JP2001033450A - Stabilized antigen-containing aqueous solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To stabilize antigen activity of a pulmonary surfactant protein by coexisting the protein and a bivalent metallic ion. SOLUTION: A pulmonary surfactant protein and a bivalent metallic ion of a calcium, barium, magnesium or the like are coexisted to stabilize antigen activity of the pulmonary surfactant protein. The metallic ion is not always necessary to be an aqueous solution state, but may be coexisted with the pulmonary surfactant protein. Not only a solution-like state but also the pulmonary surfactant protein are bonded to a plate, and even if most moisture is removed, if the bivalent metallic ion is coexisted, the surfactant protein can be stabilized. A concentration of the coexisting bivalent metallic ion is in a range of about 0.01 to 100 mM. Thus, a standard stock solution of the surfactant protein needing a low-temperature preservation can be preserved and fed at an ambient temperature. A reaction temperature when measured can be executed at the ambient temperature, and operability of the measurement is remarkably improved.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for stabilizing antigen activity in a lung surfactant protein, a stabilized antigen-containing aqueous solution, and a kit for measuring a lung surfactant protein containing the stabilized antigen-containing aqueous solution as a constituent reagent. It is about.

[0002]

2. Description of the Related Art Pulmonary surfactant proteins are apoproteins (SP) specific to pulmonary surfactant, and up to now, four types of hydrophilic SP-A and SP-D and hydrophobic SP-B and SP-C. Types are reported. Among them, SP-D is considered to play an important role in the host defense mechanism in the airway-alveolar system, and SP-D in a sample (such as serum) is determined by an immunological technique utilizing an antigen-antibody reaction. Has been reported to be able to diagnose pulmonary diseases such as idiopathic interstitial pneumonia (see Medicine and Pharmacy 36 (4): 803-808 (1996)).

[0003]

The method reported in the above report uses a recombinant SP-D (rSP-D) obtained by a recombinant DNA technique as a standard substance. If the temperature exceeds 25 ° C, the antigenicity is gradually lost.Therefore, the standard antigen solution must be stored at a low temperature and the reaction temperature during measurement must be performed at a low temperature such as 4 ° C. There was a desire for improvement.

[0004]

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, surprisingly, only by coexisting SP-D and divalent metal ions such as calcium, 37 The present inventors have found that the antigen activity is stabilized without inactivation even under the condition of ° C., and thus completed the present invention.

That is, the present invention relates to a method for stabilizing the antigen activity of a lung surfactant protein by allowing a lung surfactant protein and a divalent metal ion to coexist. The present invention also relates to a stabilized antigen-containing aqueous solution comprising a lung surfactant protein and a divalent metal ion. Furthermore, the present invention provides a kit for measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction, wherein the kit contains an antigen comprising a lung surfactant protein and a divalent metal ion as a standard antigen solution. The present invention relates to a kit for measuring a lung surfactant protein, which comprises an aqueous solution as a constituent reagent.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The pulmonary surfactant protein in the present invention includes SP-A, SP-B, SP-C and S
Any of PD may be used, but unless otherwise specified, it means SP-A or SP-D whose usefulness has been confirmed as a diagnostic item.

First, the present invention relates to a method for stabilizing the antigen activity of a lung surfactant protein by allowing a lung surfactant protein and a divalent metal ion to coexist. Examples of the coexisting divalent metal ions include Group 2a of the periodic table.
It is sufficient that the ions be a divalent metal ion of a subgroup element, and specific examples thereof include calcium, barium, and magnesium. Particularly, calcium ions are preferable. Such a divalent metal ion only needs to coexist with the pulmonary surfactant protein, and does not necessarily need to be in an aqueous solution state. As shown in the Examples below, let alone the solution, let the lung surfactant protein bind to the plate,
Even when most of the water is removed, it is possible to stabilize the pulmonary surfactant protein by coexisting the divalent metal ion. The concentration of the coexisting divalent metal ion is not particularly limited, but is usually 0.02.
01-1000 mM, preferably 0.01-100 mM
May be appropriately selected from the range.

Next, the present invention relates to a stabilized antigen-containing aqueous solution comprising a lung surfactant protein and a divalent metal ion. As the lung surfactant protein and the divalent metal ion, those described above can be used. Taking SP-D and calcium ions as an example, specifically, a metal inorganic salt such as calcium chloride is dissolved in water or an appropriate buffer, and the recombinant SP-D (prepared by recombinant DNA technique) is added thereto. The stabilized antigen-containing aqueous solution of the present invention can be prepared by adding rSP-D). Although the concentration of the divalent metal ion is not particularly limited, it is usually 0.001 to 100.
The concentration may be appropriately selected from 0 mM, and preferably from 0.01 to 100 mM.

Further, the present invention provides a kit for measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction, wherein the kit comprises a standard antigen solution comprising a lung surfactant protein and a divalent metal ion. The present invention relates to a kit for measuring a lung surfactant protein, which comprises an antigen-containing aqueous solution as a constituent reagent. Such a kit is exemplified by a kit for measuring SP-D by ELISA, and specifically described, for example, the following kits can be exemplified. (Kit A) Immobilized anti-SP-D antibody reagent Enzyme-labeled anti-SP-D antibody reagent SP-D standard antigen solution (containing calcium ions) Further, an enzyme reaction substrate usually attached to the above-mentioned kit in a diagnostic kit A solution, an enzyme reaction stop solution, a washing solution, and the like may be attached. In particular, cleaning solutions containing divalent metal ions,
Specifically, 0.001 to 1000 mM, preferably 0.1 to 1000 mM.
By attaching a washing solution containing 01 to 100 mM calcium ions, the antigen activity of SP-D during the measurement can be more firmly stabilized, so that the measurement accuracy can be improved and the operability can be improved. The use of such kits is described, for example, in Medicine and Pharmacy 36 (4), 1996 804.
The measurement may be performed according to a known measurement method described in -808 (1996) or the like.

[0010]

EFFECT OF THE INVENTION The pulmonary surfactant protein (for example,
It has been found for the first time by the present inventors that the coexistence of SP-D) and a divalent metal ion (for example, calcium ion) stabilizes the antigen activity of the lung surfactant protein. As a result, the standard antigen solution of lung surfactant protein, which had conventionally been required to be kept at low temperature, can be stored and distributed at room temperature, and the reaction temperature during measurement can be performed at room temperature. The operability and versatility of measurement can be dramatically improved. Further, not only the stability of the liquid but also the stability of the lung surfactant protein bound on the plate can be improved by coexisting a divalent metal ion, and the measurement of the lung surfactant protein with improved accuracy and operability can be performed. It became possible for the first time.

[0011]

EXAMPLES The present invention will be further described with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Investigation of stabilization in a solution state (1) A final concentration of 10 mM was added to a buffer (10 mM HEPES, 150 mM sodium chloride, 1.0% bovine serum albumin, 0.5% Triton X-100, pH 7.2). Add calcium chloride, magnesium chloride or potassium chloride so that
EDTA was added so that This buffer is used as a dilution buffer. Further, a certain amount of rSP-D was added to the dilution buffer, and the mixture was incubated at 4 ° C. or 37 ° C. for 3 hours to obtain a sample solution. Using such a buffer for dilution and sample solution, SP- manufactured by Yamasa Shoyu Co., Ltd.
Medicine and Pharmacy 36 (4), 804-8
SP-D in the sample solution was measured in accordance with the measurement method described below in 08 (1996) (hereinafter referred to as the standard operation method). Table 1 shows the results. As is clear from Table 1, it was revealed that divalent metal ions, particularly calcium ions, were effective for stabilizing the antigen activity of SP-D.

[Table 1]

Example 2 Investigation of Stabilization in Solution (2) Calcium-added dilution buffer (10 mM calcium chloride, 10 mM
mM HEPES, 150 mM sodium chloride, 0.5% Triton X-10
0, 1.0% bovine serum albumin) to dilute rSP-D to prepare a standard antigen solution. After incubating the standard antigen solution and the calcium-free standard antigen solution at 4 ° C. or 37 ° C. for 3 hours, SP-D in the standard antigen solution was measured according to a standard operation method. The result is shown in FIG. As a result, as shown in FIG.
It was confirmed that the antigenicity equivalent to that at 4 ° C. was maintained after the treatment for 3 hours. On the other hand, 37 without calcium
At 50 ° C. for 3 hours, 50% or more inactivation of antigenicity was confirmed.

Example 3 Temperature Stability in Calcium-Added Standard Solution Using the standard antigen solution to which 10 mM calcium chloride was added and the standard antigen solution to which calcium was not added, prepared in Example 2
After incubation at 25 ° C., 30 ° C. or 37 ° C. for 1, 3, 6 hours or overnight, SP-
D was measured. Heat stability was significantly improved by the addition of calcium, and it was revealed that 90% or more of the antigen activity could be retained even when treated at 30 ° C and 37 ° C for 6 hours and at 25 ° C overnight.

Example 4 Stability on Plate An anti-SP-D monoclonal antibody was bound to a 96-well microplate, and the immobilized antibody and the calcium-containing standard antigen solution prepared in Example 2 were incubated at 4 ° C. overnight. Reacted. Next, a washing solution (10 mM HEPES, 150 mM sodium chloride, pH 7.
2) and each well was washed three times with a calcium-added washing solution obtained by adding 10 mM calcium chloride to the washing solution, and then water was completely removed on a paper towel or the like to make it dry. After leaving it in that state for 5 minutes or 10 minutes, SP-D was measured according to the standard operation method. The results are shown in FIGS. When using a calcium-free cleaning solution
Absorbance decreased by 10% after standing for 5 minutes and by 15% after 10 minutes. However, when using a calcium-added cleaning solution,
The decrease was only 5%, and it was revealed that SP-D was stabilized on the plate in the presence of calcium ions.

[Brief description of the drawings]

FIG. 1 shows the stability of SP-D antigen activity in a solution depending on the presence or absence of calcium ions. The vertical axis indicates the relative activity (%) when the antigen activity at 4 ° C. is defined as 100%, and the horizontal axis indicates the concentration of the antigen solution.

FIG. 2 shows the stability of SP-D antigen activity on a plate when a calcium ion-free washing solution was used. The vertical axis indicates the relative activity (%) when the antigen activity when the standing time immediately after washing is 0 minutes is defined as 100%, and the horizontal axis indicates the concentration of the antigen solution.

FIG. 3 shows the stability of SP-D antigen activity on a plate when a calcium ion-added washing solution was used. The vertical axis indicates the relative activity (%) when the antigen activity when the standing time immediately after washing is 0 minutes is defined as 100%, and the horizontal axis indicates the concentration of the antigen solution.

Claims (10)

[Claims] 1. A method for stabilizing the antigen activity of a lung surfactant protein by coexisting a lung surfactant protein and a divalent metal ion. 2. The method according to claim 1, wherein the pulmonary surfactant protein is pulmonary surfactant protein D (SP-D). 3. The method according to claim 1, wherein the divalent metal ion is a calcium ion. 4. An antigen-containing aqueous solution comprising a lung surfactant protein and a divalent metal ion. 5. The antigen-containing aqueous solution according to claim 4, wherein the pulmonary surfactant protein is pulmonary surfactant protein D (SP-D). 6. The antigen-containing aqueous solution according to claim 4, wherein the divalent metal ion is a calcium ion. 7. A kit for measuring a lung surfactant protein in a sample by an immunological technique utilizing an antigen-antibody reaction, comprising an antigen-containing aqueous solution comprising a lung surfactant protein and a divalent metal ion as a standard antigen solution. A kit for measuring a lung surfactant protein, comprising: 8. The kit according to claim 7, further comprising a washing solution containing a divalent metal ion as the washing solution. 9. The kit according to claim 7, wherein the pulmonary surfactant protein is pulmonary surfactant protein D (SP-D). 10. The kit according to claim 7, wherein the divalent metal ion is a calcium ion.