JPS603551A - Reagent for measuring lipase - Google Patents

Abstract

PURPOSE:To enable enzyme immunomeasurement of lipase in a human bodily fluid by forming a sandwich type conjugate of an antibody-antigen labeled antibody and measuring the enzyme activity of the labeled antibody. CONSTITUTION:An antihuman puncreatogenic lipase is manufactured by sensitizing refined human puncreatolipase to a domestic rabbit then the antibody is processed with an insoluble carrier such as a polystyrene bead, glass bead or nylon bead to adsorb the same by covalent bonding or physically on such carrier, by which the insolubilized antibody bound with the antihuman puncreatogenic lipase antibody is obtd. On the other hand, a previously manufactured antihuman puncreatogenic lipase antibody is brought into reaction successively with the compd. optimum for the enzyme used then enzyme to obtain an enzyme labeled antihuman puncreatogenic lipase antibody. The lipase in the patient's serum is measured by using the antihuman puncreatogenic lipase antibody bound carrier and the enzyme labeled antihuman puncreatogenic lipase antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent for measuring lipase in human body fluids by an enzyme immunological method using a sandwich method.

Traditionally, the measurement of lipase secreted in body fluids such as serum
It is carried out using a method based on enzyme activity. However, a specific method for measuring lipase has not yet been established due to the influence of various factors in body fluids or because its content is minute.

On the other hand, immunological methods for antigen retrieval (in-gel immunodiffusion method, complement fixation method, hemagglutination reaction, etc.) have been widely used as a means of measuring trace components in body fluids. However, radioimmunoassay (radioimmunoassay) has come to be used because of its improved measurement sensitivity. However, radioimmunoassay is not satisfactory due to environmental contamination and effects on the human body caused by radioactive substances.

Enzyme immunoassay using the reagent of the present invention utilizes an antigen-antibody binding reaction, uses an antibody labeled with an enzyme to generate an antigen-antibody conjugate, and measures the enzymatic activity of the resulting substance. The sandwich method is one of the methods used to determine the amount of unknown antigen. This sandwich method takes advantage of the fact that multiple antibodies bind to an antigen that has multiple antigen groups, generates a sandwich-type combination of antibody and antigen-labeled antibody, and measures the enzymatic activity of the labeled antibody. This is a method that attempts to determine the amount of antigen, and compared to other immunoassay methods, it has a
It has the advantage of being 10,000 times more sensitive.

Therefore, the present inventors conducted extensive research on enzyme immunoassay methods for lipase in human body fluids in order to detect changes in lipase in various diseases, and succeeded in obtaining the reagent of the present invention.

The reagent of the present invention can be produced as follows.

That is, an anti-human pancreatic lipase antibody is produced by sensitizing purified human FR-visceral lipase to a rabbit, and then this antibody is treated with an insoluble carrier such as polystyrene beads, glass beads, or nylon beads. An insolubilized antibody bound to an anti-human pancreatic lipase antibody is obtained by covalently bonding or physically adsorbing it to a phase.

On the other hand, to the anti-human pancreatic lipase antibody produced in the same manner as above, a compound most suitable for the enzyme used (for example, for β-galactosidase, m-male(3) midobenzoyl-N-hydroxysuccinimide ester, peroxidase glutaraldehyde, etc.)
Then, enzymes are reacted sequentially to obtain an enzyme-labeled anti-human pancreatic lipase antibody.

In this case, the above-mentioned compound and enzyme may be reacted first, and then this reaction product may be combined with the above-mentioned antibody.

Using the thus obtained anti-human pancreatic lipase antibody-conjugated carrier (insoluble antibody) and the enzyme-specific anti-human pancreatic lipase antibody, lipase in the serum of patients with acute pancreatitis was measured. It was found that there was an unusual increase in

Therefore, the reagent of the present invention is useful for diagnosing pancreatitis.

Next, the present invention will be explained in more detail with reference to examples.

Example 1 Production of anti-human pancreatic lipase antibody Add Freund's complete adjuvant to one rabbit and a solution containing 151 pancreatic lipase and 571
(Complete adjuvant) 1- Mix (4) Inject the prepared emulsion subcutaneously on the back and between the nails of the four legs. Two weeks after the first immunization, the same emulsion as the first immunization was subcutaneously injected into the rabbits. After repeating the same immunization procedure 4 times, blood was collected from the carotid artery of the rabbit and kept at 37℃ for 30 minutes.
After warming for 3. Centrifuge for 15 minutes at OOOrpm to obtain serum. This serum was heat-treated at 56°C for 30 minutes to immobilize complement, then 20mM phosphate buffer solution (pHs, o ) was added in the same amount as the serum, and saturated ammonium sulfate of the same volume as the serum was added. The resulting precipitate is collected by centrifugation at 12.00 rpm for 15 minutes, dissolved in a small amount of the same buffer, and then dialyzed using the same buffer to completely remove ammonium sulfate. The booklet obtained here was diluted with 20mMjl phosphoric acid warm solution (pH 8,0).
A DEAE-cellulose column (2,
5x15m), and collected the two fractions that did not adsorb to the column and flowed out with the same buffer, diluted with the same buffer so as to contain the most protein of zowhi/me, and used this as an anti-human pancreatic lipase antibody. Dispense, lyophilize, and store at -30°C.

Example 2 Production of insoluble antibodies (1) Preparation of anti-human pancreatic lipase antibody-conjugated polystyrene beads 401 kJ of the lyophilized antibody of Example 1 was dissolved in 0.1 M sodium phosphate buffer (p
H7゜0) 400-, add 000 to 10,000 polystyrene beads (about 6.5 m in diameter) that have been thoroughly washed (with a strong detergent cape) to this solution.
Shake at 0°C for 2 hours. After removing the antibody solution, 0°1
After washing the beads with the same buffer containing % bovine serum albumin, the same buffer is added and further shaken at 30°C for 2 hours. After further washing with the same buffer, the thiomersal concentration was reduced to 0.
0.01% and store the beads in the same buffer.

(2) Preparation of anti-human pancreatic lipase antibody-bound glass beads Glass beads (diameter approximately 4 wn) 5,000-10.
000 pieces were added to 200 ml of a toluene solution containing 10% γ-aminopropyl triethoxysilane, and after reacting at room temperature for 30 minutes, the solution was removed with a glass filter.
After thorough washing with toluene and drying the alkyl aminated glass beads, 20mM containing 0.2% anti-human pancreatic lipase! J acid buffer (1) H7°4) 50
After stirring well, glutaraldehyde was added to a final concentration of 0.0 mt to 1%, followed by a shaking reaction at 4° C. for 12 hours. The anti-human pancreatic lipase antibody-conjugated glass beads obtained here were mixed with Q, 001M sodium phosphate buffer containing 1% bovine serum albumin (
After washing thoroughly at pH 7.0), store at 4°C in the same buffer solution 500°C containing 0.1% sodium azide.

(3) Preparation of anti-human pancreatic lipase antibody-bound nylon beads Nylon beads (about 3 mm in diameter) 1,000-10.
000 [wo'12゜5% triethyl oxonilla (7
) Add to 100 ml of a dichloromethane solution containing Mu-tetrafluoroborate and react at 25°C for 15 minutes, then wash with 500ml of dichloromethane from which water has been removed with calcium hydride. The above 〇-alkylated nylon beads were added to diaminomethane 10〇-, and after reacting at room temperature for 2 hours, 0.
＼500II+7 for 2M boric acid powder solution (pH 8°5)!
After washing at room temperature, the mixture was added to 100 ml of the same solution containing 5% glutaraldehyde and allowed to react at room temperature for 15 minutes. These beads were further washed with 500 ml of 20 mM potassium phosphate binding solution (pH 7°O), added to 500 tnl of the same buffer containing 1η/- antibody, and reacted overnight at 4°C.
After washing thoroughly with 20mM potassium phosphate buffer solution (pH 7.0) containing 1% bovine serum albumin and 0.1% sodium azide, it is stored at 4°C in the same buffer.

! mN3.

Production of enzyme-labeled antibody (1) β-galactosidase-labeled anti-human pancreatic antibody (8
) Preparation of base antibody 1°5 wf of the antibody of Example 1 which had been dried was added to 0.1 M sodium phosphate buffer containing 50 mM sodium chloride (p
H7,5) Dissolve 1.5 m/m and add 2% m-maleimidobenzoyl-N-human succinimide.
Dioxane solution of ester (MBS)15. Add M,
After reacting for 1 hour at 30°C, chromatography was performed on a 30-volume Sephadex G-25 column buffered with 50 mM sodium phosphate buffer (pH 7,5) containing 50 mM magnesium chloride and IM sodium chloride. Free MBS is removed to obtain an MBS-modified antibody. E, Co11M live β-galactosidase 1.5!9 is added to this MBS-conjugated antibody solution, and after reaction at 30° C. for 1 hour, mercaptoethanol is added to give a final concentration of 1 mM. Furthermore, β-galactosidase-labeled antibody was added to 0
．． 10mM sodium phosphate buffer (p) containing 1% bovine serum albumin, 0.1% sodium azide, 0°IM sodium chloride, mM magnesium chloride (p) 17.0)
Sepharose 4B column (1°5!40
m) to separate the β-galactosidase-bound antibody. The peak of the antibody fraction having β-galactosidase activity is collected and stored at 4°C as a β-galactosidase labeled antibody.

(2) Preparation of peroxidase-labeled anti-human #visceral hash antibody Peroxidase (POD) purified from horseradish
OoI containing 20■ 1.25% glutaraldehyde
Dissolved in MIJ acid buffer (pH 7,5) 0.3-,
React for 18 hours at room temperature.

Glutaraldehyde POD was loaded onto a Sephadex G-25 column (1
5 x 6 oOfn) to remove free glutaraldehyde. 10 wf of the antibody of Example 1 was dissolved in the above aldehyde POD solution, and then 1M carbonate buffer (p
H9,5) 0.2- was added, and after reacting at 4°C for 24 hours, 0.
Add 2M L-lysine and react at room temperature for 2 hours.

The above reaction solution was mixed with 10ml of 00154M sodium chloride.
Sephadex G-20'O column (1,5 x 70 on) buffered with M phosphate buffer (pH 7,2)
to obtain a POD-labeled antibody. Antibody fractions having POD activity are collected and thiomersal is added to the solution to give a final concentration of 0.01%, and stored at 4°C.

Experimental Example 1 (1) Method using anti-human pancreatic lipase antibody-conjugated polystyrene beads (Method 1) 101 Lt of the sample to be measured or a standard human pancreatic lipase solution of known concentration (0, 25, 50, 100, 250, 5
00,750,1,000,2,500,5,000n
9/wit) Take 10 g and make the final volume 2101Lt.
10mM'J acid buffer (pH 7,
0) (Buffer E), and the antibody-conjugated polystyrene beads obtained in Example 2 (1) were added to 1 (1
1) After reacting at 37°C for 2 hours, wash twice with 1.5-buffer E. Add 200 μt of this Heathney buffer E and β-galactosidase Mukuryoku antibody 5ILt from Example 3 (1), and react at 37° C. for 200 hours.
Wash the beads twice with 1.5 rnt of buffer E. Take the washed beads into another test tube, add 200 μt of buffer E, and prewarm at 30°C for 5 minutes.
Add 200 μt of M 4-methylumbelliferyl-β-galactoside solution, react at 30°C for 5 to 20 minutes, and add 0°1M glycine-sodium hydroxide solution (pH 1
After adding 2.5-0,3), the excitation wavelength is 35 Q ra
The enzyme activity of β-galactosidase is measured by measuring fluorescence at a fluorescence wavelength of 45011771. Figure 1a shows a standard curve for human pancreatic lipase according to its nature.

(2) Method using anti-human pancreatic lipase antibody-conjugated polystyrene beads (Method 2) Ability, -0°. 6a 6.4-1. , 1 quasi-human pancreatic lipase solution (0, 25, 50, (12) 100, 250, 500, 7 5 0.1..000.
2. Take 1 μt of 500 n9/+7 and add 0.1% bovine serum albumin to make a final volume of 210 μiK.
Add 10mM phosphate buffer (pH 7.0) (buffer P) containing 0.01% thiomersal and 10mM sodium chloride, add one antibody-conjugated polystyrene bead obtained in Example 2 (1) to each, After reacting at 37°C for 2 hours, wash twice with 1.5-buffer P.

200 μt of Buffer P and 5 M of the POD Makoto Muku antibody solution from Example 3 (2) were added to the beads, and the mixture was heated at 37°C for 2 hours.
Time reaction, 1.57! Wash twice with buffer P. Take the washed beads in another test tube and add 0.3%
Add 0.3-IM citric acid-phosphate buffer (pH 6°O) containing orthophenylenediamine dihydrochloride, 0.02% hydrogen peroxide and 0001% thiomersal, and heat at 30°C for 5 to 20 minutes. After carrying out the reaction and adding 2.5 g of IN hydrochloric acid, the absorbance is measured at 492 nm.

Figure 2a shows the standard curve of human pancreatic lipase according to its nature.

Experimental Example 2 (1) Method using anti-human pancreatic lipase antibody-conjugated glass beads (Method 1) Experimental example except that the anti-human pancreatic lipase antibody-conjugated glass beads prepared in Example 2 (2) above were used. 1 (1
). However, the standard curve of human pancreatic lipase according to its nature is shown in Figure 1.

(2) Method using anti-human pancreatic lipase antibody-conjugated glass beads (Method 2) Experimental example 1 (2) except that the anti-human pancreatic lipase antibody-conjugated glass beads prepared in Example 2 (2) were used.
Perform the same operation as . However, the standard curve of human pancreatic lipase according to its nature is shown in Figure 2.

Experimental Example 3 (1) Method using anti-human pancreatic lipase antibody-conjugated nylon beads (Method 1) Experimental Example 1 except that the anti-human pancreatic lipase antibody-conjugated nylon beads prepared in Example 2 (3) above were used. of (1
). However, the standard curve for human pancreatic lipase obtained by this method is shown in FIG. 1C.

(2) Method using anti-human pancreatic lipase antibody-conjugated nylon beads (method 2) The method (2) of Experimental Example 1 was used except that the anti-human pancreatic lipase antibody-conjugated nylon beads prepared in Example 2 (3) were used.
). However, the standard curve for human pancreatic lipase obtained by this method is shown in FIG. 2C.

Tables 1 and 2 show the simultaneous reproducibility and daily variation in standard human pancreatic lipase and serum in each experimental example.

Table 1: Reproducibility of the enzyme immunoassay method for human pancreatic lipase using a β-galactosidase labeled antibody Table 2: Reproducibility of the enzyme immunoassay method for human pancreatic lipase using a POD labeled antibody β-Gait β-galactosidase labeling Antibody PODj
POD recognition antibody 1 (15) 1゜ Simultaneous reproduction of each sample is the result of repeating the experiment 10 times with five different concentrations of the sample.

2゜The daily variation of each sample is 1 sample with 581 different concentrations.
These are the results of repeated experiments for 0 days.

Measurement results of human pancreatic lipase in the serum of patients with various diseases by enzyme immunoassay The amount of human pancreatic lipase in the serum of patients with various diseases is shown in FIG. The numbers within 0 in the figure represent the number of cases. As a result, serum human pancreatic lipase levels were specifically elevated in acute pancreatitis.

[Brief explanation of drawings]

Figures 1 and 2 show the amounts of standard human pancreatic lipase at known concentrations in Experimental Examples 1 to 3 of the present invention plotted on the horizontal axis as a logarithm, and β
- This is a graph in which the vertical axis is the enzyme activity when galactosidase is used as the labeling enzyme, and the absorbance is plotted on the vertical axis when POD is used (16). FIG. 3 is a graph showing the amount of human pancreatic lipase in the serum of patients with various diseases using the reagents of the present invention. Patent applicant Sugiura New Drug Development Institute Co., Ltd. Marco Pharmaceutical Co., Ltd. Agent Patent attorney Hara 1) Shinichi City::, 21

Claims (1)

[Claims] 1. An enzyme-labeled antibody obtained by covalently bonding an anti-human pancreatic lipase antibody obtained using purified human pancreatic lipase as an antigen to an enzyme, and an insoluble carrier by covalent bonding or physical adsorption of the purified anti-human pancreatic lipase antibody. A reagent for measuring lipase in human body fluids, comprising an anti-human pancreatic lipase antibody-bound carrier (insoluble antibody) bound to .