JPS63158463A - Monoclonal antibody to human pancreatic amylase, preparation thereof and quantitative analysis of human pancreatic amylase using said antibody - Google Patents

Abstract

PURPOSE:To easily perform the fractional quantification of the amylase derived from the pancreas and the amylase derived from a salivary gland, by using a novel monoclonal antibody specifically reacting with human pancreatic amylase. CONSTITUTION:A hybridoma is formed between the antibody producing cell of a mouse immunized with human pancreatic amylase and the myeloma cell of the mouse and cultured. An anti-human pancreatic amylase monoclonal antibody is sampled from this cultured substance. This anti-human pancreatic amylase monoclonal antibody has specificity to human pancreatic amylase. Therefore, by using this monoclonal antibody, the amylase occurring from the pancreas can be fractionally quantified with respect to the amylase occurring from a salivary gland.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention provides a novel monoclonal antibody that specifically reacts with human pancreatic amylase (hereinafter referred to as anti-P-amylase monoclonal antibody), a method for producing the same, and an anti-P-amylase monoclonal antibody.
- This relates to an immunological method for quantifying human pancreatic amylase using an amylase monoclonal antibody, and more specifically, an anti-P-amylase monoclonal antibody and an extremely effective method for distinguishing between isozymes, such as the differential quantification of human amylase. The present invention relates to a manufacturing method thereof and an immunological quantitative method using the anti-P-amylase monoclonal antibody for quantifying human pancreatic amylase, which is extremely important in clinical tests such as diagnosis of pancreatic diseases.

α-amylase exists in human blood and urine, and it has long been known that it increases in pancreatic and salivary gland diseases9.
, is currently an important measurement item in clinical tests.

There are two isozymes of amylase: amylase derived from the pancreas (hereinafter referred to as P-amylase) and amylase derived from the salivary glands (hereinafter referred to as S-amylase), and it is important to separate and quantify them in order to improve the accuracy of diagnosis. That's true. In particular, the determination of P-amylase is an important measurement item indispensable for the diagnosis of pancreatic diseases, and the development of a method for quantifying it in the clinical setting is desired.

By using the anti-P-amylase monoclonal antibody invented this time, it is possible to easily measure P-amylase by using it in endime immunoassay or radioimmunoassay.

Therefore, it is easy to immunologically quantify P-amylase in human blood, and it can be used in clinical tests.

(Prior art) There are several methods for measuring amylase isozyme: electrophoresis, a method using amylase inhibitors, a method using Dull filtration, and a method using mudgraphy with affinity.Among these, electrophoresis is the method that has been put to practical use. and a method using amylase inhibitors. However, both methods have drawbacks, such as the electrophoresis method requiring special equipment and complicated operations, and the amylase inhibitor method not having sufficient measurement accuracy because the specificity of the inhibitor is not perfect. Therefore, although amylase activity measurement itself is performed very frequently, isozyme measurement is not performed very often.

In addition, an isodime-specific assay method using a monoclonal antibody that inhibits only the enzyme activity of S-amylase without inhibiting the enzyme activity of P-amylase has also been reported (hereinafter referred to as immunoinhibition method). However, 5.% (183098/1989) has not been put into practical use.

In this immunoinhibition method, the activity of S-amylase is inhibited using an S-amylase specific antibody, and the enzymatic activity of the remaining P-amylase is measured.

There is a risk of inaccuracy due to the influence of impurities and the cross-reactivity of antibodies with P-amylase (Problem that the invention aims to solve) Electrophoresis and amylase are actually used for clinical diagnosis. This method uses an inhibitor.

Electrophoresis requires special equipment and has the disadvantage of being complicated and time-consuming, and methods using amylase inhibitors have imperfect specificity, i.e.
Since the amylase inhibitor not only reacts with S-amylase, but also cross-reacts with P-amylase by about 20%, there are drawbacks such as insufficient measurement accuracy.

In addition, all methods, including the immunoinhibition method, measure amylase isozyme if enzyme activity, and cannot measure protein activity.

These drawbacks can be overcome by performing enzyme immunoassay or radioimmunoassay using a single P-amylase monoclonal antibody.

(Means for solving the problem) The amino acid sequence of P-amylase is very similar to the amino acid sequence of S-amylase excluding the sugar chain portion, so until now, P-amylase has not been immunologically analyzed. It was thought to be impossible to identify. In fact, there have been no reports yet of obtaining anti-P-amylase antibodies that do not have reactivity with S-amylase.

If it does not react at all with S-amylase, but P-
If an antibody that specifically reacts with amylase is obtained, P-amylase can be easily quantified by performing enzyme immunoassay or radioimmunoassay using the antibody. Based on these ideas, the present inventors developed the conventional amylase isozyme ti
In order to eliminate the shortcomings of the ll fixed method, as a result of extensive research, P
It was possible to prepare a monoclonal antibody that has high reactivity with -amylase but little reactivity with S-amylase. If an enzyme immunoassay is performed using the invented anti-P-amylase monoclonal antibody, P-amylase can be easily and accurately detected.
It can also be measured as a protein broom.

Published by 5hler and C. Milstein in α (
Nature, 256.495-497.1975
) since.

Many reports have been made regarding the production of hybridomas, but in addition to these methods, the present inventors used highly pure P-amylase as an antigen in order to obtain an anti-P-amylase monoclonal antibody with excellent characteristics. The fetal bovine serum used as a culture medium is lot-checked, and those with high efficiency in forming hybridomas are carefully selected and used.Myeloma cells are selected from those that are in a complete logarithmic growth phase. 2. A large number of cell fusions and screenings were carried out while paying attention to sufficient observation of the proliferation of hybridomas and ensuring that the timing of screening, cloning, etc. was not delayed.

The lot check for fetal bovine serum in (b) above was performed in view of the fact that the quality of fetal bovine serum, which is an important factor in cell culture, varies greatly depending on the manufacturer and loft. The method is [monoclonal antibody-2, hybridoma and ELISAJ, Tatsuo Iwasaki et al., Kodansha, 105-107
According to the method on page.

R, PMI 1640 medium containing 15% fetal bovine serum is prepared for each fetal bovine serum. Myeloma cells (X
6 B-Ag8-6.5.3 strain) or a self-prepared noibridoma was diluted with each medium to a 96-well culture plate so that the number of cells per well was 0.5, 1, 2, 3. Seed and culture (in a carbon dioxide incubator, 37
℃). After culturing for about 14 days, each flea well was observed with an inverted microscope, the wells in which colonies had formed were counted, and 25 to 100 μf of P-amylase, which was highly purified from pancreatic juice with a colony formation efficiency of 9, was added to physiological saline with 0. Dissolve in 1 ml and mix and emulsify with Complete 70 Indoor Adjuvant at a ratio of 1:1.

In order to obtain a complete emulsion, it is necessary to put the antigen into one of two syringes connected by a connecting needle and the adjuvant into the other, and to mix the contents by alternating the pistons of both syringes. The prepared emulsion is injected intraperitoneally into 5-10 m old BALB/c mice. 3~
After rearing for 54 days, an emulsion of P-amylase and incomplete Freund's adjuvant was prepared in the same manner and injected into the peritoneal portal. One to two additional boosters are given in the same manner at 8-5 minute intervals. 2-4 weeks after boosting 25-50 μ2 of P-amylase in 0.1 saline
ml and injected into a vein. Eight days after the final immunization, the spleen is aseptically removed using clean pliers, loosened and suspended in RPMI-164.0 medium, and used for cell fusion.

On the other hand, mouse BALB/c myeloma cells P3-X
63-Ag 8-U 1 strain (hereinafter referred to as P8Ul).

or X 63-Ag8-6.5.3 strain (hereinafter referred to as X-653
) or Pa-N5 I-1-Ag4-1 strain was removed from cryopreservation, washed, and incubated with HT (+FC8) medium (RP containing hypoxanthine, thymidine, and 15% tallow serum).
Suspend the cells at 5 x 105 cells/me in MI-1640 medium) and culture in a Petri dish or culture bottle. A medium is added every day according to the growth of #I cells, and the cells are cultured for 5 days to obtain cells in the logarithmic growth phase. Observe the cells in each Petri dish or culture bottle, and select those with good proliferation and cell condition to use for cell fusion.

1-2×10 splenic cells and 2×10 myeloma cells
The cells were collected by centrifugation, and then added to RPMI-1640 medium for 8 hours.
Wash twice. Centrifuge again to remove the supernatant, and thoroughly mix the spleen cells and myeloma cells. Next, 100% polyethylene glycol of 50% was added and stirred gently for 1 minute.

Add 1 ml of RPMI-1640 medium at 80 second intervals,
Add lO- in about 5 minutes. Cells were collected by centrifugation and processed into 60me in HT (+FC8) medium. Dispense 0.1 to 0.15 μl into each well of a 96-well cell culture plate and culture in a carbon dioxide culture finger.The next day, add HAT (+FC8) medium (hypoxanthine/aminopterin) to each well - RPMI-1640 medium containing thymidine and 15% beef tallow serum) is added at a rate of approximately 0°lme. After 8 or 6 days, aspirate half of the medium and replace with new H.
Add AT (+FC8) medium in increments of about 0°1. 7-14
After several days, as soon as the hybridoma colonies reach a size visible to the naked eye, EL I S using peroxidase is performed.
A (Enzyme Li-nked Irrmun
Hybridomas producing anti-P-amylase antibodies are screened by the osorbent As5ay) method.

In the ELISA method, commercially available horseradish peroxidase-labeled goat anti-mouse Ig is used as the labeled antibody.
ABTS [2-2'-ad
ino-di-(8-ethylbenzothya
zol in-5ulfonate)), S-7 Mit-Seha commercial product was used as it was as the antigen, and P-
Amylase purified from pancreatic juice is used.

Select only hybridomas that produce antibodies that are weakly reactive to S-amylase and highly reactive to P-amylase, and subjected to limiting dilution twice or more using thymocytes or lung cells as feeder cells. Perform cloning to obtain a monoclonal hybridoma cell line.

From the obtained cell lines, those with no cell proliferation and antibody specificity are selected by subculturing, intraperitoneal culture, cryopreservation, etc. to obtain the desired cell line.

The following operations are performed to isolate and purify the antibodies produced by each cell line.

After culturing the hyperdomas in dishes.

Mice were previously injected intraperitoneally with pristane (0.51 nl/mouse) and were injected with 5 X 10 Ibridomas per mouse.
'; Inject 2xlO intraperitoneally. Breed the mice and collect ascitic fluid when sufficient ascites has accumulated.

Centrifuge the obtained ascites, collect the supernatant, and discard the precipitate.

After adding sodium azide to the supernatant to a ratio of 0.1, sodium sulfate was slowly added at a ratio of 0.18 t to 1 of ascites while stirring at room temperature. After the addition is complete, stir for an additional 80 minutes to ensure sufficient precipitation. Centrifuge (10,00 m, 10 minutes) and collect the precipitate in $40 mM sodium phosphate buffer (8
Redissolve in buffer A containing 0 mM sodium chloride, pH 8.0. The ligated material was dialyzed against buffer A at 4°C overnight, and the resulting precipitate was removed by centrifugation (IOlooorpm, 10 minutes). When the supernatant was passed through a DEAE-cellulose column previously equilibrated with buffer A and eluted with the same buffer, peak I
r get. Subsequently, elution with 4QmM sodium phosphate buffer (containing 1M sodium chloride, pH 8.0) yields a peak (2).Peak I is an IgG fraction, and peak (2) is an IgM fraction. Each fraction @
After condensation, Sephacryl 5-ao, which had been equilibrated with 0°1M sodium phosphate buffer (pH 7,5), was used.
Gel filtration is performed using an o column. □IgM is eluted in the void porium, and IgG is eluted at a position corresponding to a molecular weight of 150,000. Each fraction is concentrated and used as purified antibody.

Each purified antibody was tested for reaction specificity to S-amylase and P-amylase using a method similar to the ELISA method used in hybridoma screening,
It shows a strong color reaction with P-amylase, but it shows a strong color reaction with S-amylase.
It was confirmed that there was no or almost no color reaction with amylase.

EIA using anti-P-amylase monoclonal antibody, such as Santoy assay method or competitive method. By performing RIA, amylase isodeme can be easily quantified. In clinical diagnosis, low concentrations of amylase isodeme must be measured very accurately and without being affected by serum. Therefore, the labeled antibody used in Cytoi and Chi EIA was obtained by purifying anti-amylase antiserum using a conventional antibody purification method and using the method of Ishikawa et al. (JournaA of I
mmunoassay+ 4(3)+ 209-327
(1983), POD-labeled and purified by affinity chromatography using a column immobilized with KS-amylase.

Anti-P with strong P-amylase specificity obtained by the present inventors
-7 amylase monoclonal antibody is used as a solid phase, and POD-labeled Fab produced by affinity chromatography is used as a labeled antibody. Sandwich ELA is used to completely react with S-amylase in a wide concentration range including blood level P-amylase concentration. A standard curve with good linearity can be obtained.

The measurement limit is filf mol per analytical tube (1f mol/150
μt), it is possible to detect up to 1 fmol when 10 μt of serum is used as a specimen. In other words, the limit of detectable P-amylase concentration in serum is 100f.
mole/g (corresponding to 5.4 ng assuming the molecular weight of P-amylase to be 54,000). The concentration of P-amylase in the serum of a normal person is said to be 55 to 250 ngAl (see page 59 of "Immunological measurement and clinical application of blood enzymes" by author Michio Ogawa). It can be said that the sensitivity is sufficient for quantitative determination.

In addition, this system can specifically measure only P-amylase, and has no or almost no cross-reaction with S-amylase over a wide concentration range, which was previously thought to be unavoidable. - Not affected by amylase measurements.

Furthermore, using this system, a so-called addition recovery test was conducted in which P-amylase was added to serum and the recovery rate of P-amylase was measured, and recovery rates of 89 to 116% were obtained. This shows that P-amylase in serum can be accurately quantified using this system.

Until now, in sandwich EIA, it has been helpful to use antiserum or monoclonal antibodies in the same phase and use antiserum as the labeled antibody, but in order to obtain antibodies of stable quality and reproducible results, It is desirable to use monoclonal antibody 2 for both in-phase and labeled antibodies.

Therefore, a monoclonal antibody with high P-amylase specificity was coated on the same phase of polystyrene beads or microassay plates, etc., and the labeled antibody was used as a labeled antibody that reacts with both P-amylase and S-amylase in addition to affinity-purified rabbit Fab. Monoclonal antibody without P-amylase specificity? The system used was also tested with Tentoy, Cheer, and Sei, and it was found that a good standard curve could be obtained.

(Example) The present invention will be explained in more detail with reference to Examples below.

Example 1 Purified P-amylase used as an antigen was produced by Matsuu
The method described by ra et al. (Journal of Bioc
hemistry 83329-332 (1978)
) was obtained by producing 'ffII using the following method.

After adding 5 μl of aprotinin to 200 WLl of pancreatic juice, centrifugation was performed to remove the precipitate. Add 20% corn starch solution [20% kl Q (ie ammonium solution) to the supernatant]
PI-18,0) 300m/, 60t of cornstarch
1 was heated at 75° C. for 30 minutes with occasional stirring] 125- was added, and the mixture was gently stirred at 5° C. for 22 hours. Thereafter, filtration is performed to collect the cornstarch to which P-amylase has been adsorbed. In order to extract the adsorbed P-amylase, 5mM calcium acetate buffer (pH 6,0) containing 5mM sodium chloride was added to the obtained corn starch, and the mixture was stirred at room temperature for 1 hour.

The filtrate was collected by filtration. This extraction operation was repeated seven times to obtain FN825m/ containing P-amylase. After concentrating the filtrate to about 100 rnl by ultrafiltration,
For 1.5 t of 5mM calcium acetate buffer (pH 7,0) containing 5mM sodium chloride.

Dialysis was performed at 4°C for 20 hours. After dialysis, insoluble materials were removed by centrifugation to obtain a colorless and transparent solution 108-. The obtained solution was preliminarily diluted with 5mM sodium cyclide.
D equilibrated with calcium acetate buffer (7,0)
EAE-cellulose column (diameter 2.6 crn, height 8
1/-F+) to obtain 115+ ng of a non-adsorbed fraction containing P-amylase.

It is processed by ultrafiltration for about 12. After concentrating to d.

Dialysis was performed at 5°C for about 20 hours against 1 t of 1mM calcium acetate buffer (pH 7,0) containing 1mM sodium chloride. After dialysis, a Sephadex G-100 column (diameter 2.6
cm, height 71cW1).

Fraction 68- containing P-amylase was obtained.

After concentrating by ultrafiltration, the obtained P-amylase was frozen and stored at 105,000 units (according to amylase measurement kit Amylase Test Wako).
, 87! It is a protein and showed a single band at a molecular weight of approximately 60,000 in SDS poly-noacrylamide gel electrophoresis.

After physiologically emulsifying the protein with 50μ of the purified P-amylase obtained, it was intraperitoneally administered to 6-week-old BALB/c mice. After 4 weeks, incomplete Freund's azimuth
The mixture was mixed and emulsified with a band (manufactured by Difco), and a second immunization was performed. After another 3 weeks, P-amylase 50 μf was added to physiological saline 0.17! It was dissolved in and injected intravenously. Three days after the final immunization, the tile 111i was aseptically removed and treated with RPMI.
The cells were loosened and suspended in -1640 medium and subjected to cell fusion.

On the other hand, mouse BALB/c myeloma cells P8
Ul (manufactured by Flow Laboratories) was removed from cryopreservation, and after washing, the number of cells was 5x in HT (+FC8) medium.
The cells were suspended at a density of 10 cells/- and cultured in Vetridissie. Add medium every day.

Culture was carried out for 5 days to obtain cells in the logarithmic growth phase.

The cells were suspended in RPMI-1640 medium and subjected to cell fusion.

Approximately 2 x 10 pancreatic cells and 2 x 10' myeloma cells were collected by centrifugation and washed three times with RPMI-1640 medium. The supernatant was removed by centrifugation again, and the pancreatic cells and myeloma cells were mixed with light. Then 50%
After adding 0% polyethylene bricola OOQI + l me and stirring gently for 1 minute, 1- RPMI-1640 medium was added at 80 second intervals, and 10 me was added for 5 minutes. The cells were collected by centrifugation, suspended in 60 ml of HT (+FC8) medium,
0 in each hole of a 96-hole plate (manufactured by Pekton Ditsuyaun)
．． 12 mt each was dispensed and cultured. The next day, HAT each hole.
(+FC8) medium was added in Q and l me doses. After 3 and 6 days, discard about half of the medium in each hole and replace with new H.
AT (+FC8) medium 0°17! 08 days after adding the solution, select a well with a large colony of hybridomas,
Hybridomas producing anti-amylase antibodies were screened by ELISA using peroxidase. Weak reactivity towards S-amylase<, P
- Select a hybridoma clone with strong reactivity to amylase, perform limiting dilution twice and clone it,
Hybridoma cell lines 8Gl and 5D4 were obtained.

Each hybridoma was cultured and expanded in a dish, and blistane was injected intraperitoneally (0.5 d/min) in advance.
One BAL B /c mouse was injected intraperitoneally with 1 x 10 rehydromas.

The mice were kept for 2 weeks and the accumulated ascitic fluid was collected.

The obtained ascites was centrifuged, the supernatant was collected, and sodium azide was added to the supernatant to give a concentration of 0.1%. After adding 0.92 □ of the sodium acid, the mixture was further stirred for 30 minutes to cause sufficient precipitation. Centrifugation (10,00Orp
m, 10 minutes) and collect the sediment.

Redissolved in buffer A5-. The resulting solution was placed in a dialysis tube and dialyzed against buffer A at 4°C overnight. The resulting precipitate was centrifuged (l 0.00Q rpm,
l Q minutes) and removed. The supernatant was pre-equilibrated with buffer A on a DEA g-cellulose column (
(inner diameter 1.0 ctn, length 12 m) and eluted with the same buffer to obtain a peak ■. Next, 4QmM sodium phosphate buffer (containing 1M sodium phosphate, pH 8)
, 0) to obtain a peak ■.
Pass through an aoo column (inner diameter 1.5 cm, length 45 mm),
Gel filtration was performed. IgM is in the void volume, I
gG is eluted at a position with a molecular weight of 150,000.

1 from mice injected with the hybridoma cell line 3G1.
8 fnt of ascitic fluid was obtained per animal, and 52 IgM of protein was obtained by purification using the above purification method. 8 fnt of ascitic fluid was obtained from each mouse injected with the hybridoma cell line 5D4, and 58 WIg protein IgG was obtained by purifying it.

Each purified antibody was tested using the same ELISA method used in hybridoma screening.
- When the reaction specificity for amylase and P-amylase was tested, antibody 3G1 showed a strong color reaction to P-amylase, but almost no color reaction to S-amylase. Furthermore, antibody 5D4 showed a strong color reaction with P-amylase, but no color reaction with S-amylase.

Example 2 X-658 (Flolabora) I as mini 0-ma cells
Hybridoma cell line 8C12 and antibody 8C12 manufactured by Isei Co., Ltd. were obtained in the same manner as in Example 1, except that the hybridoma cell line 8C12 was used.

Hypuridoma 3Gl obtained in Example 1 and Example 2
, 5D hand and 8C12 characteristics are shown in Table-1.

Table 2 shows the characteristics of the antibodies produced by this hybridoma.
It was shown to.

[Margin below]

In Table 2, the reaction specificity was expressed by the color intensity determined by the rJLISA method.

The fact that it reacted with P-amylase in the ELISA method indicates that an antigen-antibody reaction occurred.

The classes and subclasses were determined as follows.

Assay plate encoding P-amylase (
Becton Dickinson Company 1! ! ), dispense 50 μt of each diluted antibody solution into 5 wells. After standing at 37° C. for 2 hours, each well is washed with phosphate buffered saline (hereinafter abbreviated as PBS). Peroxidase-labeled anti-mouse subclass antibody ff, (IgG+, IgG2a, Ig
G2b, IgG3. IgM (both manufactured by Zymed) was added and reacted at 37°C for 2 hours. After washing each well at least 5 times with PbS and thoroughly draining the water,
Add BTS solution to develop color. The subclass can be determined from one well that developed color among the wells to which each subclass antibody was added.

Molecular weight was determined by gel filtration using Sephacryl 5-3oo.

Example 3 Preparation of anti-S-amylase antiserum S-amylase (manufactured by Sigma) 0.51vt-dissolved in 0.5d of physiological saline and mixed with complete 70 indoor adjuvant (containing myeobacterlum: manufactured by Dlfco) and 1=1 The mixture was emulsified and injected into the rabbit's dorsum.

After 1 month, add 0.5 rv of S-amylase to 0 saline.
．． 5d and then injected into the ear vein of the same rabbit. After 8 days, stratified whole blood was collected and 35 days of serum was obtained.

3.69 g of sodium sulfate was gradually added to 20 d of serum at room temperature with stirring to form a precipitate. Centrifugation (10,
00Orpme for 10 minutes), collect the precipitate, and add 40mM
The solution was redissolved in a sodium chloride buffer (containing 30 mM sodium chloride, pH 8.0, hereinafter abbreviated as buffer N). The lysate was dialyzed against buffer N at 4°C overnight, and the resulting sediment was centrifuged (10.00 rpm, 1
0 minutes) and removed. The supernatant was passed through a DEAE-cellulose column equilibrated with buffer N and eluted with the same buffer to obtain an IgG fraction. After concentrating the obtained IgG fraction by ultrafiltration, it was added in advance to 0.1M sodium phosphate buffer (pH 7,
Gel filtration was performed using a Sephacryl S-300 column equilibrated in 5). The target IgG was eluted at a position corresponding to a molecular weight of 150,000. Fractions were concentrated by ultrafiltration.

Preparation of POD conjugate Fab Rabbit anti-S which also reacts equally with the obtained P-amylase
- After dialyzing amylase IgG against 0.1M sodium acetate buffer (pH 4, 5) overnight, add Begusin in a protein amount corresponding to 5% of the IgG protein amount, and store at 37°C for 24 hours.
Time incubation was performed. The resulting reaction solution was pre-mixed with 0.1M sodium phosphate buffer (pH 7).
, 0) equilibrated with Ultroglu AcA 44 column (
2.5 cm in diameter and 45 cm in height (manufactured by LKB) to obtain an F(ab)2 fraction. After concentrating it to about 101Q/go by ultrafiltration, 0.5
MIJ sodium dihydrogen acid? In addition, - was adjusted to 6.0. It was dissolved in 0mM mercaptoethylamine and incubated at 37°C for 90 minutes.
It became AB. In order to remove unreacted mercagutoethylamine, 0.1M sodium phosphate buffer (f
'16. The mixture was applied to a Cefazo, Kusu G-25 column (diameter 1 cm e height 453) equilibrated with o) to obtain a Fab fraction. Approximately 2 minutes using Centricon (Amicon tA)
It was concentrated to 00μt.

On the other hand, a maleimide group was introduced into POD. 0.925
9 N -(g -maL@1m1do-eaproy
A solution of 0.0511 Ll of dimethylformamide and 61n9 PO
D to 0.95aA! A solution of 0.1M sodium phosphate buffer (-7) was mixed and incubation was performed at 30°C for 30 minutes. In order to introduce maleimide groups and separate 7'j POD and unreacted EMC8, 0.1M sodium phosphate buffer (PI(6,0
) equilibrated with a Sephadex G25 column (diameter 1c
The reaction solution was applied to the POD fraction (rIM, height 453) to obtain a POD fraction into which a maleimide group had been introduced. Approximately 200μ with Sentricon
It was concentrated to t and used for reaction with Fab.

Equimolar numbers of Fab and maleimide groups were introduced and mixed with nine PODs, and ink, page, and printing were performed at 30°C for 1 hour.
OD and Fab are reacted. In order to block unreacted maleimide groups, 10mM mercagtoethylamine was added and inking, page printing was performed at 30°C for 10 minutes. The reaction solution was prepared in advance with 0.1M sodium phosphate buffer (
P.O.
A D-Fab conduit and dirt fraction was obtained.

POD condi & f-) Fab's 7 Finitei chromatographically purified S-amylase 10%, f-purinffer (0,
After dialysis against 1M sodium bicarbonate (pH 8.0) overnight, the mixture was centrifuged (3,000 rpm, 19 minutes) to obtain a supernatant containing S-amylase. This S-
The amylase solution was added to CNB r-activated Sepharose 4B (
7 (manufactured by Almasia) and stirred at room temperature for 3 hours.

More than 90 S-amylases were immobilized on Sepharose 4B by this method. Procking pa, fur (
Soaked overnight in Tris-HCl buffer, PI(8,0). Next, the glue was washed 4 to 5 times alternately with Cassilling buffer and acetate buffer (containing 0.5M sodium chloride, pH 4.0), and then washed with 0.01M sodium phosphate buffer (0.1% bovine serum). albumin containing pi4
7. O).

Glu 0,13WL immobilized with prepared S-amylase
1 was packed in a column (3 cm in diameter, 7 cm in height) and mixed with 10% phosphate buffer (containing 0.1 M sodium chloride and 0.1 T bovine serum albumin, pH 7.0, hereinafter abbreviated as Buffer B). Washed.

Next, add the previously prepared POD conshugo) tab solution (
9.08 m1i110.58 m) was flowed at a flow rate of 1 mJ/hr to adsorb it. After washing with buffer 810d,
The Fab was eluted by running one drop of 3.2mM hydrochloric acid (pH 2.5) over 10 minutes.

The eluent was immediately diluted with 1M sodium phosphate buffer (pH
7,0) was neutralized.

Affinity chromatography purified POD condi, goo)
Fab was an Ultroda # AcA 44 column (diameter 1.
The POD Fab was fractionated using a centrifugal tube (5 mm, 100 cut in height), concentrated using a centricon, and then used for sand germination.

Preparation of monoclonal antibody P-amylase monoclonal antibody 5D obtained in Example 1
The 4-producing hybridoma strain was injected intraperitoneally into mice that had been previously injected with pristane in the same manner as in Example 1.
The animals were kept for about two weeks to allow them to develop ascites. Ascitic fluid was collected, and monoclonal antibodies were purified by treatment with a protein A column (Pai Daini Tsudo Co., Ltd.).

A coating solution was prepared by dissolving the sandwich assay monoclonal antibody in 0.1M sodium phosphate buffer (PH7.0) to a ratio of 0.1147/WLl. Polystyrene sol (diameter 3.2 ml, thoroughly washed with distilled water)
(purchased from Ichikoyoshi Co., Ltd.) was immersed in the coating solution at 4° C. overnight, taken out, and immersed in buffer A for one day or more for blocking.

The length of the Delistin L/ball (hereinafter abbreviated as antibody coated ball) coated with a monoclonal antibody is 10 m.
After washing with M phosphate buffer 7 (p) (7-0), dilution series of S-amylase and P-amylase prepared using buffer B (1 to 1.000 fmol/1
50#t) and -next reaction? I made it happen. Next, after washing with 10 mg sodium phosphate buffer (-7), the affinity-purified POD conduit was adjusted to a POD concentration of 50 nV 150 μt with buffer B, 'y' -).
It was immersed in a Fab solution to perform a secondary reaction. After the reaction -
10mM sodium phosphate buffer (Fk47
).

The activity of POD bound to the antibody coating by antigen-antibody reaction was measured using 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) as a coloring agent.
(abbreviated as ABTS) to perform a color reaction and wavelength 4
This was carried out by measuring the absorbance at 10 nm. The measurement results are shown in Table 3 and Figure 1.

Table 3 Absorbance (41°nm) measured values in Sand German chia and Sei using monoclonal antibody 5D4 ■ and the value obtained by subtracting the blank value ■ Example 4 Anti-P-amylase monoclonal antibody obtained in Example 2 and stored frozen Santoi and Chia were treated in the same manner as in Example 3, except that the antibody 8C12, which had previously been used, was used.

Said.

The measurement results are shown in Table 4 and Figure 2.

Table 4 Santoi using monoclonal antibody 8C12,
Measured value of absorbance (41°nm) in Chia and Sei ■ and the value after subtracting the blank value ■ Example 5 A labeled antibody that recognizes P-amylase and S-amylase almost equally as shown in Table 5. A sandwich assay was conducted in the same manner as in Example 3, except that the clonal antibody 7F8 was used and O-7 enylenediamine, which is more sensitive than ABTS, was used as the chromogenic substrate.

The measurement results are shown in Table 6 and Figure 3.

Monoclonal antibody 7F8, like other monoclonal antibodies, was obtained by immunizing mice with P-amylase and screening among hybridomas obtained by cell fusion.

Table 5 Characteristics of Monoclonal Antibody 7F8 In Table 5, the reaction specificity is shown by the color intensity measured by ELISA.

The fact that it reacted with P-amylase in the ELISA method indicates that an antigen-antibody reaction occurred.

Determination of class and subclass was performed using ELISA as a labeled antibody using an yl oxidase-labeled anti-mouse subclass antibody solution (IgG1+IgG2atIgG2b+IgG3e).
IgM+ (both manufactured by Zymed) was used.

Molecular weight was determined by filtration with Sephacryl 5-soo.

Table 6 Absorbance (492 nm) measurements in Santoi and Chi assays using monoclonal antibody 5D4 as a solid phase and monoclonal antibody 7F8 as a labeled antibody Furthermore, in order to prove that P-amylase in serum can be measured with this measurement system, 281 Serum A showing different P-amylase concentrations.

A sandwich assay was performed on B and P-amylase added at a concentration of 3s, and the recovery rate of P-amylase was determined (a so-called addition recovery test was conducted).

The results showed a good recovery rate throughout, as shown in Table 7.

Functions and Effects of the Invention If an enzyme immunoassay kit or radioimmunoassay kit is prepared using the anti-P-amylase monoclonal antibody invented this time, it will be easy to detect it in blood or urine. P-
Amylase can be quantified separately. That is, amylases include P-amylase, which increases in the blood due to pancreatic diseases, and S-amylase, which increases in patients with parotitis.

To measure P-amylase, the pneumophoresis method, which requires complicated operations, or the inhibitor method, which inhibits S-amylase activity and measures the remaining P-amylase activity, was often used. Each had drawbacks. Measuring using an anti-P-amylase monoclonal antibody has the following advantages: (1) Unlike electrophoresis, special equipment is not required.

Furthermore, measurements can be easily performed.

■Anti-P-amylase monoclonal antibody directly reacts only with P-amylase, instead of inhibiting S-amylase activity and using the remaining activity as the amount of P-amylase, as in the inhibitor method and immune inhibition method. Since it can be quantified,
Can be measured accurately and precisely.

(2) The amount of P-amylase can be measured not as the enzyme activity that has been conventionally measured, but as the amount of protein. Therefore, it seems that it can be accurately regarded as a disease.

■This antibody can be used in various measurement systems.

For example, it is effective for measuring P-amylase in blood if used in endime immunoassay, radioimmunoassay, or latex method.

■ Compared to the enzyme activity in the specimen, which is easily deactivated, the antigenicity is stable, so the specimen can be easily stored and handled when measured by the assay method using this antibody.

[Brief explanation of the drawing]

FIG. 1 shows a standard curve in a sandwich assay using monoclonal antibody 5D4. FIG. 2 shows a standard curve in a sandwich assay using monoclonal antibody 8C12. FIG. 3 shows a standard curve in a sandwich assay using monoclonal antibody 5D4 as a solid phase and monoclonal antibody 7F8 as a labeled antibody. Patent applicant Kojin Co., Ltd. Figure 1 Amylase concentration (fmol/150ul) Figure 2

Claims (3)

[Claims] (1) An anti-human pancreatic amylase monoclonal antibody that has specific reactivity with human pancreatic amylase but has no or almost no reactivity with human salivary amylase. (2) A hybridoma is formed between antibody-producing cells of a mouse immunized with human pancreatic amylase and mouse myeloma cells, the hybridoma is cultured, and an anti-human pancreatic amylase monoclonal antibody is collected from the culture. A method for producing an anti-human pancreatic amylase monoclonal antibody. (3) An immunological method for quantifying human pancreatic amylase, which comprises quantifying human pancreatic amylase using a monoclonal antibody specific for human pancreatic amylase.