JPS58131566A - Lyophilized matter of red blood cell - Google Patents
Lyophilized matter of red blood cellInfo
- Publication number
- JPS58131566A JPS58131566A JP57072253A JP7225382A JPS58131566A JP S58131566 A JPS58131566 A JP S58131566A JP 57072253 A JP57072253 A JP 57072253A JP 7225382 A JP7225382 A JP 7225382A JP S58131566 A JPS58131566 A JP S58131566A
- Authority
- JP
- Japan
- Prior art keywords
- red blood
- blood cells
- albumin
- lyophilized
- blood cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
- G01N33/555—Red blood cell
- G01N33/556—Fixed or stabilised red blood cell
Abstract
Description
【発明の詳細な説明】 本発明は、安定なる赤血球凍結乾燥物に関する。[Detailed description of the invention] The present invention relates to stable lyophilized red blood cells.
赤血球の凍結乾燥物は、これを長期保存した場合、赤血
球が変性する。この変性が問題となるのは、特に赤血球
に抗原又は抗体を感作させた感作赤血球、赤血球會フォ
ルムアルデヒド、グルタルアルデヒドなどで処理した固
定化赤血球である。If freeze-dried red blood cells are stored for a long period of time, the red blood cells will denature. This degeneration is particularly problematic in sensitized red blood cells obtained by sensitizing red blood cells with antigens or antibodies, and in fixed red blood cells treated with red blood cell formaldehyde, glutaraldehyde, or the like.
ところで感作赤血球等は、抗原又は抗体を検出するため
の試薬（例えは、受身赤血球凝集反応試薬、逆受身赤血
球凝集反応試薬、赤血球凝集阻止反応試薬等）などどし
て利用されているが〔ビアｙ、、 Ｑ、 Ｎ、及ヒシ
ュル−ｖｙ、 Ｎ、 Ｒ，（Ｖｙａａ。By the way, sensitized red blood cells are used as reagents for detecting antigens or antibodies (for example, passive hemagglutination reagents, reverse passive hemagglutination reagents, hemagglutination inhibition reagents, etc.). Vyaa.
Ｇ、Ｎ、ａｎｄ Ｓｈｕｌｍａｎ、Ｎ、Ｒ，）：サイエ
ンス（Ｓｃｌｅｎｅａ）、 １７０．３３２． １９
７０及び奇弁光信、高橋隆、外「受身赤血球凝集反応に
よるＡｕ抗体の検出」、医学のあゆみ７８，７５９゜１
９７１〕、かかる検出試薬に使用される赤血球、特に感
作赤血球は不安定である。特に、水浮遊液の状態では短
期間で使用に耐えない程度にまで抗原又は抗体検出感度
が低下する。これを防止するためには感作赤血球の水浮
遊液ｔ−凍結乾燥することが望ましい。しかし凍結乾燥
したものであっても依然として保存安定性に欠け、また
凍結乾燥時の安定性にも欠ける。G, N. and Shulman, N. R.: Science, 170.332. 19
70 and Mitsunobu Kiben, Takashi Takahashi, et al. “Detection of Au antibodies by passive hemagglutination reaction”, History of Medicine 78,759゜1
[971], red blood cells used in such detection reagents, especially sensitized red blood cells, are unstable. In particular, 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 a suspension of sensitized red blood cells in water. However, even when freeze-dried, it still lacks storage stability and also lacks stability during freeze-drying.
そこで、本発明者らは赤血球、特に感作赤血球、固定化
赤血球の凍結乾燥物の安定化全図るべ（研究を重ねた。Therefore, the present inventors have conducted extensive research to fully stabilize lyophilized red blood cells, particularly sensitized red blood cells, and fixed red blood cells.
その結果、赤血球１重責部に対してアルブミン０．４Ｍ
量部以上管添加してなる赤血球凍結乾燥物が長期保存に
よっても変性を来たさず安定であること、しかも赤血球
の５％（Ｗ／ｖ）濃度水浮遊液（就中、感作赤血球水浮
遊液、固定化赤血球水浮遊液）にアルブミンが２％（Ｗ
／Ｖ）濃度以上となるような割合でアルブミンを加えて
凍結乾燥することにより、凍結乾燥時に赤血球が変性す
ることがなく、特に感作赤血球にあっては凍結乾燥時に
尚該感作赤血球による検出感度の低下全米たすことがな
いことを見出した。As a result, albumin was found to be 0.4M per 1 red blood cell.
The lyophilized red blood cells obtained by adding more than one part to the tube must be stable without denaturation even during long-term storage, and must be 5% (W/v) suspension of red blood cells in water (in particular, sensitized red blood cell water). 2% albumin (W
/V) By adding albumin at a ratio higher than the concentration and freeze-drying, the red blood cells will not be denatured during freeze-drying, and especially in the case of sensitized red blood cells, detection by the sensitized red blood cells will still be possible during freeze-drying. We found that there was no decrease in sensitivity across the board.
本発明りかかる知見に基づいて児成されたものであり、
赤血球ｌＴＬ量部に対してアルブミン０．４Ｘ−を部以
上會含有してなる赤血球凍結乾燥物に関する０
本発明に関して、赤血球は、その動物種の由来に制限は
なく、例えは咄乳類（例えは、ヒト、ウシ、ウサギ、マ
ウス、ウマ、ヒツジ）、鳥類（例えば、ニワトリ）、は
虫類などの由来のものがあけられる。また、当該赤面球
社抗原（例えは、ＨＢ＠抗原、ＨＢｅ抗原、サイログロ
ブリン）、抗体（例えは、抗インターフェロン抗体、抗
ウロキナーゼ抗体、抗アルファフェトプロティン）全感
作した感作赤血球、フォルムアルデヒド、クルタルアル
デヒドなどで処理した固定化赤血球などの修飾された赤
血球であってもよい。The present invention was developed based on the knowledge related to the present invention,
Regarding the red blood cell freeze-dried product containing 0.4 parts or more of albumin based on the amount of red blood cell TL. Examples include those derived from humans, cows, rabbits, mice, horses, sheep), birds (e.g. chickens), reptiles, etc. In addition, the sensitized red blood cells, formaldehyde, antibodies (e.g., anti-interferon antibody, anti-urokinase antibody, anti-alphafetoprotein), antibodies (e.g., anti-interferon antibody, anti-urokinase antibody, anti-alphafetoprotein), Modified red blood cells such as fixed red blood cells treated with cultaraldehyde or the like may also be used.
本発明で使用されるアルブミンは、その動物種の由来に
特に制限はなく、一般的には赤血球について例示した如
き動物種由来のものが使用される。The albumin used in the present invention is not particularly limited in its origin from the animal species, and albumin derived from the animal species exemplified for red blood cells is generally used.
アルブミンの添加量は、赤血球１重量部に対して０．４
重量部以上、好ましくは０．６重量部以上である。当該
アルブミン祉、赤血球凍結乾燥物の製造前に添加してお
くことが好ましい０即ち、本発明の赤血球凍結乾燥−社
一般に赤血球水浮遊液を凍結乾燥して製するが、その際
赤血球水浮遊液中に、５％（Ｗ／Ｖ）濃度の赤血球水浮
遊液を使用する場合には、アルブミンが２％（Ｗ／マ）
１１度こｎを凍結乾燥して製造することが好ましい。ア
ルブミン絡加愈の上限については特に制限性ないが、経
済的見地からは１．６重量部、好ましくは１゜２重量部
である。The amount of albumin added is 0.4 per part by weight of red blood cells.
The amount is at least 0.6 parts by weight, preferably at least 0.6 parts by weight. It is preferable to add the albumin to the lyophilized red blood cell product before producing the lyophilized red blood cell product.In other words, the lyophilized red blood cell product of the present invention is generally produced by freeze-drying a red blood cell water suspension. When using a red blood cell water suspension with a concentration of 5% (W/V), the albumin content is 2% (W/V).
Preferably, it is produced by freeze-drying 11°C. There is no particular restriction on the upper limit of albumin binding, but from an economic standpoint it is 1.6 parts by weight, preferably 1.2 parts by weight.
本発明に侮る赤血球凍結乾燥物は、一般にアルブミン添
加赤血球水浮遊数音凍結乾燥することによって製造され
、当該アルブミン添加赤血球水浮遊液の凍結乾燥は自体
既知の操作にて行えばよい。The lyophilized red blood cells used in the present invention are generally produced by freeze-drying a suspension of albumin-added red blood cell water, and the freeze-drying of the albumin-added red blood cell water suspension may be performed by a known procedure.
赤血球の水浮遊液としては、蒸留水浮遊液、生理食塩溶
液遊液、等張化リン酸緩衛液などがあげられる。当該浮
遊液は自体既知の操作にて調製される。Examples of the red blood cell suspension in water include a distilled water suspension, a physiological saline solution, and an isotonic phosphate buffer solution. The suspension is prepared by a procedure known per se.
本発明に係る赤血球の凍結乾燥物は長期間保存に対して
も安定であるにかりでなく、凍結乾燥時にアルブミンを
存在させて製したものは凍結乾燥時にも変性全米たさな
い。特に、感作赤血球にあっては、凍結乾燥時に検出感
度の低下がみられず、長期保存に対しても安定なもので
あるから、各種の赤血球凝集反応試薬に利用することが
できるものである。The lyophilized red blood cells according to the present invention are not only stable during long-term storage, but also those prepared in the presence of albumin during lyophilization do not undergo denaturation during lyophilization. In particular, sensitized red blood cells do not show any 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. .
実施例１
ＨＢａ抗原陽性血漿よシ、硫安分画法、イオン交換クロ
マトグラフィー法および超遠心分離法等を組合せた方法
によりｎ製ＨＢｓ抗原會得た０ヒツジ赤面球を生理食塩
溶液で４回洗浄し友後、藺述の奇弁らの文献［医学のあ
ゆみＪ７８，７５９，１９７１に記載の方法に準じてグ
ルタルアルデヒドで固定し、タンニン酸処理１行い精製
Ｈ］３ｍ抗原と反応せしめ、ＨＢａ抗原感作ヒツジ赤血
球管得た。Example 1 HBs antigen-prepared sheep bulbs obtained by a method combining HBa antigen-positive plasma, ammonium sulfate fractionation, ion exchange chromatography, ultracentrifugation, etc. were washed four times with physiological saline solution. After fixing with glutaraldehyde according to the method described in the literature of Chiben et al. of Shiyugo and Isho [Igaku no Ayumi J78, 759, 1971], HBa antigen was purified by 1 treatment with tannic acid] and reacted with HBa antigen. Produced sheep red blood cell tubes were obtained.
生理食塩溶液又は等帰化リン酸緩衝液で洗浄し余分な刊
り抗ｉｖ除去した後、５％（Ｗ／マ）濃度の浮遊液とし
た０この浮遊液にそれぞれＭｌ（ｗ／ｖ）８度にクリシ
ン（コントｔ：Ｉ −ル） １に％ １％（ｗ／ｖ）
６１度にグルタチオン（コントロール）を又は、１〜８
％（Ｗ／Ｖ）Ｉｌ［にウシアルブミンを添加し、１７ず
つ分注後凍結乾燥を行った。After washing with physiological saline solution or naturalized phosphate buffer to remove excess anti-IV, a suspension solution with a concentration of 5% (W/MA) was added. Chrysin (conte:I-le) 1% 1% (w/v)
Glutathione (control) at 61 degrees or 1 to 8
Bovine albumin was added to % (W/V) Il[, and lyophilized after dispensing 17 portions.
かくしてそれぞれ赤血球１１１を部に対してグリシン０
．２１ｃｆＮ＋、グルタチオン０．２重量部、アルブミ
ン０．２〜１．６３１量部を含有する赤血球凍結乾燥物
會待だ。これらに、等帰化リン酸緩衝液１０ｍ１ケ加え
、再浮遊液（０，５％（ｗ／ｖ）ｌＩＩ度）とし赤血球
＃集反応試験の検出感度および隘性像の大きさ會観察し
た。Thus, each red blood cell has 111 parts and glycine 0 part.
．． A lyophilized red blood cell composition containing 21 cfN+, 0.2 parts by weight of glutathione, and 0.2 to 1.631 parts by weight of albumin. To these, 10 ml of naturalized phosphate buffer was added, and a resuspension solution (0.5% (w/v) lII degree) was used to observe the detection sensitivity and the size of the obscurity image of the red blood cell collection reaction test.
凍結乾燥前の浮遊液も等帰化リン酸緩衝液で０゜５％（
Ｗ／Ｖ）濃度にｖ！４製し同時に試験した。The suspension before freeze-drying is also diluted with naturalized phosphate buffer at 0.5% (
W/V) Concentration v! 4 were made and tested at the same time.
ｊた凍結乾燥品は４℃に保存し３ケ月ごとに同様の試験
を行った。The freeze-dried products were stored at 4°C and the same tests were conducted every 3 months.
表１に示す如くアルブミン無添加のもの及び０゜２重量
部以下含有のものでは凍結乾燥直後の検出感度はｌ／８
〜１／１６に低下するとともに隘性像も太きくなり、凝
集判定は困難となったｏしかしアルブミンｋＯ，４Ｎ象
部以上含有のものは、いずれも凍結乾燥直後の検出感度
は変らず安定であった。As shown in Table 1, the detection sensitivity immediately after freeze-drying is 1/8 for those without albumin and those containing 0.2 parts by weight or less.
As the concentration decreased to ~1/16, the obscurity image also became thicker, making it difficult to determine agglutination. However, for albumin kO, containing more than 4N quadrants, the detection sensitivity did not change immediately after freeze-drying and remained stable. there were.
凍結乾燥直後に検出感度が低下しなかったアルブミン０
．４重を部以上含有群につき４℃に保存し、更に長期安
定性試験上行い、その結果を表１に併り己した。Albumin 0 whose detection sensitivity did not decrease immediately after freeze-drying
．． Groups containing 4 parts or more were stored at 4°C, and a long-term stability test was conducted, and the results are summarized in Table 1.
（余 白）
実施例２ ゛
実施例１と同様にして精製ＨＢｓ抗原會、グルタルアル
デヒド処理およびタンニン酸処理したニワトリ赤血球に
感作した。(Blank) Example 2 Chicken red blood cells treated with purified HBs antigen, glutaraldehyde treatment, and tannic acid were sensitized in the same manner as in Example 1.
このＨＢａ抗原感作ニワトリ赤血球の５ ％ （Ｗ／マ
）浮遊液にヒトアルブミンｔ５％（ｖ／マ）濃度に加え
凍結乾燥して赤血球１重量部に対してアルブミン１重量
部を含有する赤血球凍結乾燥物會得た０凍結乾燥前後の
検出感度および陰性儂の大きさ岬に差は認められなかっ
た０
この凍結乾燥後の感作赤血球は４℃で１２ケ月間保存し
た場合でも検出感度の低下は認められなかった０
実施例３
実施例１と同様にしてヒ）Ｏ型赤面球會用いてＨＢｓ抗
原感作ヒ）Ｏ型赤面球浮遊数音得た。A 5% (w/ma) suspension of HBa antigen-sensitized chicken red blood cells was added with human albumin at a concentration of 5% (v/ma) and lyophilized to freeze red blood cells containing 1 part by weight of albumin per 1 part by weight of red blood cells. No difference was observed in the detection sensitivity before and after freeze-drying and the size of the negative one. Even when the sensitized red blood cells after freeze-drying were stored at 4°C for 12 months, the detection sensitivity decreased. Example 3 In the same manner as in Example 1, H) HBs antigen sensitization was carried out using type O blush bulbs.
このＨＢｓ抗原感作ヒト０型赤血球の５ Ｓ （Ｗ／マ
）浮遊液にウシアルブミンｔ４９６（ｗ／マ）濃度に加
え凍結乾燥して、赤血球１重量部に対してアルブミン０
．８重量部全含有する赤血球凍結乾燥物會イ与り。Bovine albumin T496 (w/ma) concentration was added to this 5S (w/ma) suspension of HBs antigen-sensitized human type 0 red blood cells and lyophilized to give a concentration of albumin 0 to 1 part by weight of red blood cells.
．． A lyophilized red blood cell solution containing 8 parts by weight was used.
凍結乾燥前後の検出感度および陰性像の大きさ等に差は
認められなかった。No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.
この凍結乾燥後の感作赤血球は４℃で１２ケ月間保存し
た場合でも検出感度の低下り認められなかった。Even when the freeze-dried sensitized red blood cells were stored at 4°C for 12 months, no decrease in detection sensitivity was observed.
実施例４
ＨＪ３ｅ抗ＷＬを用釦抗体結合セファロ〜スで精製し、
以下実施例１と同様にしてＨＨｅ抗原感作ヒツジ赤血球
浮遊液金得た。Example 4 HJ3e anti-WL was purified using button antibody-conjugated sepharose,
Thereafter, in the same manner as in Example 1, an HHe antigen-sensitized sheep red blood cell suspension was obtained.
このＨＢｅ抗原感作ヒツジ赤血球の５９６ （ｗ／ｖ
）浮遊液にウシアルブミン１に：５％（Ｗ／マ）濃度に
加え凍結乾燥して赤血球１重量部に対してアルブミン１
車景部會含有する赤血球凍結乾燥物會得た。596 (w/v) of this HBe antigen-sensitized sheep red blood cell
) Add 1 part of bovine albumin to the suspension at a concentration of 5% (W/ma) and freeze-dry to add 1 part of albumin to 1 part by weight of red blood cells.
A lyophilized red blood cell containing carcinogen was obtained.
凍結乾燥前後の検出感度および陰性像の大きさ等に差は
認められなかった。またこの凍結乾燥後の感作赤血球は
４℃で１５ケ月間保存した場合でも検出感度の低下は認
められなかった。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.
実施例５
抗ＨＪ３ｓモルモット血清より硫安分画法およびＤＥ
Ａ Ｅ −＋ ＃ ｏ−スイオン交換吸着法にょシ精製
刊り抗体を得た。Example 5 Ammonium sulfate fractionation and DE from anti-HJ3s guinea pig serum
AE −+ #O-purified antibody was obtained using ion exchange adsorption method.
この抗体を用い実施例１と同様にして抗ＨＢｓ抗体感作
ヒツジ赤血球浮遊液を得た。Using this antibody, an anti-HBs antibody-sensitized sheep red blood cell suspension was obtained in the same manner as in Example 1.
この抗ＨＢ ｓ ＃作ヒツジ赤血球の５％（Ｗ／Ｖ）浮
遊液にウシアルブミン會３チ（Ｗ／マ）濃度に加え凍結
乾燥して赤血球１重量部に対してアルブミン０．６重量
部を含有する赤血球凍結乾燥物を得た０
凍結乾燥前後の検出感度および陰性像の大きさ等に差Ｆ
ｉ認められなかった。またこの凍結乾燥後の感作赤血球
は４℃で１８ケ月間保存した場合でも検出感度の低下Ｆ
ｉ認められなかった。A 5% (W/V) suspension of this anti-HBs #-produced sheep red blood cells was added with a concentration of bovine albumin (W/M) and lyophilized to give 0.6 parts by weight of albumin per 1 part by weight of red blood cells. A lyophilized red blood cell containing 0 was obtained. Differences in detection sensitivity and size of negative image before and after lyophilization
i was not recognized. Furthermore, even when this freeze-dried sensitized red blood cell is stored at 4°C for 18 months, the detection sensitivity decreases.
i was not recognized.
実施例６
抗ヒトインターフェロン（α）ウマ血消よ５、ＤＥＡＥ
−セファデックスおよびヒトインターフェロン結合セフ
ァロース４Ｂ會用いて抗ヒトインターフェロンウマ抗体
を精製した。Example 6 Anti-Human Interferon (α) Equine Blood Antioxidant 5, DEAE
- Anti-human interferon horse antibodies were purified using Sephadex and human interferon-conjugated Sepharose 4B.
この精製抗体音用い実施例１と同様にして抗ヒトインタ
ーフェロン感作ヒツジ赤血球會得た。Using this purified antibody, anti-human interferon sensitized sheep red blood cells were obtained in the same manner as in Example 1.
この抗ヒトインターフェロン感作ヒツジ赤血球の５％（
Ｗ／ｖ）浮遊液にヒトアルブミン會５チ（Ｗ／Ｖ、ｌ
８度に加え凍結乾燥して赤血球１重量部に対してアルブ
ミン１！Ｅ量部を含有する赤血球凍結乾燥物ケ得た。5% of this anti-human interferon sensitized sheep red blood cells (
W/v) 5 h of human albumin (W/V, l) was added to the suspension.
In addition to 8 degrees of freeze-drying, 1 part by weight of red blood cells contains 1 part of albumin! A lyophilized red blood cell product containing 1 part E was obtained.
凍結乾燥前後の検出感度および陰性像の大きさ等に差は
認められなかった。No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.
また、この凍結乾燥後の感作赤血球は４℃で１８ケ月間
保存した場合でも検出感度の低下は認められなかつ友。In addition, even when this freeze-dried sensitized red blood cell was stored at 4°C for 18 months, no decrease in detection sensitivity was observed.
実施例７
抗ヒトウロキナーゼウサギ血清よシ、硫安分画、ポリエ
チレングリコール分画およびＤＥＡＥ−セルロースクロ
マトグラフィー法ケ用いて抗ヒトウロキナーゼ抗体ケ梢
製した。この精製抗体音用い実施例１と同様罠して、抗
ヒトウロキナーゼ感作ヒツジ赤血球浮遊液を得た。Example 7 Anti-human urokinase antibodies were prepared using rabbit serum, ammonium sulfate fractionation, polyethylene glycol fractionation, and DEAE-cellulose chromatography. This purified antibody was used in the same manner as in Example 1 to obtain an anti-human urokinase-sensitized sheep red blood cell suspension.
この抗ヒトウロキナーゼ感作ヒツジ赤血球の５％（Ｗ／
ｖ）浮遊液にウシアルブミンｋ ２ 懺ｔ （ｗ／ｖ）
１１１度に加え凍結乾燥して赤血球１重量部に対してア
ルブミン０．４ｊｉＪｉ：＃に含有する赤血球凍結乾燥
物會得た。5% (W/
v) Bovine albumin k2 (w/v) in suspension
The mixture was heated to 111 degrees and lyophilized to obtain a lyophilized red blood cell containing 0.4 jiJi:# of albumin per 1 part by weight of red blood cells.
凍結乾燥前後の検出感度および陰性像の大きさ等に差は
認められなかった。No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.
ま几、この凍結乾燥後の感作赤血球は４℃で１８ケ月間
保存した場合でも検出感度の低下は認められなかつ友。However, even when this freeze-dried sensitized red blood cell was stored at 4°C for 18 months, no decrease in detection sensitivity was observed.
実施例８
アルファフェトプロティン陽性ヒト腹水よシイオン交換
吸着法、ゲルろ適法および抗アルファフェトプロティン
抗体結合セファロース吸着法會用いて精製アルファフェ
トプロティンを得た０このアルファフェトプロティンを
用い実施例１と同様にしてアルファフェトプロティン感
作ヒツジ赤血球浮遊液會得た。Example 8 Purified alpha-fetoprotein was obtained from alpha-fetoprotein-positive human ascites using the ion-exchange adsorption method, gel filtration method, and anti-alpha-fetoprotein antibody-conjugated Sepharose adsorption method. Using this alpha-fetoprotein, the same procedure as in Example 1 was carried out. An alpha-fetoprotein sensitized sheep red blood cell suspension was obtained.
このアルファフェトプロティン感作ヒツジ赤血球の５１
（ｗ／ｖ）浮遊液にウシまたはとトアルブミン′に５％
（Ｗ／マ）１度に加え凍結乾燥して赤血球１重量部に対
してアルブミン１重量部を含有する赤血球凍結乾燥物′
に得た。51 of this alpha-fetoprotein-sensitized sheep red blood cell
(w/v) 5% bovine or toalbumin in suspension
(W/Ma) Freeze-dried red blood cells containing 1 part by weight of albumin per 1 part by weight of red blood cells after being lyophilized once
I got it.
凍結乾燥前後の検出感度および陰性像の大きさ等に差Ｖ
ｉｇめられながった。There is a difference in detection sensitivity and size of negative image before and after freeze-drying.
ig was disappointed.
この凍結乾燥後の感作赤血球１４℃で１２ケ月間保存し
た場合で本検出感度の低下は認められなかった。No decrease in detection sensitivity was observed when the sensitized red blood cells after freeze-drying were stored at 14° C. for 12 months.
実施例９
ヒト甲状腺全細切した上清液より硫安分画によりサイロ
グロブリンを精製した。この精製抗原を用い実施例１と
同様にしてサイログロブリン感作ヒツジ赤血球浮遊液を
得た。Example 9 Thyroglobulin was purified from the supernatant of whole human thyroid gland sliced by ammonium sulfate fractionation. Using this purified antigen, a thyroglobulin-sensitized sheep red blood cell suspension was obtained in the same manner as in Example 1.
この５％（Ｗ／Ｖ）浮遊液にヒトアルブミンを５％（Ｗ
／ｖ）＋！’Ｉｉに加え凍結乾燥して赤血球１重量部に
対してアルブミン１ｍ−を部を含有スる赤血球凍結乾燥
物を得た。Add 5% (W/V) human albumin to this 5% (W/V) suspension.
/v)+! In addition to 'Ii, lyophilized red blood cells were obtained containing 1 m-part of albumin per 1 part by weight of red blood cells.
凍結乾燥前後の検出感度および陰性像の大きさ等に差は
昭められなかつ友。The difference in detection sensitivity and size of negative image before and after freeze-drying remains unchanged.
この凍結乾燥後の感作赤血球は４℃で１２ケ月間保存し
た場合でも検出感度の低下は認められなかつ友。Even when this lyophilized sensitized red blood cell was stored at 4°C for 12 months, no decrease in detection sensitivity was observed.
実Ｍ＠世月０
抗ヒト胎盤性ゴナドトロピンヒツジ血清より硫安分画、
ＤＥＡＥ−セルロースクロマトグラフィー法會用いて抗
ヒト胎盤性ゴナドトロピン抗体全精製し友。この！′Ｉ
！！製抗体を用い実施例１と同様にして抗ヒト胎盤性ゴ
ナドトロピン抗体感作赤血球浮遊液を得た。Mitsu M @ Segeki 0 Anti-human placental gonadotropin Ammonium sulfate fraction from sheep serum,
Anti-human placental gonadotropin antibodies were totally purified using DEAE-cellulose chromatography method. this! 'I
! ! An anti-human placental gonadotropin antibody-sensitized red blood cell suspension was obtained in the same manner as in Example 1 using the produced antibodies.
このＦｓ９ｂ （Ｗ／Ｖ）浮遊液にウシアルブミンを５
％（Ｗ／Ｖ、）濃度に加え凍結乾燥して赤血球１重量部
に対してアルブミン１３１量部を含有する赤血球凍結乾
燥物會得た。Bovine albumin was added to this Fs9b (W/V) suspension for 5 minutes.
% (W/V) concentration and lyophilized to obtain a lyophilized red blood cell containing 131 parts of albumin per 1 part by weight of red blood cells.
凍結乾燥前後の検田＃度および陰性像の大きさ等に差は
認めらｎなかつた〇
この凍結乾燥後の感作赤血球１４４℃で１２ケ月間保存
した場合でも検出感度の低下は認められなかった。No difference was observed in the degree of detection and the size of negative images before and after freeze-drying. No decrease in detection sensitivity was observed even when the sensitized red blood cells after freeze-drying were stored at 144°C for 12 months. Ta.
実施例１１
抗ヒツジ赤血球ウサギ血清より硫安分画、ＤＥＡＥ−セ
ルロースクロマトグラフィー法を用いて抗ヒツジ赤血球
ウサギ抗体を精製し次。このｎＩ製抗体を用い実施例１
と同様にして抗ヒツジ赤面球つサギ抗体ヒ）０型光Ｉｌ
′１１球浮遊液會得た。Example 11 Anti-sheep red blood cell rabbit antibodies were purified from anti-sheep red blood cell rabbit serum using ammonium sulfate fractionation and DEAE-cellulose chromatography. Example 1 using this nI antibody
Similarly to
'11 A ball suspension was obtained.
この５％（Ｗ／Ｖ）浮遊液にヒトアルブミンを５％（Ｗ
／ｖ）１１度に加え凍結乾燥して赤血球１乗１部に対し
てアルグミン１重景部を含有する赤血球凍結乾燥物紮得
た。Add 5% (W/V) human albumin to this 5% (W/V) suspension.
/v) at 11 degrees and lyophilized to obtain a lyophilized red blood cell containing 1 part of argumin per 1 part of red blood cells.
凍結乾燥前後の検出感度および陰性像の大きさ等に差は
認められなかった。No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.
この凍結乾燥後の感作赤血球は４℃で１２ケ月間保存し
た場合でも検出感度の低下は閣められなかった。Even when this freeze-dried sensitized red blood cell was stored at 4°C for 12 months, there was no decrease in detection sensitivity.
実施例Ｉ２
ヒトバセドウ病患者の甲状腺組織よシ超高速遠心分画法
によりミクロゾームに分離し友。このミクロゾーム會実
施例１と同様にしてバセドウ病のミクロゾーム感作ヒツ
ジ赤血球浮遊液ケ得た。Example I2 Thyroid tissue of a human Graves' disease patient was separated into microsomes by ultra-high-speed centrifugal fractionation. A microsomal sensitized sheep red blood cell suspension for Graves' disease was obtained in the same manner as in Example 1.
この５％（Ｗ／ｖ）浮遊液にウシアルブミンを５％（ｗ
／ｖ）濃度に加え凍結乾燥して赤血球１乗量部にｒｊＬ
でアルグミン１重景部を含有する赤血球凍結乾燥物ケ得
た。Bovine albumin was added to this 5% (w/v) suspension at 5% (w/v).
/v) In addition to the concentration, lyophilize and add rjL to 1 part of red blood cells.
A lyophilized erythrocyte containing a large amount of algumin was obtained.
凍結乾燥前後の検出感度および陰性像の大きさ等に差は
認められなかった。No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.
またこの凍結乾燥後の感作赤血球は４℃で１２ケ間保任
し７′Ｃ場合でも検出感度の低下は認められなかった。The sensitized red blood cells after freeze-drying were kept at 4°C for 12 days, and no decrease in detection sensitivity was observed even at 7'C.
実施例１３
溶血性連鎖球菌の培養ろ液より硫安分画法奮用いストレ
プトリジン０、ストレプトキナー−Ｖｔ−１１１製した
。これらの精製抗原を用い実施例１と同様にしてストレ
グトリジン０感作ヒツジ赤血球浮遊液およびストレグト
キナーゼ感作ヒツジ赤血球浮遊液を得た。Example 13 Streptolysin 0 and Streptokiner-Vt-111 were prepared from the culture filtrate of hemolytic streptococci by ammonium sulfate fractionation. Using these purified antigens, a stregtolysin 0-sensitized sheep red blood cell suspension and a stregtokinase-sensitized sheep red blood cell suspension were obtained in the same manner as in Example 1.
この５％（Ｗ／Ｖ）浮遊液にヒトアルブミン會５％（Ｗ
／ｖ）＃ｉに加え煉結乾録して赤血球ｌｉｔ部に対して
アルブミンｌＪｆ量部會含有する凍結乾燥物を得た。This 5% (W/V) suspension contains 5% (W/V) human albumin.
/v) In addition to #i, a lyophilized product was obtained by calcination to obtain a lyophilized product containing lJf of albumin per lit part of red blood cells.
凍結乾燥前後の検出感度および陰性像の大きさ等に差扛
認められなかった。No difference was observed in detection sensitivity or size of negative image before and after freeze-drying.
この凍結乾燥後の感作赤血球は４℃で１２ケ月間保存し
た場合でも検出感度の低下は認められなかった０
実施例１４
ブドウ球菌、緑膿菌、肺炎双球菌、肺炎桿菌、太１１＃
ｌ菌、インフルエンザ菌、マイコプラズマ、トキソプラ
ズマなどの菌体膜成分を破音波破壊法、フェノール水加
温法などで抽出し、また破傷風菌、ガス壊迫菌の菌体外
毒素を硫安分画法で精製した。これら全実施例１と同様
にして菌体膜あるいｔ′ｉ菌体外毒素感作ヒツジ赤血球
浮遊液を得た。Even when this freeze-dried sensitized red blood cell was stored at 4°C for 12 months, no decrease in detection sensitivity was observed.
Bacterial membrane components such as Haemophilus tetanus, Haemophilus influenzae, Mycoplasma, and Toxoplasma are extracted using the sonic destruction method and phenol water heating method, and exotoxins of Clostridium tetani and Clostridium gastrogenus are extracted using the ammonium sulfate fractionation method. Purified. In the same manner as in Example 1, a bacterial cell membrane or a t'i bacterial exotoxin-sensitized sheep red blood cell suspension was obtained.
この５％（Ｗ／Ｖ）浮遊液にウシアルブミンを５％（Ｗ
／マ）濃度に加え凍結乾燥して赤血球１重蓋部に対して
アルブミン１重量部を含有する赤血球凍結乾燥物會得た
。Bovine albumin was added to this 5% (W/V) suspension at 5% (W/V).
/m) In addition to the concentration, lyophilization was performed to obtain a lyophilized red blood cell product containing 1 part by weight of albumin per 1 layer of red blood cells.
凍結乾燥前彼の検出感度および陰性像の大きさ等に差は
認められなかった。No differences were observed in the detection sensitivity and size of negative images before freeze-drying.
この凍結乾燥後の感作赤血球は４℃で１２ケ月間保存し
た場合でも検出感度の低下は認められなかった。Even when the freeze-dried sensitized red blood cells were stored at 4°C for 12 months, no decrease in detection sensitivity was observed.
実施例１５
ヒツジ赤血球、ニワトリ赤血球、ヒト０型赤血球、ガチ
ョウ赤血球、ウシ赤血球およびウサギ赤血球を生理食塩
溶液で４回洗浄後、フォルムアルデヒド又はグルタルア
ルデヒドで処理し残余のフォルムアルデヒド又祉グルタ
ルアルデヒドを遠心分離法で除去した。これら固定赤血
球のｌｓをタンニン酸処理し、残余のタンニン酸會遠心
分離法で除去した。これら固定赤血球およびタンニン酸
処理赤血球の４チ浮遊液に、ヒトアルブミン、ウシアル
ブミン又はウマアルブミンを終濃度が２％（Ｗ／マ）お
よび５％（Ｗ／Ｖ）になるように添加し凍結乾燥して赤
血球１重量部に対してアルブミンをそれぞれ０．５重量
部及び１゜２５重量部含有する赤血球凍結乾燥物を得た
。この凍結乾燥物音、等帰化リン酸緩衝液を加えて再浮
遊せしめ、同じ等帰化リン酸緩衝液に対する反応を赤血
球沈降像の直径全測定することにより比較した。２〜５
％（Ｗ／マ）のアルブミン添加凍結乾燥後のそれぞれの
赤血球はいずれも凍結乾燥前のそれと比べ変化は認めら
れなかったが、アルブミン無添加の状態で凍結乾燥した
ものはいずれも赤血球沈降像の直径が大きくな夛、凍結
乾燥によ）性状が変化していることが推１ｊｔｌｌ 烙
れた。Example 15 Sheep erythrocytes, chicken erythrocytes, human type 0 erythrocytes, goose erythrocytes, bovine erythrocytes, and rabbit erythrocytes were washed four times with physiological saline solution, then treated with formaldehyde or glutaraldehyde, and the remaining formaldehyde or glutaraldehyde was centrifuged. Removed by separation method. The ls of these fixed red blood cells were treated with tannic acid, and the remainder was removed by tannic acid centrifugation. Human albumin, bovine albumin, or horse albumin was added to the 4-cell suspension of these fixed red blood cells and tannic acid-treated red blood cells to a final concentration of 2% (W/MA) and 5% (W/V) and freeze-dried. Lyophilized red blood cells containing 0.5 parts by weight and 1.25 parts by weight of albumin per 1 part by weight of red blood cells were obtained. This lyophilized sample was resuspended by adding naturalized phosphate buffer, and the response to the same naturalized phosphate buffer was compared by measuring the total diameter of the erythrocyte sedimentation image. 2-5
% (W/ma) of each red blood cell after lyophilization with the addition of albumin, no change was observed compared to that before lyophilization, but the erythrocyte sedimentation image of each erythrocyte lyophilized without the addition of albumin was It was speculated that the properties had changed due to the large diameter and freeze-drying.
筐た、この凍結乾燥後の感作赤血球は４℃で１２ケ月間
保存した場合でも検出感度の低下は認められなかった。However, no decrease in detection sensitivity was observed even when the sensitized red blood cells after freeze-drying were stored at 4°C for 12 months.
一］ ・−Ｓｌ 刊 第１頁の続き ０発 明 者 船越哲 交野市青山１丁目１６番５号one] ・-Sl Published Continuation of page 1 0 shots Akira Funakoshi 1-16-5 Aoyama, Katano City
Claims (1)
以上を含有してなる赤血球凍結乾燥物。 （２） 赤血球の５チ（Ｗ／ｖ）濃度水浮遊液を使用
した場合にはアルブミンが２％（ｗ／マ）＃度以上とな
るような割合でアルブミン全添加した赤血球水浮遊液を
凍結乾燥して製造して第（１）項記載の赤血球凍結乾燥
物。 （４）赤血球が任意の抗原又は抗体を感作せしめた感作
赤血球である特許請求の範囲第（１）項記載の赤血球凍
結乾燥物。[Claims] (11 Lyophilized red blood cells containing 0.4 parts by weight or more of albumin per 1 part by weight of red blood cells.) (2) Use of a suspension of red blood cells in water at a concentration of 5% (W/v). In this case, the red blood cell freeze-dried product according to item (1) is produced by freeze-drying a red blood cell aqueous suspension to which albumin has been completely added at a ratio such that the albumin content is 2% (w/ma) or more. (4) The lyophilized red blood cell according to claim (1), wherein the red blood cell is a sensitized red blood cell sensitized with any antigen or antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57072253A JPS58131566A (en) | 1982-04-28 | 1982-04-28 | Lyophilized matter of red blood cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP57072253A JPS58131566A (en) | 1982-04-28 | 1982-04-28 | Lyophilized matter of red blood cell |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57013862A Division JPS58131913A (en) | 1982-01-29 | 1982-01-29 | Preparation of lyophilized erythrocyte |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS58131566A true JPS58131566A (en) | 1983-08-05 |
Family
ID=13483940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP57072253A Pending JPS58131566A (en) | 1982-04-28 | 1982-04-28 | Lyophilized matter of red blood cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58131566A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5340592A (en) * | 1988-05-18 | 1994-08-23 | Cobe Laboratories, Inc. | Lyophilization of erythrocytes |
| US5425951A (en) * | 1988-05-18 | 1995-06-20 | Cryopharm Corporation | Method of reconstituting lyophilized cells |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4921052A (en) * | 1972-06-16 | 1974-02-25 | ||
| JPS5423119A (en) * | 1977-07-25 | 1979-02-21 | Takeda Chem Ind Ltd | Erythrocyte for haemagglutination test |
-
1982
- 1982-04-28 JP JP57072253A patent/JPS58131566A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS4921052A (en) * | 1972-06-16 | 1974-02-25 | ||
| JPS5423119A (en) * | 1977-07-25 | 1979-02-21 | Takeda Chem Ind Ltd | Erythrocyte for haemagglutination test |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5340592A (en) * | 1988-05-18 | 1994-08-23 | Cobe Laboratories, Inc. | Lyophilization of erythrocytes |
| US5425951A (en) * | 1988-05-18 | 1995-06-20 | Cryopharm Corporation | Method of reconstituting lyophilized cells |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
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