspension of red blood cells in water is freeze-dried and this is used as a freeze-dried product, the red blood cells are denatured.This denaturation is particularly problematic for sensitized red blood cells and red blood cells that have been sensitized with antigens or antibodies. Fixed red blood cells treated with formaldehyde, glutaraldehyde, etc.

By the way, sensitized red blood cells and the like are used as reagents (for example, passive hemagglutination reagents, reverse passive hemagglutination reagents, hemagglutination inhibition reagents, etc.) for detecting antigens or antibodies. [Pierce, G.N., and Shulman I.N.R. (Vyaa.

G, N-and Shulmin, N= R,)
:Science, 170.33
2.1970 and Mitsunobu Joint Venture, Takashi Takahashi, et al. “Detection of Au antibodies by passive hemagglutination reaction”, History of Medicine 78,
759.1971).

However, the red blood cells used in these detection reagents, especially sensitized red blood cells, are unstable. Particularly in the state of aqueous suspension, the antigen or antibody detection sensitivity decreases in a short period of time to the extent that it cannot be used. In order to prevent this, it is desirable to freeze-dry the aqueous suspension of sensitized red blood cells. When stabilizing the freeze-drying of cells such as sub-awakening and other cells, 0.5 to 1% of albumin, amino acids, and sugars KTLJs are added and freeze-dried after freezing.0 (Isao Ota et al., Vacuum Technology Course 8, Vacuum Drying ,
Nikkan Kogyo Shimbun, 1962) If these conditions are applied to a sensitized red blood cell aqueous suspension, the detection sensitivity of sensitized red blood cells after freeze-drying will be 1/8 to 1/8 as shown in Table 1.
It decreases by l/16.

In addition, fixed red blood cells may be used, for example, as the above-mentioned sensitized red blood cells.
The fixed red blood cells are denatured (1) as shown in Example 15 if the red blood cells are used in water suspension tubes or freeze-dried.

Therefore, the present inventors have conducted extensive research to fully stabilize red blood cells, particularly sensitized red blood cells, and fixed red blood cells during freeze-drying. As a result, red blood cell water suspension (in particular, sensitized red blood cell water suspension,
2% (w/v) of albumin' in fixed red blood cell aqueous suspension)
By adding it to a concentration higher than that and freeze-drying it,
Red blood cells do not denature during freeze-drying, and detection sensitivity of sensitized red blood cells does not decrease during freeze-drying. Also, degeneration 1 did not come,
Does it have to be stable? I found it.

The present invention has been completed based on such knowledge,
The red blood cells used in the present invention are as follows: There is no restriction on the origin of the animal species, and for example, those derived from mammals (for example, humans, cows, rabbits, mice, horses, and sheep), birds2 (for example, chickens, etc.) are used. In addition, the red blood cells are infected with antigens (for example, HBa anti-JQ, HB eFC
with modified red blood cells such as t-sensitized sensitized red blood cells, fixed red blood cells treated with formaldehyde, glutaraldehyde, etc. There may be.

Examples of red blood cell suspensions in water include distilled water suspensions, physiological saline suspensions, and isotonic phosphate buffer solutions. The suspension is prepared by a procedure known per se.

The origin of the albumin used in the present invention is not particularly limited as to the animal species, and those derived from the animal species numerically exemplified for erythrocytes can be used.

The amount of albumin added is such that the concentration of albumin in the suspension is 2% (W/v) or more, preferably 3qb (W/v).
V) The amount corresponding to the concentration or higher. When the albumin content is 3% (W/V) or more, it not only prevents denaturation during freeze-drying but also significantly increases storage stability. There is no particular restriction on the upper limit of the amount of albumin added, but from a salmon management perspective, it should be 8% (w/v) concentration, preferably 6% (w/v).
/v) concentration.

Freeze-drying of the albumin-added red blood cell water suspension can be carried out using known procedures.

The lyophilized product thus obtained does not cause denaturation of red blood cells by lyophilization and is stable even during long-term storage. In particular, sensitized red blood cells do not show a decrease in detection sensitivity during freeze-drying and are stable even during long-term storage, so they can be used in various reagents for red blood cell agglutination reactions.

Example 1 Cloth HBa antigen was obtained from HBa anti-*+W plasma by a combination of ammonium sulfate fractionation, ion exchange chromatography, and ultracentrifugation. After washing 4 times with physiological saline solution on sheep red blood cells, it was identified with curtaraldehyde according to the method described in the above-mentioned paper by Chiben et al., ``The History of Medicine, J 78. HBs antigen-sensitized sheep red blood cells were obtained by reacting with the antigen.

Wash with saline or isotonic phosphate buffer to remove excess H.
After removing the Bs antigen, it was made into a 5% suspension at 11°C. Each of these floating threads (W/V) llk to yv
Curtathione (control) at a concentration of 1% (W/V) and 1-8% (W/V)
) Bovine albumin was added to ml and freeze-dried after ordering ld.

After freeze-drying, 10 d'i of isotonic phosphate buffer was added, and the suspension was resuspended (0.5% (w/v) at 111 degrees), and the detection sensitivity and size of the porcelain image were all observed.

The suspension before freeze-drying is also adjusted to 0°51 with isotonic phosphate buffer.
(w/v) Ilf i, 1l11 were tested simultaneously.

The freeze-dried product was stored at 4°C and the same test was conducted every three months.

As shown in fil, those without albumin addition and 1% (w
/v) Detection immediately after freeze-drying for the following JIJf#i
As the temperature decreased from 1/8 to 1/16, the porosity image also increased, making the determination of agglutination a national problem.But albumin? l1
-2% (W/Ma) or more added was stable without any change in detection sensitivity immediately after freeze-drying.

Albumin 2 whose detection sensitivity did not decrease immediately after freeze-drying
The groups containing 56 (W/m) or more were stored at 4°C and further subjected to long-term stability tests.

When albumin was added at 2s (W/V), the detection sensitivity decreased to 1/2 at the 9th month;
) was completely stable even after several months of UlB. Therefore, the amount of albumin added should be Id 2 To (w/v) or more for long-term stability during freeze-drying, and 3 T (w/v) for long-term stability immediately after and after freeze-drying. W/V) or more was found to be preferable.

(Left below) Example 2 Implementation? ! Just like I 1, it's 1t! HBa antigen was sensitized to glutaraldehyde and tannic acid treated chicken red blood cells.

51 (W/v) of this HBa antigen-sensitized chicken red blood cell
5% (w/v) concentrated fK in human albumin was added to the suspension and freeze-dried.

There is a difference in detection sensitivity and size of negative image before and after freeze-drying.
i was not recognized.

Even when this freeze-dried sensitized red blood cell was stored at 4'C for 12 months, no decrease in detection sensitivity was observed.

Example 3 In the same manner as in Example 1, a suspension of type O red blood cells sensitized to HBa antigen using human type 0 red blood cells was obtained. Bovine albumin T496 (w/v) concentration was added to the sample and lyophilized.

No difference was observed in detection sensitivity and size of negative image before and after freeze-drying. Even when the freeze-dried sensitized red blood cells were stored at 4°C for 12 months, no decrease in detection sensitivity was observed.

Example 4 HBe antigen t-HBe antibody conjugated with Sepharose,
Thereafter, an HBt4 cool sensitized sheep red blood cell suspension was obtained in the same manner as in Example 1.

59b (w/v) mW of bovine albumin was added to a 5% (w/v) suspension of the HBe antigen-sensitized sheep red blood cells and lyophilized.

No difference was observed in detection sensitivity or size of negative image before and after freeze-drying. Furthermore, no decrease in detection sensitivity was observed even when the sensitized red blood cells after freeze-drying were stored at 4° C. for 15 months.

Example 5 Ammonium sulfate fractionation and DEAE from anti-HBs guinea pig serum
- Purified HBa antibody was obtained by cellulose ion exchange adsorption method.

Is this carried out using this antibody? ! An anti-HBs antibody-sensitized sheep red blood cell suspension was obtained in the same manner as in Il.

Bovine albumin was added to 3% (w/v) allfK to this 5% (w/v) suspension of anti-HBa sensitized sheep red blood cells and freeze-dried.

No difference was observed in detection sensitivity or size of negative image before and after freeze-drying. In addition, this sensitized red blood cell F after freeze-drying
No decrease in detection sensitivity was observed even when stored at 4°C for 18 months.

Example 6 Anti-human interferon (RO) DEAE from horse serum
Anti-human interferon horse antibodies were purified using Sephadex and HiF interferon-conjugated Sepharose 4Bt.

Using this purified antibody, anti-human interferon sensitized sheep red blood cells 1 were obtained in the same manner as in Example 1.

5% of this anti-human interferon sensitized sheep red blood cells (
w/v) human albumin t-5% (v/v) 1 in suspension
11 degrees and lyophilized.

No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.

Example 7 Anti-human urokinase antibody was purified from anti-human urokinase rabbit serum using ammonium sulfate fractionation, polyethylene glycol fractionation, and DEAE-cellulose chromatography. Using this purified antibody, an anti-human urokinase-sensitized sheep red blood cell suspension was obtained in the same manner as in Example 1.

5% (w/
v) Bovine albumin was added to the suspension at a concentration of 2% (W/V) and freeze-dried.

No difference was observed in the detection If&degree and the size of negative Keyaki before and after freeze-drying.

Example 8 Alpha-fetoprotin-positive human ascites was purified using ion-exchange adsorption, gel filtration, and anti-alpha-7 ethoprotene antibody-conjugated Sepharose adsorption to obtain Mli! Full 7a7 ethoprotene was obtained.

Using this alpha-fetoprotin, a suspension of alpha-fetoglot-sensitized sheep red blood cells was obtained in the same manner as in the tHJ1 experiment.

This alpha-fetoprotin sensitized sheep has 5% of blood cells (
51 (w/v) bovine or human albumin was added to the suspension.
/v) concentration and lyophilized.

No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.

The sensitized red blood cells after freeze-drying were stored at 4° C. for 12 months, and no decrease in detection sensitivity was observed even in the case of fc.

A thyroglobulin-sensitized sheep red blood cell suspension was obtained in the same manner as in Example 1 using the *0 cleft antigen gold produced by thyroglobulin VS.

To this 5% (w/v) suspension, add 5% human albumin (
W/V) concentration and lyophilized.

No difference was observed in the detection degree of IIA and the size of negative images before and after freeze-drying.

Example IO Anti-human placental gonadotropin ammonium sulfate fraction from sheep serum,
Anti-human placental gonadotropin antibodies were purified using DEAE-cellulose chromatography method. Using this purified antibody, an anti-human placental gonadotropin antibody-sensitized red blood cell suspension was obtained in the same manner as in Example 1.

To this 5% (w/v) suspension, all 5 pieces of bovine albumin (w) were added.
/v) once and lyophilized.

No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.

Example 11 An anti-sheep red blood cell rabbit antibody t-ha was produced using ammonium sulfate fractionation from anti-sheep red blood cell rabbit serum and DEAE-cellulose chromatography. Example 1 using this purified antibody
In the same manner as above, an anti-sheep red blood cell suspension (human type O red blood cell suspension) was obtained.

To this 5% (w/v) suspension, 5 h (w) of human albumin was added.
/v) (11 degrees plus freeze-dried.

No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.

Example 12 Thyroid tissue from a human Graves' disease patient was isolated by ultrahigh-speed centrifugal fractionation. This ξcroseam was used in the same manner as in Example 1 to obtain a suspension of microsomal sensitized sheep red blood cells with Graves' disease.

Bovine albumin ts% (
w/v) concentration and lyophilized.

There is a difference in detection sensitivity and size of negative image before and after freeze-drying.
i was not recognized.

Example 13 Streptolysin 0 and streptokinase were purified from a culture filtrate of hemolytic streptococci using an ammonium sulfate fractionation method. Using these purified antigens, a streptolysin 0-sensitized sheep red blood cell suspension and a streptokinase-sensitized sheep red blood cell suspension were obtained in the same manner as in Example 1.

Five grams of human albumin (w/w) was added to this 5% (w/v) suspension.
/v)ml and freeze-dried.

No differences were observed in detection sensitivity or size of negative images before and after freeze-drying.

Example 14 Staphylococcus, Pseudomonas aeruginosa, Diptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli,
Bacterial membrane components of influenza, Leucoplasma, Toxoplasma, etc. were extracted by sonic destruction method, phenol water heating method, etc., and extracellular toxins of Tetanus and G. . A bacterial membrane or an exotoxin-sensitized sheep red blood cell suspension was obtained in the same manner as in Example 1.

Bovine albumin was added to this 5% (w/v) suspension at 5% (
w/v) concentration and lyophilized.

No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.

Example 15 Sheep erythrocytes, chicken erythrocytes, human type 0 erythrocytes, goose erythrocytes, bovine erythrocytes, and rabbit erythrocytes were washed four times with whole physiological saline solution, treated with formaldehyde or glutaraldehyde, and the remaining formaldehyde or glutaraldehyde centrifugation. removed by law. A portion of these fixed red blood cells was treated with tannic acid, and the remaining tannic acid was removed by centrifugation. Human albumin, bovine albumin, or horse albumin was added to a suspension of these fixed red blood cells and tannic acid-treated red blood cells at a final concentration of 2 s (W/ma) and 5% (W/V) and freeze-dried. fC,) After lyophilization, the samples were resuspended in an equal silver phosphate buffer solution, and the reaction to the same silver phosphate buffer was compared by measuring the total diameter of the erythrocyte sedimentation image.02-5
% (w/v) of each erythrocyte after lyophilization with the addition of fulbumin, no change was observed compared to that before lyophilization, but the erythrocyte sedimentation image of each erythrocyte lyophilized without albumin was It was inferred that the properties changed due to selective freeze-drying with a larger diameter.〇

Claims (3)

[Claims] (1) Is there albumin in the water suspension of red blood cells? 2%
(v/ma) Addition to a concentration higher than that and lyophilization t-Method for producing lyophilized red blood cells characterized by 0 (2) Claim No. 1 in which the red blood cells are fixed red blood cells
) The method for producing a lyophilized red blood cell as described in section 2. (3) The method for producing lyophilized red blood cells according to claim (1), wherein the red blood cells are sensitized red blood cells sensitized with any antigen or antibody.