JPS59134763A - Secretin-relating derivative - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (X is a group which corresponds in the form of X-NH2 to secretin or a fragment devoid of 10 amino acids from its N-terminal; Y is residue corresponding in the form of Y-SH to beta-galactosidase; Z is group of formula II-formula IV). USE:An enzyme-labeling antigen for the enzymatic immunoassay of secretin. Secretin can be determined in high specificity and sensitivity with a simple and easy operation with easy calibration. PROCESS:The compound of formula I can be prepared by (1) reacting the crosslinking agent of formula V with secretin or secretin fragment of X-NH2 to introduce m-maleimidobenzoyl group to the N-terminal of secretin or secretin fragment, and (2) reacting the resultant compound of formula VI with beta-D- galactosidase of formula Y-SH thereby adding the galactosidase to the maleimide group via the SH group of the galactosidase.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a secretin-related derivative, a method for measuring secretin, and a reagent for measuring secretin, which are characterized by using the derivative as an enzyme-labeled antigen.

To measure secretin, the biological activity of secretin has traditionally been measured in vitro or in vivo.
Measurements have been made using various methods. However, these conventional methods require special skill in handling animals and can lead to inaccurate results when a large number of samples need to be processed in a short period of time. There are some easy drawbacks. Due to these circumstances, the implementation of immunoassay methods has been proposed, especially for R.
Radioimmunoassay using IA kits is recommended. However, secretin labeled with a radioactive isotope is unstable and does not necessarily result in high measurement sensitivity, and the method for measuring secretin has not yet reached a satisfactory state.

In view of this situation, the present inventor has realized that the present invention can be carried out by simple and convenient operations without requiring any special techniques.
In addition, we conducted studies to provide a method for measuring secretin that enables measurement with high calibrator, specificity, and sensitivity. As a result, the inventors learned that the desired purpose could be achieved by performing an enzyme immunoassay using an enzyme-labeled antigen in which secretin and a desired fragment of secretin were labeled with a cross-linking agent. reached.

That is, the first object of the present invention is to provide a method for measuring secretin that enables measurement with high calibrability, specificity, and sensitivity through simple and convenient operations, and a measuring reagent prepared in advance for the same. To achieve this, a technical means is disclosed that uses an enzyme-labeled antigen in which secretin or a desired fragment of secretin is labeled with an enzyme using a cross-linking agent.

The configuration of the present invention will be explained in more detail below.

The enzyme-labeled antigen of the present invention is a secretin-related derivative represented by the following formula.

Here, in the formula, X means a residue in which X-NH2 is secretin or a fragment lacking amino acids from the N-terminus to the 10th amino acid of secretin, and Y is a residue in which Y-3H is β-
Denotes a residue that is D-galactosidase. A more specific explanation is as follows.

First, secretin is shown by the following primary structural formula.

H-His-Ser-Asp-Gly-Thr-ph
e-Thr-8er-Glu-Leu-8er-Arg
-Leu-Arg-Asp-8er-Ala-Arg-
Leu-Gin-Arg-Leu-Leu-Gln-G
ly-Leu-ValNH2 Furthermore, a <7 fragment lacking amino acids from the N-terminus to No. 10 of secretin (hereinafter abbreviated as secretin fragment) is represented by the following primary structural formula.

H,Set-Arg-Leu-Arg-Asp-8e
r-Ala-Arg-Leu-Gln-Arg-Leu
-Leu-Gin-Gly-Leu-Val-NH.

In addition, -NH, described in the above-mentioned X -NH, means the most basic primary amine group (N-terminal amine group) present in the secretin or secretin fragment molecule, and C-terminal V
It does not mean an amide group that forms an acid amide bond to al.

-SH described in Y-3H means a thiol group present in the β-D-galactosidase molecule. β-D-galactosidase is a hydrolase of β-D-galactoside.

For example, 4-methylumbelliferyl-β-D as a substrate
- If galactosides are used, 4-methylumbelliferone and β-D-galactose are liberated.

The secretin-related derivatives exhibit secretin antigenic activity and β-D-galactosidase activity, and therefore can be used as enzyme-labeled antigens in secretin enzyme immunoassay.

The secretin-related derivative of the present invention may be prepared as follows when using, for example, m-maleimidobenzoyl N-hydroxysuccinimide ester as a crosslinking agent.

First, m-
Maleimidobenzoyl N-hydroxysuccinimide ester (abbreviated as MBS) is reacted to introduce an m-maleimidobenzoyl group to the N-terminus of secretin, secretin flag, or mento. Here, a gel is passed through to separate the reaction products and unreacted MBS. Then the reaction product has β
-D-galactosidase is reacted and attached to the maleimide group via the SH group in the galactosidase.

After concentration by ultraviolet filtering, the gel is filtered again.

Predetermined fractions may be collected by measuring the absorbance at 225 nm and β-D-galactosidase activity. The secretin-related derivative of the present invention obtained by the above preparation has X-NH
When 2 is secretin (abbreviated as S), the secretin derivative of the present invention is abbreviated as 5-MBS-β-D-galactosidase. Similarly, X NH2 is a secretin fraclin (
The secretin-related derivative of the present invention is abbreviated as -MBS-β-D-galactosidase.

Succinimidyl 4-(1
)-maleimidophenyl)butyrate or hassuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1
The secretin-related derivative of the present invention can also be obtained by preparing in the same manner as in 1. except that -carboxylate is used. The secretin-related derivatives obtained in these cases are those in which Z is the respective one in the above formula.

Next, the measuring method of the present invention will be explained.

The measurement method of the present invention is an enzyme immunoassay method, which is a "competitive method" (
solid phase). Therefore, as an example of the measurement method of the present invention, secretin (secretin in a test sample) as an antigen system and secretin-related derivatives (enzyme-labeled antigen) and a secretin antibody (first antibody) as an antibody system are used.
There is also a measurement method that uses immobilized IgG (second antibody) as a component. In particular, this method will be described in detail as follows.

First, as the secretin antibody (first antibody), antiserum from a rabbit immunized with secretin was used.
IgG ion-exchange chromatographed with hadex
Calbiochem-Behr
Secretin antiserum, which is commercially available from Ing, may be used as is. This secretin antiserum is an antiserum from a rabbit immunized with synthetic pig secretin, and was lyophilized from a phosphate buffer solution at pH 7.6.

As the second antibody, for example, an antiserum from a goat immunized with rabbit IgG is used, and the IgG side is separated by affinity chromatography. An example of an antiserum for a goat immunized with rabbit IgG is Cappel Lab.

Freeze-dried products that are commercially available can be used. As a column for affinity chromatography, for example, a column filled with a resin in which rabbit γ-globulin is coupled to Sepharose 4B activated with BrCN may be used. Affinity chromatography can be performed using 5Q mM Tris-containing 0.1 M NaCl.
Dissolve in HCI buffer (pH 7, 8) and pass through the column.
Next, 50 mM glycine-HCI buffer (pH
2, 3).

As the solid phase for homophasing, those commonly used in the art may be used.2 For example, Sepharose 6MB, silicone pieces, etc. can be used. For example, solid phase formation may be performed as follows. The second antibody is added to BrCN-activated Sepharose 6MB and stirred to bind, and then stirred and washed with an ethanolamine-containing buffer.

Alternatively, after thoroughly washing the silicone piece, the second antibody is added and stirred to adsorb it.

Since the present invention is a measuring method and a measuring reagent characterized by using a secretin-related derivative and a secretin-related derivative, the resin and method for affinity chromatography or the solid phase and method for immobilization are described in 2. It can be freely selected as long as it does not impair the purpose of the present invention. Therefore, the description herein and the examples below are merely illustrative of preferred embodiments.

It is not necessary to limit the present invention.

Now, the measurement method is specifically implemented as follows.

After mixing the mixture and leaving it for example at 4°C overnight, add immobilized IgG and incubate at 37°C for 2 to 6 hours.

The solid phase is separated and the activity of the enzyme bound to the solid phase is measured.

Note that the enzyme activity may be measured as follows.

Substrate solution (4-methylumbellifer lilu β-
D-galactoside) was added, and after reacting at 37°C for a certain period of time, one reaction was stopped. Next, the amount of 4-methylumbelliferone produced is measured by fluorescence analysis (excitation wavelength 360 nm, fluorescence wavelength 450 nm).

Next, the measurement reagent of the present invention is a reagent containing a secretin-related derivative as an enzyme-labeled antigen as an essential component. Therefore, specific embodiments of the reagent for the enzyme immunoassay method exemplified above are as follows. That is, the measuring reagent of the present invention is a set comprising a secretin-related derivative itself or a combination of the same derivative and a secretin antibody (first antibody) and/or immobilized IgG (second antibody).

Here, immobilized IgG is used as its production raw material 1, for example, anti-rabbit Ig.
G-goat serum or its IgG fraction may be freely provided as a side and/or in solid phase.

In this case, for measurement, in-phase 1, gG
Prepare and use immediately. In addition, suitable dilution buffers, substrates (e.g. 4-methylumbelliferyl-β-D-galactoside), etc. may be included in the set for the convenience of measurement operations, and these may be included in the present invention. It is not limited to.

The present invention will be explained in detail using the experimental examples described below.

Example B/Bo (%) was determined for the test sample secretin by the measurement method described in Examples 7 to 10 below, and a calibration curve was drawn with B/BO (%) on the vertical axis and secretin concentration on the horizontal axis. Created for each measurement method.

The results are shown in Figures 1-4. Figure 1 shows a calibration curve determined by the measurement method described in Example 7, in which the IgG fraction of anti-secretin rabbit serum was diluted 5x10', 5-MB5-β-D
- Galactosidase diluted 2X iO', substrate 1
．． '5 Xl0-5M. FIG. 2 shows a calibration curve determined by the measurement method described in Example 8.

Anti-secretin rabbit serum IgG fraction side diluted 5x10' times + Sl + 1. , -MBS-β-D-galactosidar, anti-secretin rabbit serum diluted 1000 times.

S o-,71VI B S-β-D-galactosidase diluted 2 x 104 times, substrate 1.5 x 10"'
It is M. FIG. 4 shows a calibration curve determined by the measurement method described in Example 10, in which the anti-secretin rabbit serum was diluted 200 times.

S,,27-MB5-β-D-galactosidase is 2×
104-fold dilution, substrate is 1.5 x 10''M.

It is clear from FIGS. 1 to 4 that the measurement reagent of the present invention and the measurement method of the present invention each have high calibration performance. Also, the measurement sensitivity is 51)! / tube, and is found to exhibit high measurement sensitivity.

Example: Secretin, secretin fragment (
S11-+-27) + Taking glucagon and VIP, B/B for each test sample was determined by the measurement method described in Example 7 and the measurement method described in Example 1.
o (%) was determined, and the same test results as in Experimental Example 1 are shown in FIGS. 5 and 6. FIG. 5 shows a calibration curve according to the measurement method described in Example 7. FIG. 6 shows a calibration curve according to the measurement method described in Example IO. In both figures, the solid line with ○ indicates secretin, the dotted line indicates secretin fragment, the dotted line with mu indicates Hakurkagon, and the dotted line indicates VIP.
Shows what is about.

From FIGS. 5 and 6, the assay reagent of the present invention and the assay method of the present invention are highly specific for secretin, including the secretin fragment (So-27), and the cross-reactivity with similar peptides is 5 to 200 pf/ It turns out that it cannot be seen within the measurement range of the tube.

EXAMPLE Secretin and secretin to which large serum was added were taken as test samples, and the B/B of each test sample was determined by the measurement method described in Example 1o.

(%), and a calibration curve similar to that in Experimental Example 1 was created. In addition, the dog serum is Peagle dog serum with Charco.
The one treated with al-dextran to make it secretin-free was used.

The results are shown in FIG. In the figure, the circle mark line shows the calibration curve when secretin alone is used, and the @ mark line shows the calibration curve when dog serum is added to secretin.

□ From FIG. 7, it is clear that the measuring reagent of the present invention and the measuring method of the present invention have high calibration performance as a measuring reagent and method for measuring secretin in serum.

The present invention will be explained in more detail with reference to Examples described below.

Example 1 One inch of secretin (abbreviated as S) was dissolved in 21 nl of 50 mM phosphate buffer (pH 7.0). In addition, 501.
MBS-dioxane solution of Le (2077+f MBS
/ml) was added and reacted at room temperature for 1 hour. This reaction solution was passed through a Sephadex G-25 column to remove unreacted MBS. Elution is 50mM! J acid buffer.

The test was carried out at pH 7.0.

This column chromatography is shown in FIG. In the figure, ■ indicates the S-MBS reaction product, and ■ indicates each peak of unreacted MBS.

Next, the obtained 5-MB5 reaction product D, 2,5rrMi
β-D-galactosidase was added for coupling. The coupling reaction was carried out for 1 hour at room temperature.

The test was then carried out by allowing it to stand overnight at 4°C.

Next, this reaction solution was concentrated by ultrafiltration (molecular weight cut off = 50,000) and passed through a column of Sephadex G-75. Elution was performed using 5QmM IJ acid buffer (pH
7,0) to obtain the desired 5-MB5-β-D-galactosidase.

This column chromatography is shown in FIG.

In the figure, the solid line is determined by the absorbance value at 225 nm, and the dotted line is determined by the β-D-galactosidase activity.

Example 2 Secretin fragment (SIl, 27) 0.9~
was dissolved in 2-50mM phosphate buffer (pH 7,0).

To this, 100 μe of MBS-dioxane solution (6”l
'IBs/m/li was added and reacted at room temperature for 1 hour.

The subsequent operations were performed in the same manner as described in Example 1.

So-*-MBS-β-D-galactosidase was obtained.

Column chromatography corresponding to FIGS. 8 and 9 of Example 1 is shown in FIGS. 10 and 11.

Example 3 The 5-MBS-β-D-galactosidase obtained in Example 1 and the reagents ① to ① below were combined to prepare a reagent set for secretin measurement.

■ Immobilized IgG (immobilized IgG obtained by immobilizing an IgG fraction of anti-rabbit IgG goat serum on Sepharose 6MB)
Raw materials for preparation ■ IgG fraction of anti-secretin rabbit serum ■ Dilution buffer Note that solid-phase IgG was prepared at the time of use from the raw materials for manufacturing solid-phase IgG through the following two steps a and 2. .

a, Purification of second antibody against BrCN-activated Sepharose 4B:59 100)'2? Affinity chromatography was performed using a resin coupled with rabbit γ-globulin.

Freeze-dried goat anti-rabbit IgG serum (containing about 24 m2 as IgG) was added at 4 mA' (7) 0.1
M NaC/? 50mM Tris HC1! It was dissolved in a buffer solution (pH 7, 8) and passed through a column filled with the above-mentioned broken fat. After washing the column with the same buffer to remove unadsorbed substances, add 50mM G containing 0.1M Na Ce.
Using lycine-HCe buffer (pH 2,5),
Antibodies that specifically bound to rabbit γ-globulin were eluted.

Immediately add IMTri8 to the eluate and adjust the pH to 7.9.
And so. The obtained specific antibody was used as a second antibody.

b. Binding of 5epharose 6MB and second antibody. Sepharose 6MB::1 activated with BrCN was added to 1 m
After swelling in MHCl for 15 minutes, it was washed on a glass filter with the same 1mM HCe. Tsuite, 0.5
M NaC1! The second antibody 6 was added to the resin obtained by washing with 0.1M NaHCOs buffer (pH 8,3) containing
(2) dissolved in the same buffer solution 10- was added and left at room temperature for 2 hr)! Stirred.

After the coupling reaction, wash the Sepharose with the same buffer on a glass fin letter, and add 1M to the same buffer.
A solution containing ethanolamine was added at 10 mA' and stirred at room temperature for 2 hours.

Next, on a glass filter, 0.5. MNaCe
0.1M NaHcOs buffer (pH 8.3) containing
) and an acetate buffer (pH 4) containing 0.5M NaC/
, 0) to wash away the adsorbed proteins.

The obtained second antibody Sepharose was
The suspension was suspended in 5QmM IJ phosphate buffer (pH 7,0) containing 5QmM IJ and stored at 4°C.

The 111 IgG fraction of anti-secretin rabbit serum was prepared by subjecting the anti-secretin rabbit serum to ion exchange chromatography using DEAE-Sephadex and separating the IgG fraction. The elution is 20mM! J acid buffer (p
H7,6), and the NaC/' concentration was changed from 0 to 1.
．． I used OM's linear gradient. The color chromatography here is shown in FIG.

The dilution buffer was 50mM IJ acid buffer (pH 7.4).
) with 150 mIV NaC/, 0.2% BSA (
bovine serum albumin), 1 mM MgCe2.2
50 KIU/ml aprotinin and 0.05% Tw
It was prepared by containing een 80.

Example 4 So-27-MBS-β-D obtained in Example 2
- Galactosidase and ■~■ used in Example 1
This reagent and the same reagent were combined to form a reagent set for secretin measurement.

Example 5 SI+-27MB S- obtained in Example 2
β-D-galactosidase, the same reagents as ■ and ■ used in Example 1, and Calbioch
A reagent set for measuring secretin was prepared by combining the anti-secretin rabbit serum obtained from em-Behring.

Example 6 S obtained in Example 2, -MBS-
A reagent set for measuring secretin was prepared by combining β-D-galactosidase and the reagents ① to ① below.

■ Raw materials for manufacturing immobilized IgG (immobilized IgG obtained by immobilizing an IgG fraction of anti-rabbit IgG goat serum on a silicone piece) ■ Anti-secretin rabbit serum ■ Dilution buffer In addition, immobilized IgG Solid-phase IgG was prepared at the time of use from the production raw materials through the following two-stage operations a and s.

a. Purification of second antibody The same procedure as described in section a of Example 1 was carried out.

b. Adsorption of the second antibody to the silicone piece The silicone piece was soaked with distilled water in advance, and then 150mfvl of Na
It was used after washing with 5Q mM phosphate buffer (pH 7.0) containing Ce.

The second antibody 5rnIi+ was dissolved in 50ml of the same buffer,
500 silicone pieces were added thereto, stirred at room temperature for 2 hours, and then left at 4°C for several days before being used for measurement.

In addition, the anti-secretin rabbit serum is Calbiochetn-
Anti-secretin rabbit serum obtained from Behring was also used, and the same dilution buffer as in Example 1 (2) was used.

Example 7 A test sample was measured using the reagent set described in Example 3. That is, the test sample, 5-MB5-β-ri-galactosidase on the IgG fraction side of anti-secretin rabbit serum, was diluted with a dilution buffer, and 100 ue and 500 ue, respectively, were diluted with a dilution buffer.
1001 tl of Be was mixed and left overnight at 4°C. Add 100 u of the immobilized IgG prepared before use in Example 3 diluted with dilution buffer #, shake at 37°C for 2 hours + centrifuge at 800 rpm for 3 minutes at room temperature, and remove the supernatant. , and washed once with dilution buffer.

Sepharose 6MB was collected by centrifugation, and the enzymatic activity CB) of β-D-galactosidase bound to Sepharose 6M, B was measured.

Enzyme activity was measured as follows.

On the solid phase, 100 tLe of substrate solution (4-methyl
mbelli-feryl-71-1)-gala
ctoside to 1.5 Xl0-M or 1.5 X 10-'M, lmM
5QmM IJ acid solution (pH 7) containing MgCe2
, 0) dissolved in
2.5 az of 0.1 M Glyci
The reaction was stopped by adding ne -NaOH (pH 10,3).

The amount of methylumbelliferone produced was analyzed by fluorescence analysis. Fluorescence was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 450 nm.

Separately, the test sample was removed, 5-MB5-β-D-galactosidase from the IgG fraction of anti-secretin rabbit serum was diluted with a dilution buffer, and then bound to Sepharose 6MB by the same procedure as above. The enzymatic activity of β-0-galactosidase (B,) was measured.

Finally, find B/B (%) using the following formula.

B/ Bo (%) = x 100B.

Example 8 B/B (%) was determined for the test sample using the reagent set described in Example 4 in the same manner as in Example 7.

Example 9 B/BQ (%) was determined for the test sample in the same manner as in Example 7 using the reagent set described in Example 5.

Example 10 A test sample was measured using the reagent set described in Example 6. That is, the test sample, anti-secretin rabbit serum, Sn-2
7MB5-β-D-galactosidase was diluted with a dilution buffer, 100 μe of each was mixed, and the mixture was left at 4° C. overnight. Add the immobilized 1gG prepared before use in Example 6, and incubate at 37°C for 6 hours.
Shake for an hour, take out the silicone piece, wash with dilution buffer, collect the silicone piece, and collect the enzyme activity of β-D-galactosidase bound to the silicone piece (B
) was measured.

Separately, the test sample was removed, namely anti-secretin rabbit serum, So-==MBS-β-D-galactosidase was diluted with a dilution buffer, and the β- The enzymatic activity (B,) of D-galactosidase was measured.

Finally, B/B (%) was determined using the following formula.

[Brief explanation of the drawing]

1 to 4 correspond to FIGS. 1 to 4 described in Experimental Example 1, and show calibration curves with B/B (%) on the vertical axis and secretin concentration on the horizontal axis. Figure 51 Figure 6 corresponds to Figures 5 and 6 described in Experimental Example 2, with B/go (%) on the vertical axis and secretin, secretin fragment, and glucagon on the horizontal axis. Each calibration curve for each concentration of VIP is shown. FIG. 7 corresponds to FIG. 7 described in Experimental Example 3, and shows a calibration curve with B/BO (%) on the vertical axis and secretin concentration on the horizontal axis. FIG. 82 and FIG. 9 correspond to FIGS. 8 and 9 described in the example section, respectively, and show column chromatography. Figure 101 corresponds to Figures 10 and 11 described in Example 2, each showing column chromatography. FIG. 12 corresponds to FIG. 12 described in Example 3,
Ion exchange chromatography is shown. Patent application Artificial-Zai Co., Ltd. Figure 1 to, 50100. 50010001-Kleben (r+g/m], ) 'Figure 2 Seven Reign (ng/m1., 1oo1+1.j1
713 71/ノ>'(ng/m, 1.+1007i, 1
．． ) Figure 4 B/B. Secretin (pg/ml, + ooν)
[・15 IQ 50 100 500
1000 concentration (ng/m),) Figure 6, a degree pg/ml C100JII-a
, ssa, y) 7 25 50 100 250 5
00' 1000 2000 se ri 1no
Chin (pg/ml, , lCK) 7
i1) Figure 8 0 10 20 F Xitton, NaP (2,5m], /lut+e
) Figure 9 0 70
/lo OF'r++, ctaon%
(2,15m', 1./1uba) and 110 0 - 10 20Fr, cti
On, no! Otsu 2ml / juleno [187111 FractionNo, (2-2m], /l, uhe)
Figure 12

Claims (1)

[Claims] A secretin-related derivative represented by the formula (1). [However, in the formula, X means a residue where X・-NH2 is secretin or a fragment lacking amino acids from the N-terminus to secretin up to number 10, and Y means a residue where Y-3H is β-D-galactosidase. means. In addition, Z tastes] (2) A patent characterized in that a crosslinking agent represented by the formula % is reacted with X-NH2 to form (), and the product is reacted with Y-3H. A method for producing secretin derivatives. [However, x, y, and z mean the same residues as stated in the proviso in claim 1.] (3) When secretin is measured by enzyme immunoassay, a claim is made as an enzyme-labeled antigen in the measurement. range 1
(4) A method for measuring secretin characterized by using the secretin-related derivative set forth in Claim 1. A reagent for measuring secretin by enzyme immunoassay, which is used as an enzyme-labeled antigen in said measurement. A reagent for measuring secretin, characterized in that it contains a secretin-related derivative of