JPS60123427A - Monoclonal anti-insulin antibody and purification of insulin using the same - Google Patents

Abstract

PURPOSE:To provide the titled antibody produced by the hybridoma formed by the fusion of the cell from the tumor line of mouse and the pancreatic cell of a mouse immunized with insulin. CONSTITUTION:The cell from the tumor line of mouse (e.g. P3-X63) is fused with the pancreatic cell of a mouse immunized with insulin, and the formed hybridoma is selected. The hybridoma is cloned twice or more to obtain a prefect monoclone. When the clone is established, the cell is cultured in vitro or in vivo to obtain a monoclonal anti-insulin antibody. The antibody has high specificity to insulin, and exhibits excellent effect when applied to the purification of insulin. High-purity insulin can be produced easily, in high yield, without necessitating a long and complicated process.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a single roan anti-I//Yuri/antibody with high specificity for insulin, a hybridoma producing the same, and a single roan anti-I//Yuri/antibody. This invention relates to a method for purifying Yin/Yuri/ using.

Inotyulin U is a pegutide hormone secreted by islet β cells in the pancreas, and regulates sugar, fat, and chorion in tissues such as the liver. When insulin production in the body is insufficient, it becomes difficult for sugar to accumulate in the liver and the liver, resulting in a rapid increase in blood sugar and the ability of the kidneys to absorb glucose to be unable to keep up with the increase, leading to diabetes.

Therefore, Ino/Nirin is very useful as a treatment for diabetes.
Therefore, the development of an inexpensive production method for I/Noyuri/ (1) has extremely important significance for human health and medical development.

Industrially, insulin is generally extracted and purified from animal pancreatic tissue, and K P
, K. Because it contains many types of proteins other than insulin, it is necessary to develop a method for specifically purifying insulin.

Conventionally, the method of Fisher et al. has been used for the industrial purification of Ing.
ndI+Jngineering Chemistry
32 908 (1940)) (.

This pre-preparation method rf, pancreas (orchid) → mincing → hydrochloric acid acidic ethanol extraction → centrifugal separation → − to clear Pl (adjustment (pH 8,0) → filtration → filtrate PII adjustment (sulfuric acid acidity) → vacuum concentration → pH adjustment (PH
2-25) → 50°C heating filtration → filtrate salting out (salt 25%
) → Precipitate redissolution (water) → PH adjustment (pt (1,8~22
)→filtration→filtrate salting out (salt 15% precipitate redissolution (pH 2)
~25) → PH adjustment (PH5, Q ~ 54) → Left at low temperature for 48 hours → Filtration → Re-dissolution of precipitate (Fuji 1 acid) → Acetate/addition → P1-1 adjustment (pH 5, 9 ~ 60) → Room temperature for 2 hours Leaving → Filtration → Dilution of filtrate → Addition of zinc acetate → Leave at room temperature for 24 hours →
Leave at low temperature for 24 hours → Filtration → Re-dissolve the precipitate (dilute acetic acid) → Acetic acid! 111 Lead addition → P11 adjustment (Pl-16, l~66)
→ PH readjustment (pH 5, 9) → Standing at room temperature for 48 hours → Zinc acid/Shuri/crystallization As shown in this process, it is a long and complicated process, so improvement was desired. Since then, various improved methods have been proposed for the extraction and purification process, but the essential principles have remained largely unchanged and the 11 previous methods have been followed, resulting in long and troublesome production processes. That hasn't changed even now. For this reason, innovation/
Deterioration in quality due to denaturation of Nilin, especially the formation of desamide insulin, is unavoidable, and there is still room for improvement from the economic point of view.

In order to solve this problem, the inventors focused on the specificity of antibodies in order to simplify the step of producing σ) after insulin extraction.1. I thought about applying this.

However, using antiserum may interfere with Ino/Nirin purification because the antiserum contains other proteins, impure antibodies, or anti-insulin antibodies with low affinity. Ta. Therefore, we thought that this problem could be solved by using a single IJ-on substance, ``7.'', which has a strong affinity for insulin 2.As a result of intensive research, we completed the present invention. reached.

The first step in achieving the object of the present invention is to produce a novel monoclonal hybridoma that produces antibodies. Although specific details will be shown in Examples, the process simply consists of the following three steps.

1. Immunity 2. Cell fusion 3. Hybridoma selection and monoclonal 7-conjugated immunization antigens,
1. Uses Yin/Lily/ obtained from animals commonly used for insulin production.

In general, the raw material animal species for i7ru4//lily/ used in antidiabetic drugs include pigs and pigs, so it is desirable to use them as the immunizing antigen1.For insulin, physiological saline, Alternatively, it is preferable to dissolve it in a buffer or the like and apply 3001 tg of It19 to each mouse or rat at a time. stomach. Immunization f-1 is carried out in several doses, but the first immunization is generally carried out together with horse mackerel and J bunt. , Froy/Froy as an adjuvant
Toajuba/to, Myoba/, etc. are used. Immunization is carried out at intervals of 2 to 1 week, and the final immunization is with Ajiyuba/
Dissolve the drug in physiological saline or the like and administer it intraperitoneally or intravenously. Rats and mice are commonly used as immunized animals. This is determined by the tumor cell line used for cell fusion, and 13A I, B/c, which is an established mouse cell line that does not produce immunoglobulin, is often used. 2 to 40 days after the final immunization, the 77 segments or kites are removed, and the resulting 77 bulbs are subjected to cell fusion.

On the other hand, as tumor cell lines used for cell fusion, P3-X63-8AZ-U ] and P:, + -N S + -], which do not produce immunoglobulin, are used. During cell fusion, 5 to 20 times more lymphocytes than tumor cells are used. 4EM medium, McCoy medium, It i)
After washing with M, 11640 medium or isotonic buffer solution Ki1, lymphocytes and tumor cells are pelleted by centrifugation.

Helenoth 2A'J, <" I, A=after,) I V J
(Center Iheals) Or, add O (polyethylene glycol) and perform cell fusion.
Usually, 40-60% of LOOU ~ 8,000 is used for 05-2 me.As a fusion accelerator, I'
J・', (When adding J, a small amount of dimenal sulfoxide may be added, but it is not essential.

11 E (Add J solution to the cells and perform fusion reaction 1-1.
After about 0 minutes, add MJ No.M medium or I(P
Gradually add M11640 medium to 10~50 me to stop the reaction. 1. After stopping the fusion reaction, centrifuge immediately and remove the supernatant. 11 Fetal serum (Fe2) 5-20
% Containing Mt> M medium or PMJ] Suspend the cells in 640 medium, and place them in a 24-well culture plate with 1 m of cells so that there are 105 to 5 x 106 lymphocytes per well.
Dispense 1' each. In either case, it is preferable to add feeder cells. As feeder cells (eg, syngeneic rat or mouse thymocytes, spin cells, etc. are used, and added at a concentration of 0.5 to 2 x 10''/ml'. Next, Hibokiza/Chi/1 x] ,0'M,
, aminobutyryl/4x]0-7M, fmi):/1.6X
R4' M i]640 medium (or MEM medium) containing IO-5M, ie, HAT medium.

Generally, the method for replacing the II A T medium is to add n1 equal to the volume dispensed after fusion into a culture flute the next day, and replace half of it with 11 A i' medium on the second day. Replace half the amount with J-I Ai'' medium every 3 days. Remove aminophyterin from 10 to 14 days after fusion.
, Replace half of L with 11'1゛ medium, then fI for 1 to 3 days.
Replace half of the JK medium with normal medium that does not contain HA i'. The culture supernatant of wells in which fused cells (hybridoma) are actively proliferating is analyzed using various analysis methods, such as It I A, EL.
Select the desired antibody-producing hybridoma using ISA or the like.

We will perform cloning of hybridomas, but the method is 11J+” A CS (FIuo+
°escentActivpt, ed (-:ell
5orter), Sof tA, gar
There is a commonly used method to pick up more colonies, such as the limiting dilution method. Whichever method you use - repeat at least two times to ensure complete measurements or in vivo measurements.
By culturing the monoclone 1/1. Anti-(y
Antibodies/antibodies are detected (4fI-).

Either the in vitro method or the in vivo method may be used, but the in vivo method is preferable because it has much higher potency and antibody titer.

The novel lucuro anti-insulin antibody of the present invention (2A
3-6) is the result of analysis using the bipedal diffusion method 7J
・Confirm that it belongs to the mouse 1g01 class],
Puko. EJ using peroxidase-conjugated anti-mouse immunoglobulin as the second antibody.

l S A (1'V elementary connection f\immunity measurement method) for each in7
Examining the reactivity with anti-antibody 2 A 3-(i), the results are shown in Figure 1.This allows us to test the antibody tj1 for pigs, ``Nun Yuri/, human i/N Yuri 7, etc.''
(-anti+, 1=, position, affinity is also sufficient (lI# Rgsa7t/(,t, single clone anti-I//Yuri/antibody 6-I/Nyurin purified) It is necessary to separate the I/7 urin that binds with the antigen-antibody reaction from other types of γj, but as a means of preventing this, we immobilized the antibody and Combined /
A method of separating flea white matter, etc. that does not bind to pharyngeals using the so-called affinitake method is considered.As a method of immobilization, cellulose, tegist, acarose, polyacrylamide, porous glass, vinyl There are methods of binding water-insoluble Jl1 antibody to polymers, nylon/, boristrene, amino acid copolymers, etc., and methods of making the antibody itself insolubilized using gel polymerization method C (-). As a bonding method with a small Rr i? phase, a method using halogenation/a/, a method using periodic acid, a suberly (e.g., diazonium derivative, thiol derivative, ω-aminoargylamine derivative, etc.) are used. There are methods, etc., considering the 4/l quality of the antibody, the immobilization mechanism, and the 1・-1・quality of the in-phase 1'i, :, l-(
, I don't use a noble method;>Q It's good to be wrong.

On the other hand, anti-1) was used as an antibody, a spacer consisting of kurtararzohi l', □f nopuananate derivative, bisdiazo/di//, N,N'-ethyl/7 bismaleimito snow, 1 and 2 to form a polymer. What antigen-antibody reaction (adsorption) and binding to the immobilized antibody/
Elution of ζ1 Non-Yuri/ is performed in one container in a suitable container, and can be used for one-way flow method, one-way Hanobu method, Izu, and 11. For kelized antibodies, the Q' batch method is appropriate. Hard>
In order to combine J-engineered antibody or V'gelled antibody with test f-, Pll is 4~jO1 salt concentration is 2-1 or more 1・Ip2+ :/ Is it necessary to roll? In order to elute 1)) 1 or less than 1 or more than 10' or the salt concentration is 2M or more, or one organic solvent. For example, using 50% ethanol, 50% ethylene glycol, etc.
11 Vt-t7i ;ge 1 normal θ) method of crystallizing the produced Ino/Yuri/ from the eluate, i.e., acetic acid! 11
You can use the method of adding lead to make 477117 songs (1) salt. Since the jJ-7 anti-Ino/Yuri/ antibody according to the present invention is a highly specific 141 antibody against I/Suri/, it can be used as an antibody against In/Yuri/ as described above. When applied to 'n-made products, Hitoi/Yuri/, Butai//Yuri/
It exhibits excellent purification effects in all cases, regardless of whether it is beef lily or Japanese lily, and does not involve the long and complicated steps of conventional methods, and has the major advantage of (i).

The present invention will be explained in more detail with reference to Examples below.
Present Invention 1-1 It is needless to say that the present invention is not limited in any way by these.

EXAMPLE: Preparation of a hybridoma producing a mono-Rho/sex anti-Ino/Yuri/antibody and Kotl.

(1) Immune Tsuku 1 // Lily 710071g 1N IQI I-,
, fc O, ] I'vl acetic acid 0.1 mt pco/Preton Roy/Doerspant.
, It AIIIf/c mice (female, 6 AJ
ll+'3)'s j return 11'1 ('(-).

・1. ll! ! After 1, the insulin I O(] not,y %-(1,l 8I f!! acid(1,2m(K
Wj IQ'F L,, J also LL shooting (-7
G7, (2) Cell fusion Three days after the final immunization, the spleen of the immunized mouse was removed and
The splenic lymphocytes are loosened in a plastic Petri dish containing ml of RPM11640 medium. The splenic lymphocytes were washed three times with RPM11640 medium by repeated centrifugation (1000 rpm, 10 minutes). 1 x 108 splenic lymphocytes and 1 x 107 mouse myeloma cells P3-NS1-1 were mixed in a test tube and precipitated by centrifugation. After removing the supernatant by suction, loosen the precipitate slightly. 50% polyethylene glycol (
Add 1 ml (average molecular weight 6,000) to the loosened precipitate,
The fusion reaction was carried out for 1 minute at room temperature while rotating the test tube. , then every 30 seconds, 1 ml of 111640 medium to It, I) for 5 min 1'i,! The reaction was stopped by adding λ.

Immediately centrifuge (I OOL 1 rotation, 5 minutes) to remove the tweeter cells.
lln', J・' (-: S (tallow blood 7?t) drink-
H2I'l'VI I + (+4'
OJ'! Cells were suspended in 5Qml. , 2-
Culture the cells in a root plate of 1 well at 1 minute of 1 minute CO per hole in an inoguvator.

24 hours later, II A 'I' medium (Hibokiri/)-
I x 104 8'1, Amino Fite
li //IXI (1 7 to 1, -f- mi /7
1, (La X 1. ()-” contains 4
2 0 So δ J(” (−: S cold force III also P
M 11 6 4 0 J@ground) 1 hole afc j2,
1ml'-J" Q 7Jl+ *- Le.

20th, 3 1-4+" J et al. K 2 ]"4Q:' V
C JM Jyano'r+i'. ':+: IIAT
The medium was changed again. On the 10th day, half of the medium was mixed with Aminobutyry/6, minus/.
II 1' 7j He changed his father in the land. 21 days after the notification date, the normal tr: □i ground t) 20% F (: S addition J
, I) was replaced with 4 1 l 6 71 0 J'ir, and the 18F1 culture supernatant was treated with anti-I/
/Lily/Used for antibody production assay.

(3) ...) Free 1 - Ibritoma selection / / Yuri / Antibody production / 1 - ... Ibritoma selection /
・-me(・ε-11-poor hole neck cell culture with 1'I(+
・: 1. Analysis at ISA1. /~, l-, l'
E1. H-IQ 1r, 9/'ml' 0) in I S Al1196-well plate
'L's Ii Te (l I me each minute 11-1, l
'Cl Oll,''' Intermediate device (7)
0 (contains 0.059% of noniface-based surfactant (ti'ill, product name of Atlas Inc.) 5-
After that, add 1% bovine serum albumin 7m solution (17k) to avoid lI-specific adsorption of proteins.
1,2me each minute 74-1-. ．． 37°G 2 o'clock IM+ quiet. Next, after washing three times with washing solution, remove the cell culture soil with O,]
, me was dispensed and left to stand for 2 hours. As a negative control, 20% J=” (+8 added I(, P M
] Dispense 640 medium (1, J rne/). After further washing 13 times with washing solution, add 0, J ml of Bell Ogyisiguse-labeled anti-mouse immunoglobulinol anti-II solution;
l-,,:37'C21t;jliJ quiet j~1~.

After washing 3 times with washing solution, dispense 04 orthophenylene/diami/bath Q, ]+1iff, and react for 2 minutes at room temperature, then 6
Add 0.05 ml of N sulfuric acid to stop the reaction, and 0.1)
, 490 nm was determined. Among the Jlij11 hybridomas showing a concentration D more than twice that of the Y1-1 control, the Jlij11 hybridoma was selected as an anti-I/7 lily/antibody-producing hybridoma. Anti-In/Yuri/antibody production was observed in 3 out of 48 wells.

(4) Monoclonal 13 A l-13/C mouse (female, (1 week old) thymus J: 3-11 x 1, l Ome+, LPMi
Place 77 thymus lymphocytes in a flask Petri dish with J640 medium.
Repeat (], Ou o rotation, 10 minutes) to Ri'
l Wash 13 times with IJ040 medium.

77 balls of thymus with 20% J=”CS added, Il, J'
The cells were suspended in 50 ml of M I I 6.10 medium. In this suspension, there are 50 antibody-producing hybridomas.
(〕pcs/m(0) W4 liquid O,] rnl was added, and after mixing well, 0 per hole in 9G hole ``u'' and J1 toflate.
．． Dispense 2 me at a time for 10 minutes (:su).

Culture in an incubator/ζ. The results of analysis of the culture supernatant in which #lII cells were observed by ELISA,
A hybrid-79 clone producing anti-Japanese lily/Japanese lily/antibody was obtained. One of these clones was selected from 6 clones of hybridomas producing anti-I/Thuurin antibody.
↓I, complete the glory and transformation.

(5) Production of monoclonal antibodies (4) One step at a time: i1/kokuro 72A3
-6.2 X I Q6 pieces Il, J' M 116
40 medium Q, 2 mL (fl suspended 1) - 1 L (A
1. +13/C mice (approximately 1.6 weeks old) were injected intraperitoneally, and condensate was collected. I'm tired of 3ml of ascites]
Slowly add 100% of sulfuric acid, make the final sulfurized A1 concentration 5θ%, stir 100% of IA crystal-C21, mix 70%, and add 30% The precipitate was collected at the same time (5 ml of physiological A water).
(Bath) 8, this is a saline saline bath for 10 years.
'``Ify-1'F' blasphemy L-t[)ki E L
I S A K Te Analysis 1, , , 17i: I'+□
The permissible dilution factor of l-7J'l is II-toko/'
), 103゛de・i・・′)/ko. In addition, mouse IgG
The content is SII I 4 no θf 'tJ ii! :l>
J Yu l-k Tokoro 22 mr) / ml! Met.

(fil 2 A, 3-0 cross-reactivity 00-hole plate (-)
The mixture was incubated at 37° C. for 2 hours to stabilize the original material.

After 4 washes with OmM acid buffer (pH 7,4) containing 0.5 mm of 1% bovine serum albumin solution, dispense 0.2 ml of 1% bovine serum albumin solution. 37°C14It
4J Shizuka 1 and 7. Next, after washing four times with the same buffer solution,
2 A, 2μ of 3-6,! ;l/mt concentration solution 5CJ
tti and various insulins (Butain Yuri/, Hii・
Insulin, i,'hr-OB u-insulin) and 50 μl of Ott9/me solution were simultaneously dispensed.

: It was left standing for 7°02 hours. After washing four times with the same buffer,
Anti-mouse immunoglobulin antibody-Periogi 7 Dase original p
1＼Bath liquid 0. ] Dispense mt and 37° 01:01 iJl
Leave it still.

Further, the solution was washed four times with the same buffer solution, and 0.4" 10 orthophenic nonamine bath solution (J, 1 ml) was dispensed, and the mixture was reacted for 1 minute at room temperature. 0.05 ml of 6N sulfuric acid was added to stop the reaction.

J,i, was measured at 490 nm. The absorbance obtained when various insulins were added at a weak absorbance was divided by 7 to obtain the inhibition rate, and the relationship between this inhibition rate and each insulin concentration is shown in Figure 1. From this, 2A3-6 is a pig/I/
Not only Shurin but also Hitoi/Shurin, 1,” h r
-OB II It can be seen that there is a cross-reaction with Lee/Nyuri/.

Example 2 (1) 2 A: 3-61i! Preparation of lJ-regulated Sepharose 4133.5 g of Brominated/A/Activated Sepharose
Wash with JM hydrochloric acid many times and swell for 7 hours. Then 0.
5 M thorium monide containing 01M carbonate & value P118
3. Wash-Jru. 01M carbonated carbonated liquid P with a swelling of 1t7j
I18.3 19mt' K suspension and 3 ml of a 50% ammonium sulfate fraction bath solution of a single low/specific antibody were added and stirred overnight at room temperature to immobilize the antibody. d, 3 g of medium clone 71/1 antibody 1 immobilized on cells from the filtrate after IXI filtration.
It was counted as 1 as l1zy. Kel was treated with 1M ethanolamine to protect the active groups. After v, i () 00184 Tris-buffered tty containing 9% sodium chloride, pH
The gel was washed and equilibrated in s3.

(2) Extraction of pig incillin Minced/minced piglet pancreas pI & 2009, added 1% hydrochloric acid-containing ethanol/150 ml, extracted ia/nilin with a homogenizer, and centrifuged (IO000 rpm, 20 minutes).
One liquid was collected. Add ammonia water to pH 80 and (-7
4f% sulfuric acid was added to the filtrate to adjust the pH to 23, and the filtrate was concentrated under reduced pressure to about 1/3 of its volume. 09%
Sodium chloride containing 0.0], M) Lys buffer p
Dialysis was performed using H8,3 as an external solution. The internal liquid was taken out and defatted with 1,1,2-)dichloro-1,2,2-trifluoroethane/I/7 lily/extract 38(
I got llj.

(3) Purification of Butai/Nyuri/ 2A3-6 immobilized Cepharose prepared in (1) of Example 2 was packed in a 15 mm diameter bed body u110m/
column. Next, 200 ml of the insulin extraction obtained in (2) of Example 2 was passed through the column.
It was washed with 0.01 M Tris buffer, pH 3, 3150 mA' containing *'% sodium chloride. Then, 30 mt' of IM acetic acid solution was passed through it to elute the in/lily/ adsorbed on the column. 3Qml of the eluate was freeze-dried to obtain 2mg of purified porcine insulin.

Figures 2 and 3 show the results of purity analysis of illegally purified purified porcine insulin products and commercially available porcine insulin (manufactured by Shimizu Pharmaceutical Co., Ltd.) using disk electrophoresis and high performance liquid chromatography. Figure 2 shows the results of electrophoresis.
J 7 (in case of a) and commercially available product (in case of b) both 1tf
A main band with a value of 0.646 is obtained. The number of bands is based on the number of illegal surnames.
There are 3 bottles of 7 yurin, i [5 wood and illegal products were of better purity among commercial products. Also, Fig. 3 f': 1. '+p, r+ The results of liquid chromatography method were 73e, but the main heat was obtained in about 50 minutes during the holding period for both the illegally purified butine 7 urin (in the case of a) and the illegally sold product (in the case of b). There is. From both analytical methods, it can be seen that the purity of Ia/Nirin obtained by this purification method G (Yoto) is 90'10 or higher.Example 3 (2) of Purification Example 2 of Unoi/Shuri/ Bovine pancreas instead of pig pancreas in
Extract insulin e by operating the same Gff using 1i'&,
400ml was obtained. This I was passed through the 2A3-6 immobilized CEF column used in Example 2 (3).
+-Lys buffer'tLPI 483.20 tl ml washed with fcO, followed by 017M glycine-hydrochloric acid with human titer solution P.
630 ml of H2 was passed through to elute the I/Shuri/ adsorbed on the column. Eluate: 30 ml was lyophilized and purified to 725 mq (725 mq) (0) The obtained illegally purified fake insulin was purified by heavy-speed liquid chromatography, and the results are shown in Figure 11.

That is, the main heat was obtained at a holding time of about 20 minutes, and
It was found that the purity of the obtained purification method was over 90%.

Example 4 (1) Preparation of gelled antibody Single rho/sexual antibody (2A 3-6) 30 ml was dissolved in 2 ml of phosphate buffer pH7.
Glutaraldehyde (36 m 7
1 mg of mt:'') was added. Stirring was continued for 20 hours at 4°C to obtain a gelled antibody. 7. Wash and equilibrate the antibody.

(2) Futainsulin Essence Example 2Q) Add 100 ml of the butai/shuri/extract obtained in step 21 to the gelled antibody obtained in (1) of Example 4, and stir at room temperature for 30 minutes. , centrifuge (3000 rpm, no rotation) and remove the supernatant. Centrifuge (3000 rpm, 10
); Repeat 3 times, containing 0.09% sodium chloride.
Wash the I/Suri/conjugated antibody with 1 M +-Lis buffer P118.3. After removing the supernatant, add 3M potassium thionanate containing 〇, 0]M phosphate buffer pH
7,020 ml was added and stirred at room temperature for 30 minutes to elute insulin from the gelled antibody to obtain eluate 19me. Purified butaino/nilin by lyophilizing the eluate], 5 m9
Obtained / Hi.

Figure 5 shows the results of purity analysis of illegally purified insulin using high performance liquid chromatography. Immediately t-
), the main peak was obtained at a retention time of about 50 minutes, and the purity of Butai/7 Yuri/ obtained by this purification method was 90%.
It turns out that it is more than %.

[Brief explanation of drawings]

Figure 1 shows the results of a competitive reaction between Butaino/Nirin bound to the solid phase and various I/Nirin present in the liquid phase against antibody 2A3-6 in Example 1-(6). This is a graph showing the following. −〇−1 is Butainshrif, −
o-o- stands for human inoshrift, and 'I'hr -OBu-intuulin'. FIG. 2 shows the results of ruching the purity of the futaine/nirin purified product in Example 2 and the commercially available butaino/nirin product (manufactured by Shimizu Pharmaceutical Co., Ltd.) by disk electrophoresis. However, a is illegally refined butai/shuri/
and b were conducted using a commercially available product. FIG. 3 shows the results of purity analysis using high performance liquid chromatography for the purified futain/nilin product in Example 2 and the commercially available butaino/nilin (manufactured by Shimizu Pharmaceutical Co., Ltd.). However, test b was made using illegally purified porcine insulin and a commercially available product. FIG. 4 shows the results of purity analysis of the bovine/shuri/purified product in Example 3 by high performance liquid chromatography. Figure 5 shows the results of purity analysis of the futaine/nilin purified product in Example 4 by high performance liquid chromatography.
It's a J comb. 'l'Till Applicant: Wako Pure Chemical Industries, Ltd. No. 1
Figure +1 (1, (l) 0.1 1.0 1f1.0 Added Insulin Concentration (/Z g/ml Figure 2 1) Figure 3; Temperature (min))] 0 30 60 Retention Time (minutes) Figure 1 Figure 5 Horo time (minutes)

Claims (1)

[Scope of Claims] (1) Fusion of cells from mouse ll1lI tumor cells with tile cells from mice previously immunized with 7-C p(-J: +1 formed 'i1 /Ko Hai Fri Doma e Koyori +) 4j /l: Sano 1 bubble /i r-+
-7 anti-inone urino antibodies. (2+ -? Cells from sea urchin ku no tachi J ↓ Shirai /, P
;3-X (i, 1 (/(-Arit4/,) cow I"
i! 'l', ii' + sword
Self-published condolence [,--,'Jt(wai,17'/yu
Li/4 liter book. 1: I + 7 us (]] Ha middle J & 3 :) I/from 2
fusion (i-, J,'su1shi-sein\)1, rli-claw/subject anti-i/ /-1-Li/J No I4 bodies? ■ξ1＞+Beef゛4 high fritoma. (4) Cells from mouse tumor line are P3-X63
The hybridoma according to claim 3 belonging to . (5) Cyclonal anti-I/Suri produced in a hybridoma formed by the fusion of cells from a mouse V) lung cancer and tile cells from a mouse 7 previously immunized with Yin/Yuri/ /Ino/Yuri/Purification method using antibodies as an opportunity. (6) The method for purifying Ino/Yuri according to claim 5, which uses an immobilized anti-Ino/Yuri antibody. (47) Soybean purification method according to claim 5 using a gelatinized simple anti-infection/infection antibody.