JPS58148963A - Reagent for measuring bound material of alpha-1- antitrypsin-trypsin in human body fluid by sandwich method - Google Patents

Abstract

PURPOSE:To enable the measurement of the alpha1-antitrypsin (AT).trypsin (T) in human bodily fluid with good accuracy by using the enzyme-signaled antibody of the anti-alpha1- AT antibody and insolubilized anti-T antibody obtained respectively by sensitizing the human alpha1-AT and T in domestic rabbits. CONSTITUTION:The enzyme-signaled anti-alpha1-AT antibody wherein the anti-alpha1-AT antibody obtained by sensitizing the alpha1-AT in refined human sera in domestic rabbit is bound with peroxidase or beta-galactosidase or the like and the insolubilized anti-T antibody wherein the anti-T antibody obtained by sensitizing the trypsine of the human pancreas in domestic rabit is bound with glass beads, polystyrene, etc. are prepd. The insolubilized anti-T antibody is added to human bodily fluid (serum) and after the reaction, the enzyme-signaled anti-alpha1-AT antibody is added thereto. After the reaction, the enzyme activity of the insolubilized anti-T antibody-alpha1-AT T-enzyme-signaled anti-alpha1- AT antibody is measured by measuring the intensity of the color or fluoresence developed by addition of a suitable reagent. The measured activity is contrasted with a standard curve obtained beforehand, whereby the alpha1-AT.T in the serum are measured with good accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present BTA is a 11KM test in which the alpha-1-annatrypsin trypsin conjugate (hereinafter referred to as α1-much-trypsin) in human body fluids is measured by an enzyme immunological method using the sandwich method. Conventionally, the amount of trypsin is determined by radioactive immunity! III standard method, and its relationship with pancreatic disease was discussed.
sbav, am-ding.

W@od, Lamest Otori: 66-68e (1
977)), which is present in the autolyte of seven different tie types, including trypsinogen and alpha-1-antitrypsin (Heme I).

Binding substances with inhibitory substances such as alpha-2-macroglobulin are known (By・-rl@k).

J, W,, M, C, G＊ekas, C, La
rgmam, M. Fasutt.

and J, H, J@hns*n smukaku, J
, Pkyslsl, 237eE 474-1i:
480 (1979) and B@rgstrWm, Mu.
amdK, 0hlsion's Hopp*
-8*yl @r's Z, Physiel.

Ch@w, 1677-681 (197g)).

However, the currently measured amount of immunoreactive trypsin indicates trypsin that exhibits these complicated forms of existence as a whole, and is not necessarily considered to be a correct determination method. te, whereas the existence of immunoreactive trichons can be deduced even in normal people, α, −A
TLIT tdAlthough it is introduced in patients with pancreatic disease, it has been reported that in normal people, it exists only in an amount that cannot be leaked [Borgstr.
, and K., 0hlssonxScand, J.
, Cl1n, Lab, Invest, 36 8
09-814 (19〕6)〕,〕α, -AT-T The development of the O leakage method is l! It is thought that this will provide a useful tool for disease diagnosis.

On the other hand, although methods have been developed to quantify trypsin in serum according to its mode of existence, these methods [Brodrlmk
, J, W, , M, C, G@okas, C
, Largmn.

M, Fams*tt, and J, H, Johnson
n% Am, J, Physial.

237, E474-E480 (1979) &B
@rgstrWm, A.

s+ad K, 0hlss*n s H@pps-8
@yl@r's Z, Phymlol.

Ch@m, 359677-681 (1978))
This method uses trypsin radioimmunoassay after 1000 min by gel key method, and li! The application to the floor diagnosis method is Kokuden.

Traditionally, immunological determination methods (in-gel immunodiffusion method, complement fixation method, IIJA murine agglutination reaction) have been widely used as a means of measuring trace components in body fluids. However, radioimmunoassay (radioimmunoassay) came into use due to its improved measurement sensitivity. However, there is still some controversy regarding the effects of environmental pollution caused by radioactive substances on the human body under the Radioactive Immunization Act.

The enzyme immunotitration method using the sandwich method using the present Liu test site utilizes antigen-antibody binding and uses anti-IfjL or Shikune antibodies established with enzymes to generate antigen-antibody conjugates. The sandwich method is one of the techniques used to determine the amount of unknown antigen or #-i antibody by measuring the enzyme activity of a substance. This Nandwich method uses antigen K, which has a specific antigenic group.

Antibody-anti-wLIs
Iil! Raw antibody sandwich noodle conjugate! This method attempts to estimate the amount of antigen by 1 NII hL by measuring the enzyme activity of the sous+ antibody, and has the advantage of being 50 to 10,000 times more sensitive than other immunoassay methods. . Therefore, the present inventors aimed to search for changes in α, -^'re in each m disease.
, -We have conducted extensive research on the enzyme immunoassay method for AT@T, and have succeeded in obtaining the substance of the present invention.

Specifically, #IFi of the present invention, an enzyme swk antibody obtained by covalently bonding an anti-alpha-1-antitrypsin antibody obtained using purified human serum alpha-1-antitrypsin as an antigen, and a purified human pancreatic pancreas. Binding of alpha-1-antitrypsin to trypsin in a human body by a sandwich method consisting of an anti-trypsin antibody obtained using trypsin as an antigen and an insolubilized antibody bound to an insoluble phase by covalent binding or physical adsorption.
Concerning test mulberry for determination.

The turtle of the present invention can be manufactured as follows. That is, anti-α1-A antibody and anti-trypsin antibody were produced by sensitizing each rabbit with human α, -AT and trypsin, and then the anti-trypsin antibody was sensitized to polysprene beads, glass beads, and nylon beads. treated with an insoluble phase;
An insolubilized antibody bound to an anti-trypsin antibody is obtained. On the other hand, the most suitable enzyme for anti-α, -AT antibody is 1kijI.
The enzymes are sequentially reacted with external agents (β-galactosidase (maleimidobenzoyl-N-hydrochunacinimide ester), peroxidase (gumeraltehyde), and hyperpigmentation (hyperoxidase)). A crosslinking agent and an enzyme may be reacted first, and then the antibody may be bound to the antibody.

Using the nine anti-trizucin antibody-bound phase obtained in this way and the enzyme-anti-α1-muT antibody, α, -^ in human body fluids was used.
It became possible to measure TΦ.

Therefore, the present drug is useful for diagnosing pancreatic diseases.

Next, a more detailed explanation will be given with reference to examples.

Example (1) Preparation of α-T antibody 1 mg of a bath solution containing purified α,-AT0.7511f was added to one rabbit using Fr@unshi's complete adjuvant (C-1・T・Adju mother ant) 1 - Inject the mixed emulsion prepared subcutaneously on the back and between the nails of the extremities.

1 - 2 m after the immunization of the first rabbit, the same emulsion as the first one was injected subcutaneously into the rabbit's skin. Similar immunological manipulations were performed for 2 to 31 g I
After repeated death, blood was collected from the rabbit's hazy artery, and the blood was collected at 37°
After heating for 130 minutes, centrifuge at 3,000 rym for 15 minutes to obtain serum. 56°C130 for this serum
Heat treatment for 30 minutes to immobilize complement, add 0.9% sodium chloride solution in the same amount as the serum, and then add the same volume of saturated IL@ammonium as the serum to make it 33% saturated, where it is precipitated. The precipitate was collected by centrifugation at 12.00 rpm for 15 minutes, and a small amount of 20Fl1M phosphorus III solution (
After dissolving in pH 8,0), dialysis was performed using the same buffer,
Completely eliminates the value of annium. One minute obtained here was converted to 11 with 20mM phosphate buffer solution (pH 8.0) and placed on a nine DEAI-cellulose column (2.5X20eIIM).
Collect the fraction that does not adsorb to the column and flow out with the same buffer, dilute it with the same buffer to a protein amount of 20sf/-, and use this as anti-α, -muT antibody.
Aliquot each, lyophilize and store at -30°C.

(2) Preparation of anti-trypsin antibodies Purified from human pancreatic juice against 14 rabbits and 9 trypsin 0.
The same procedure as in (1) above was carried out except that solution 1- containing 611f was used, and the anti-trypsin antibody was added to 1-9.

(3) Anti-trinosine antibody-conjugated polystyrene beads 01
1111k lyophilized nine antibodies 200 cape was added to 0.1M sodium phosphate aronmeng containing 5011M disintegrated sodium (p
Dissolve 400ml of H7,6), add 1,000 to 2,500 polystyrene beads (approximately 6.5g in diameter) that have been thoroughly washed (with a strong detergent) into this solution, and heat at 30°C.
Shake for 2 hours. After removing the antibody droplets, the beads were washed with the same buffer solution III as above containing 0.1 ml of 0.1-glycide, then a -buffer was added, and the beads were incubated at 30°C for 2 hours. After washing with the same buffer solution No. 11, sodium azide is added to the solution so that the concentration becomes 0.01%, and the beads are stored in the same buffer solution.

(4) The Klj Fusin anti-$IML laminated glass beads
Glass peas (approx. 4cm in diameter) 5,000~10.00
0 pieces were added to 200 sd of toluene solution containing 10% r-aminopropyl triethoxysilane, and after reacting at room temperature for 30 minutes, the solution was removed with a glass filter, and after thorough washing with toluene, alkyl aminated glass beads were added. After drying and post-mortem, 20 μl containing 0.2% anti-tricine antibody was added.
mm! j y*buffer 11k (pH7,4) 500
-1,000, stir well, add glutaraldehyde to a final straining degree of 0.1-1%, and boil at 40℃ for 12 hours.
Shake for a time to react. The anti-trypsin antibody-bound glass beads obtained here were mixed with 0, IM sodium phosphate A11lli'l (p) containing 0.1% bovine serum albumin.
H7,0) After thorough washing, store at 4°C in the same solution III containing 0.1% sodium azide.

(11ll of 51 anti-trypsin antibody-conjugated nylon beads)
Made of nylon beads (approx. 3-diameter) 1,000 to 10.
000 pieces T12.5% dichloromethane solution containing triethyl oxonium tetrafluoroborate fi100
After adding mK and reacting at 25°C for 15 minutes, water was removed with calcium hydride and washed with dichloromethane 500 wIItK. The above O-alkylated nylon beads were added to shea in Tsumethane 100, and after reacting at mold temperature for 2 hours, 0.
After washing with 500 mK of 2M boric acid buffer (pH 8.5), it is added to the same buffer solution 100 containing 5≦glutaraldehyde and allowed to react at room temperature for 15 minutes. K out these beads.
20jmM potassium phosphate buffer (pH 7,0) 5
After washing with 00wd, a four-layer solution containing 1%'mO antibody was added.
Add to solution 500- and react at 4°C overnight.
% Bovine Self-cleaning Alpunun, 0. After washing thoroughly with 20111M potassium phosphate slow release* (pH 7.0) containing sodium azide, and after death, store in the same buffer solution at 4°C.

(6) Per ID 1-41! ish anti-at-h'r antibody of111m Nine peroxitase (POD) purified from horseradish (POD) 20q was dissolved in 0.3 m of 0.1 M phosphate buffer (pH 6,8) containing 1.25% glutaraldehyde and incubated at room temperature. No reaction for 18 hours. Glutaraltehyde POD was added to 154M jJI sodium chloride softened Sephatex G-25 column (1゜5X60
a+) Add K and remove free glutaraltehyde. Dissolve the antibody lO cape in the above curcuraldehyde POD solution, and then add K114 charcoal chloride alIf & (pH 9,5) 0
．． 2M was added, and after reacting at 4°C for 24 hours, 0°2M
Add L-lysine and react at room temperature for 2 hours. The above reaction solution was loosened with IOIILM phosphorus solution (pH 7°2) containing O, 154M sodium bridging, and loaded onto a Sephadex G-200 column (1.5X 70m) K g
li to obtain POD Q noodle antibody. Collect the antibodies having POD activity, add thiomersal to a final concentration of 0.01%, add a large amount of glycerin, and store at 4°C.

(7) β-Casictosidase 41 fused anti-α,-^T antibody - lyophilized 1.5q of nine antibodies in 50111M sodium chloride-containing 0.1M sodium phosphate* *-7,
5) Dissolve 1.5 mK1@, add 15 liters of 2-maleimidobenzoyl-N-hydroxy natakunimide ester (MIIB) to it, and
5073 containing 50mM magnesium chloride, 0.1M sodium chloride, 1 hour reaction time at 0°C! Free MBS was removed using a Sephadex G-25 column chromatograph using M phosphorus sodium buffer (H7), K to IIIJ, and a nine-column test #3o to obtain an MBS-formed antibody. Add 1.5 caps of this MBS-conjugated antibody solution KE, C-11-producing I-galactosidase, and incubate at 30°C for 1 hour.
After the reaction, add mercaptoethanol to a final concentration of 1mM. Additionally/- galactosidase labeled antibody was added to lOrlLM sodium phosphate buffer (pH 7.0) containing 0.1% bovine autologous albumin, 0.1% sodium azide, 0.1M sodium chloride, and 1111M magnetium chloride. Tempered Sepharose 48 force j
(1.5 x 40 cm) KfA is added to separate the β-galactosidase-bound antibodies. The peak of the antibody having β-galactosidase activity is collected and stored at 4°C as β-galactosidase labeled 1 antibody.

Experimental Example 1 (Method using 11 anti-trypsin antibody-conjugated polystyrene beads (Method 1) Purified α,-muT and purified trypsin were mixed at neutral pH.
and a standard α of known concentration by mixing at a molar ratio of 10
, -kTaT solution* (0,2,0,4,0°8.0
, 16.0.32.0.64゜0. 128,256
150 μL) or the sample to be measured for Fi, diluted with 50 liters of water, then diluted with lO containing 11% bovine serum albumin, 0.01% deomersal, and IOIIIM sodium chloride.
Abusive M phosphoric acid! I! Equilibrium (pa7.0) (buffer P)
Add 200μ of each of the seven well-bound polystyrene beads obtained in (3) above, react for 2 hours at 37°C, and wash twice with 2.0-buffer PK. To these beads, *IIk', - Perform washing. Take the washed beads into another test tube and add 0.3 orthophenylenediamine di-salt @*, 0.
0.1M citric acid-phosphate buffer (pH 6.0) containing 025G hydrogen peroxide and 0.01% Theomerdale.
2- was added and reacted at 37 °C for 15-20 minutes,
After adding 112.0-N, absorbance is measured at 492-N. Figure 1a shows the standard drawing of α, -Aτ-τ according to Kenpo.

(2) Method using anti-trypsin antibody-conjugated polystyrene beads (Method 2) Measurement sample 50ILt or a standard α1-T solution with known concentration (0, 2, 0, 4, 0, 8, 0, 16,
0°32.0.64.0. 128.256 calls 150μ
j) Take 504 and KO it so that the final capacity is 250MK,
1-Bovine serum albumin, 0.1% sodium azide,
Add IOIIM phosphate buffer solution (pH 7.0) (buffer E) containing 11mM magnesium chloride and 10+sM inoculating sodium. Add one antibody-conjugated polystyrene bead obtained in 11th century (3) to each, and heat at 37°C.
After reacting for 2 hours, wash twice with 2.0-buffer EK. Add 250 µt of Buffer E and approximately 71 β-galactosidase 411 I-antibody solution to the beads at $5 µl.
Add t, react at 370C for 3 hours, and wash with 2°0- buffer E. Take the washed beads in N0Ekt, add 200g of buffer gt,
After prewarming at 30°C for 5 minutes, add 100 I of a 0.371 M 4-methylumbelliferyl-β-galactosyde solution.
After adding Lt, one reaction was carried out at 30°C for 5 to 20 minutes.
1M glycine-sodium hydroxide buffer 11 (PH10
, 3) was added at 2.5111/C, and then the excitation wavelength was 360 l/C and the fluorescence wavelength was 450 l/C to cause a fluorescence leakage to determine the enzymatic activity of ρ-galactosidase. Figure 2 shows the standard curve of α, -AT''T by Kempo Experimental Example 2 Method (Method 1) Other than using the anti-trypsin antibody-conjugated glass and -s pre-diluted in (4) above. The operation was performed in the same manner as (1) of Experimental Example 1. However, the standard curve of α1AT-T by Kenpo is shown at t*btc.

(2) Method using anti-trypsin antibody-conjugated glass beads (Method 2) In (4) above, lol! ! The procedure is carried out in the same manner as in (2) of Experimental Example 1, except that trypsin antibody-conjugated glass beads are used. However, according to Kempo, the standard side of AT@T can be shown in Figure 2.

Experimental Example 3 () Method using anti-trypsin antibody-conjugated nylon beads (Method 1) Same as (1) of Experimental Example 1 except that the nylon beads conjugated to trypsin antibody prepared in (5) above were used. However, the standard curve of α1-AT@T by Kempo is shown in FIG.

(2) Method using anti-trypsin antibody-conjugated nylon beads (Method 2) Experimental example 1 (2) and 1IfJ beads were used except that 9 anti-trypsin antibody-conjugated nylon beads prepared in step 1 (5) were used. Perform operations. However, the Kōfu #play of α8-AT and Ding by Kenpo is shown in Figure 2C.

Tables 1 and 2 show the simultaneous reproducibility and day-to-day fluctuations in standard α, -AT·T, and serum in each experimental example.

Table 1 α,-AT using POD 11m1k antibody
・P) Visibility Table 2 of T's immunoselection method Reproducibility of Kizada immunoselection method of β-galactosidase ll1l & antibody POD :
Simultaneous reproduction of each sample with POD theory and anti-antibody is one of the most important samples with a concentration of 51m.
This is the result of repeating the experiment 0 times.

Z The daily M variation of each sample is 511 concentration Opi sample.
These are the results of repeated experiments for O days.

[Brief explanation of the drawing]

FIGS. 1 and 2 show α in experimental examples 1 to 3 of the present invention,
-MuT-Tt) II A graph showing the entrance of the bend, with a known degree of crossing-
The amount of quasi-α,-AT-T is plotted on the horizontal axis as a logarithm, and the absorbance is plotted on the vertical axis when POD is used as the standard enzyme, and the cholinergic activity is plotted on the vertical axis when β-galactosidase is used as the standard enzyme. be. Patent applicant: Marco Pharmaceutical Co., Ltd.

Claims (1)

[Claims] 1. The anti-alpha-1-antitrypsin antibody obtained by using purified nine-human alpha-1-antitrypsin in autonatant serum as an antigen was covalently linked to an enzyme and purified nine-human pancreatic trypsin antibody. A reagent for measuring alpha-1-antitrypsin/trizucin conjugates in human body fluids by a sandwich method comprising an anti-trizucin antibody obtained as an antigen and an insolubilized antibody bound to an insoluble carrier by covalent bonding or physical adsorption.