JPS58171672A - Determination of calmoduline - Google Patents

Abstract

PURPOSE:To enable the simple and quick determination of a trace of calmoduline by causing calmoduline and enzyme marked calmoduline to competitively react with a calmoduline antibody insolubilizing carrier to imeasure an enzyme activity. CONSTITUTION:The calmoduline obtained from the cerebrum of a cow is immunized in a rabbit and a calmoduline antibody is prepd. The antibody is adsorbed on a silicone resin body and is insolubilized. On the other hand, an SH group is introduced into the calmoduline, whereafter beta-galactosidase is caused to react with the SH group, and the enzyme marked calmoduline is obtd. The sample of body fluid, the above-described insolubilized calmoduline and the marked calmoduline are mixed to cause competitive reaction with each other. The enzyme activity of the marked calmoduline coupled with the insolubilized calmoduline antibody is measured by adding 4-methyl umbelliferyl-beta-D-galactosid and measuring the fluorescence thereof, whereby the quick determination of a trace of the calmoduline in the body fluid is made possible. Thus, the method is made helpful for elucidation of the interstitial distribution of the calmoduline and diseases.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the force/L' modulus 7 (Calmodul i
n: Calcium modulator prot
Abbreviation for ein, Calcium-dependent
Also called regulatory protein. ) is related to the quantitative method. In more detail, calmodyulin is determined by G. At this time, calmodyulin and enzyme-labeled calmodyulin are competitively or competitively bound to a calmodulin antibody insolubilized carrier, and the enzyme activity is measured by measuring the amount of enzyme-labeled lumodelin bound to the carrier. Alternatively, calmodulin and enzyme-labeled calmodulin are competitively or competitively bound to a calmodulin antibody, and the enzyme-labeled calmodulin bound to the antibody is bound to anti-immunoglobulin antibody insolubilized carrier G. The present invention relates to a method for quantifying calmodulin, characterized in that the amount of enzyme-labeled calmodulin bound to the carrier is quantified by measuring enzyme activity, thereby determining a constant value of calmodyulin.

Calmodeulin is widely distributed throughout the animal and plant kingdoms, and is involved in various calcium-dependent enzymes, such as the cyclic nucleus.
otide phosphodiesterase (
Brain) Adenylate Cyclase (Brain), M
yosin lightchain kinase (
smooth muscle), Phosphorylaseb kinas
e (skeletal muscle), Glycogen 5ynthase
Kinase (skeletal muscle), (Ca + Mg)
ATPase (erythrocyte), NAI) klnase
It is a calcium receptor protein with a molecular weight of approximately 17,000 that activates (plants) etc. Calmodeulin, which has various functions such as activation of various enzymes in the body, can be easily and minutely quantified in saliva for future research on calmodyulin, especially new calmodyulin-dependent biological maintenance functions. elucidation and tissue distribution of calmodyulin,
It is very useful in 1- to elucidate the relationship with diseases.

The conventional method for quantifying calmodyulin is cyclic.
cleotide phosphodiesteras
It is measured by a method that measures the activation of enzymes activated by calmodyulin G, such as cyclic nucleotide ph, which does not contain calmodyulin.
It is difficult to adjust osphodiest-erase, and the combination with 5'-nucleotidase (
As a result, cyclic nucleotides are decomposed and the amount of liberated phosphoric acid is measured, which requires a complicated operation and has low measurement sensitivity.

On the other hand, in recent years in the field of immunoassay, radioimmunoassay (RI) using radioisotopes as labeling compounds has become more popular.
A) was developed, making it possible to measure trace amounts of calmodyulin.

However, since the RIA method uses a radioisotope as a labeling compound, special equipment and technicians are required to carry it out, and strict care must be taken when disposing of measurement waste. There are drawbacks.

The inventors of the present invention took into consideration the current situation and as a result of intensive study,
By using an enzyme immunoassay method that uses an enzyme as a labeled compound, calmodyulin can be detected with high sensitivity (
We have discovered that this can be quantified. That is, in the present invention, enzyme-labeled calmodulin, a calmodulin antibody, a calmodulin antibody insolubilized carrier, and an anti-immunoglobulin antibody insolubilized carrier are used in combination, and calmodulin is quantified using an immunological competitive reaction. Since enzymes are used, special equipment like the RIA method is not required, and there are no problems with the treatment of measurement waste. Of course, the measurement sensitivity is also equal to or higher than that of the RIA method.

Examples of carriers used here include polystyrene spheres, polystyrene tubes, silicone resins, glass beads, and various insoluble polysaccharides. When using a carrier as a column, the carrier is preferably in the form of fine particles or fibers.

The carrier and the antibody can be bound by physical adsorption or by activating the carrier by various methods to form a covalent bond. As a method for activating the carrier, for example, when the carrier is an insoluble polysaccharide, there is a method using an activating reagent such as cyanogen bromide, epichlorohydrin, sodium periodate, H'-carbonyldiimidazole, or the like. Furthermore, if the covalent bond between the carrier and the antibody is made into an openable bond, for example, an S-8 bond, after using the antibody-insolubilized solid phase, the antibody can be separated and the carrier can be used again. Additionally, a suitable spacer may be introduced between the antibody and the carrier.

The antibody may be immunoglobulin G (IgG) itself, but antibodies with only the antigen-binding site isolated, such as F
a b', F (a b ')'2, etc. may be used (-Claim 4) As the immunoglobulin-binding protein, for example, Staphylococcus e aureus (5taphylococcus aureus) may be used.
eus ) (D student 1ffi-f protein A (Pr
oteinA).

When filling a column with a carrier, the column to be used should be 01 to 1. Qm/ is preferably 0. Various bifunctional reagents are used for binding the enzyme and calmodulin. For example, glutaraldehyde, carbodiimide, m-maleimidobenzoyl-N-hydroxysuccinimide, etc. are used. Calmodulin also has S-
Introduction of S)I group using acetylmercaptosuccinic anhydrideS using L,N,N-0-phenylene dimaleimide
It can also be combined with an enzyme having an R group.

If the sample is a biological body fluid in a moist system, low measurement accuracy may be observed due to the interference effect of the biological body fluid components. This interfering effect causes antibodies to become Fab' or F
The state of (ab')z (can be suppressed by straining and using it, but by further adding hydrophobic proteins and salts to the reaction solution, G can be suppressed or removed more completely. 1. Hydrophobic) Gelatin or the like is used as the protein, and table salt or the like is used as the salt.

As described in 1-1 above, the present invention is characterized by carmodulin (one-stroke operation is simple and allows fixed stitching with good sensitivity, but this will be explained by referring to an example below).

Example 1 Quantification of calmodyulin (1) Preparation of calmodyulin Calmodeulin is Teo, T, S from beef cerebrum.

The method of Wang, TH6, et al. (Journal of
Biol-logical chemistry 24
8, p. 588, 1973).

(2) Preparation of calmodyulin antibody A calmodyulin antibody was prepared by immunizing a rabbit with the calmodulin obtained in (1) combined with bovine serum albumin. Calmodeulin has little antigenicity due to its low species specificity in its primary structure, making it an extremely difficult substance to prepare antibodies against.
Abundant calmode and phosphorus adjusted by (1) for a long period (
After immunizing rabbits for 3 months, antibodies were prepared. Antibodies were prepared using the method of Kato et al. (Journal o
Adjust the IgG fraction according to fBiochemistry Volume 81, Page 1557,
Fourth Me a n s et al.'s force method (Journal o
f Biolo-gical Chemistry:Y
253, page 7515, May 1978. Lumodulin was purified using a column of insolubilized Sepharose.

(3) Preparation of anti-immunog-11 purine antibody Anti-rabbit immunoglobulin antibody (goat) was purchased from the Institute of Medical Biology. Antibodies were prepared using the method of Kato et al. (Journal
ofBiochemistry, Vol. 81, p. 1557, 1977). The obtained IgG fraction was subjected to limited decomposition with tetrazeta-pepsin and then subjected to column chromatography using Sephadex G-150 to separate the "F(ab')2 portion.

(4) Regulation of β-galactosidase-labeled calmodulin at + the method of Fuji et al. (Chemistry and Biology Vol. 14, p. 737)
(1976). That is, first, SHi was introduced into calmodulin prepared with S-acetylmercaptosuccinic anhydride (ampholytic), and this SH group-introduced calmodulin and Escherichia coli β-galactosidase, which naturally has an SH group, were mixed with N, N-o- Coupled by phenylene dimaleimide.

(5) Preparation of second antibody insolubilized carrier A piece of silicone resin (a cylinder with a diameter of 3++++++1 and a length of 4 mm) was mixed with the solution of F (a b') 2 prepared in (2) (A
280 to 0.5 to 0.7, and 0.1% of NaNa added).After soaking overnight to physically adsorb F(ab')2, the silicone resin pieces were soaked in 0.1% of NaNa. M phosphate buffer (,
After washing with buffer iA (pH 7.0) and buffer iA, this was used as a second antibody insolubilization carrier. (Buffer A: O, 1% bovine serum albumin, 0, IM Na (J'11mM MgC
e2. pi containing 0.1% NaNa (7,0 lQmM
Phosphate buffer) (6) β-galactosidase activity measurement method β-galactosidase-containing solution (pH 7,0) Q, 0 to 2Ill
．． 1 mM' 4-methylumbelliberylu β-D-
After adding 01 l of galactoside solution and reacting at 30°C, 0.1M glycy7-NaOH buffer (pH 10.8)
The enzymatic reaction was stopped by adding 2.5-7, and the generated 4-
Methylumbelliferone was quantified by fluorophotometry.

(Excitation wavelength 360n, m, measurement wavelength 450n, m) (7) Calmodulin measurement Standard calmodulin solution was mixed with buffer G (0.8M NaCe, 1mM MgCe2.0.5
% gelatin, 0.1% bovine serum albumin, 0.61% Na
10ml 1Vi of phosphate buffer of pt17.0 containing N5) & trowel diluted standard solution 0.1.7 (Calmodulin variable 0.l, 8.to, 80.100°300
, 1000 nglo, 1 rd) and buffer GQ, lme, containing 10 ng of calmodulin antibody.
Buffer GO11d containing β-galactosidase-labeled calmodulin was mixed and left at 30°C for 1 hour. One second antibody insolubilized carrier was added to the reaction mixture and the mixture was further incubated at 30°C.
There was a 3 o'clock device. After washing the antibody insolubilized carrier, add buffer AO
82-, add 0.1 mM 4-) tilumbelliferyl-β-D-galactone Q, 1 ml, and add 30
The reaction was allowed to proceed at ℃ for 30 minutes, 2.511 Ig of 0.1 M glycine-NaOH buffer (pH 103) was added to stop the reaction, and fluorescence was measured using 1xlo''M4-methylumbelliferone as a standard solution. (Excitation wavelength: 860 nm, measurement wavelength: 450 nm) The obtained standard curve is shown in Figure 1.

Example 2 Quantification of calmodulin using a calmodulin antibody insolubilized carrier Using the calmodulin antibody obtained in Example 1 (2), F(ab')2 was prepared according to Examples 1 (3) and (5). It became insoluble in the silicone resin piece. Standard solution 0, 1 d (
Karmodu') :' Pig O+ L 3+ 10+
80+100, 300.101000n”) and β-
Gala, buffer G containing tosidase-labeled calmodulin
Mix Q, l, and d, add 1 insolubilized carrier of G (anti-calmodulin) F(ab')2, and make 3.
When the enzyme activity was measured using the same method as in Example 1 (7), a standard curve almost the same as in Example 1 (7) was obtained. was measurable.

Example 3 Stationary standard of calmodyulin using a column Calmodulin solution was diluted with buffer G and a trowel.
1.0.25.0.5.1.0.2.5; 5. Oμg
10.1-t) and buffer Q, l ml containing 2.5 μg of calmodulin antibody, and buffer Q, Q, l ml containing β-galactosidase-labeled calmodulin were mixed and reacted at 37°C for 1 hour. The reaction solution was applied to a column (Q, 1 ml) of (anti-rabbit IgG) F(ab')2 insolubilized sepharose. (Anti-rabbit IgG
) The F(ab')2 separate fraction was prepared by adjusting the F(ab')2 fraction according to the method of Example 1 (8).
Add this to CNBr-activated Sepharose (Pharmacia)
and stirred overnight to adjust.

After washing the column carrying the reaction solution with buffer G, a solution of 0-nitrotsuni-β-D-galactoside (10 μ/ml.
Buffer A) 0.1 m6 was applied to the column, and after repulsion at 37°C for 2 hours, the column was washed with 0.08 M Na2CO31-, and the wavelength of the washing solution was measured at 1420 nm. Figure 2 shows the standard curve obtained.

In this measurement method, a wider range of calmodyulin could be measured compared to Examples 1 and 2.

[Brief explanation of drawings]

Figure 1 shows the calibration curve for calmodyulin, and Figure 2 shows the calibration curve when measuring calmodyulin by in-column reaction1.

Claims (4)

[Claims] (1) A method for quantifying calmodulin, which is characterized in that calmodulin and enzyme-labeled calmodulin are subjected to a competitive G-reaction with a carmodulin antibody-insolubilized carrier, and calmodulin is quantified by measuring the enzymatic activity of the enzyme-labeled calmodulin bound to the carrier G. . (2) competitively reacting calmodulin and enzyme-labeled calmodulin with a calmodulin antibody, and binding the enzyme-labeled calmodulin bound to the antibody to a carrier in which an anti-immunoglobulin antibody or an immunoglobulin-binding protein produced by a microorganism has been dissolved; A method for quantifying calmodulin, which comprises: measuring the enzymatic activity of enzyme-labeled calmodulin bound to the carrier; and placing calmodulin in place. (3) A method for quantifying calmodyulin, which is characterized by adding a hydrophobic protein and a high concentration of salt to a system in which calmodyulin and enzyme-labeled calmodyulin react competitively with a calmodyulin antibody, thereby suppressing interference by biological fluid components. (4) Claims (1) and (2) in which the antibody insolubilized carrier is packed into a column and the enzyme activity is measured within the column.
) or the quantitative method described in (3).