JP5609107B2 - Immunoassay for troponin I - Google Patents

Description

  The present invention relates to a troponin I immunoassay method. More specifically, a method for immunologically measuring troponin I present in a biological sample such as blood in the form of a monomer, in the form of an IC dimer, or in the form of an ITC trimer in the presence of an alkali metal ion. It is about.

  Troponin (Tn) is a complex protein consisting of three subunits of I (molecular weight about 23,500), T (molecular weight about 37,000) and C (molecular weight about 18,000), and striated with tropomyosin and actin. It consists of thin fibers of myofibrils and controls muscle contraction in skeletal muscles and myocardium (Non-patent Document 1).

  Troponin I (TnI) has different amino acid sequences in cardiac muscle and skeletal muscle, and cardiac troponin I (cTnI) has high myocardial specificity. Therefore, cTnI should be specifically measured based on the difference in amino acid sequence. Is useful for diagnosis of myocardial infarction and the like. Unlike other myocardial markers, cTnI is usually not present in the blood and does not show false positives due to exercise, muscle disease, etc., so it can be applied to sensitive detection of microinfarcts and mild myocardial damage. (Non-Patent Document 2). When the myocardium is damaged due to acute myocardial infarction or the like, troponin escapes into the blood and shows an abnormally high value after 3 to 4 hours. In this case, since the abnormally high value of cTnI in blood persists for about one week, it can be diagnosed even after the onset time (Non-patent Document 3).

  Immunoassay is known as a method for measuring cTnI. Blood samples such as serum, plasma, or whole blood are usually used for immunoassay, but cTnI is required to perform immunoassay within a very short time after sampling for initial diagnosis of myocardial infarction. There is. In such a case, after blood collection, immunoassay of cTnI is often performed using plasma as a biological sample because the sample can be rapidly prepared without waiting for blood coagulation time. There are several types of plasma used for immunoassays, such as EDTA plasma, citrate plasma, and heparin plasma, but heparin plasma is used because it does not chelate metal ions involved in the binding of troponin complexes. It is frequently done. However, when cTnI is immunoassayed, the presence of a heparin binding site in troponin suggests that heparin may affect the immunoassay (Non-patent Document 4).

Ganong, W.H. , Review of medical physology, 16th ed. , Appleton-Lange, East Norwalk, CT; 56-60 (1993) F. S. Apple, R.A. H. Christenson, A.M. S. Jaffe, et al. , National academy of clinical biochemistry and IFCC committee for standardization of markers of cardiac damage laboratory medicine practice guidelines: Analytical issues for biochemical markers of acute coronary syndromes, Clin Chem 53: 4; 547-551 (2007) Laure C, Calzolari C, Bertinchard JP, Leclerq F, Grolleau R, Pau B. et al. Cardiac-specific immunoenzymatic assay of troponin I in the early phase of acute myocardial information. Clin Chem 39: 972-979 (1993) T. T. et al. Brinkmann, C.I. Gotting, K.M. Kleesiek, Proteolytic degradation of cardiac troponin I dos not influencing the TOSOH AIA-PACK cTnI 2G assay, a second generation test in I. Med. 28: 1; 91-96 (2004)

  The cTnI immunoassay is required to have high sensitivity and good reproducibility in addition to being able to measure in a short time. Therefore, the possibility that the measurement result is affected by the presence of heparin as described above needs to be eliminated as much as possible even if the influence is insignificant. The impact of heparin on cTnI immunoassays can be significant, especially when large amounts of heparin are administered to treat certain cardiovascular conditions, such as before angioplasty or after myocardial infarction .

  Conventionally, in order to eliminate the effects as described above, in order to avoid the use of heparin blood collection tubes or to avoid interference with heparin, antibodies that do not affect the cTnI measurement results even when heparin is bound are used. The immunoassay used has been proposed. As another proposal, as disclosed in Patent Document 1, a method of performing immunoassay for cTnI in the presence of polybrene has been proposed. However, each method has problems such as requiring a special antibody for implementation and expensive polybrene.

  In view of the situation as described above, the present inventors examined a simple and highly effective solution capable of eliminating the influence that may be caused by the presence of heparin in the immunoassay of cTnI. The present invention applicable to the present invention has been completed. The present invention is an immunological assay for troponin I, which is carried out in the presence of an alkali metal ion, and a reagent therefor. Hereinafter, the present invention will be described in detail.

  The target of immunoassay in the present invention is troponin I. Troponin I may be any troponin I monomer form, IC dimer form or ITC trimer form present in a biological sample such as blood or plasma, among which cardiac or skeletal troponin I Can be exemplified as the measurement object of the present invention. In the present invention, the alkali metal ion that coexists in the immunoassay has the effect of eliminating the influence that can be caused by the coexistence of heparin, but the troponin I existing in the form of the dimer or trimer as described above is converted into the monomer form. It doesn't change. Therefore, in the application of the present invention to an immunoassay that does not measure troponin I in the form of dimer or trimer, for example, to selectively measure only monomeric troponin I, such identification is In the measurement of troponin I in the following form, the effect of eliminating the influence that may be caused by the coexistence of heparin is achieved.

  In the present invention, the alkali metal ion that coexists in the immunoassay of troponin I is lithium chloride, sodium chloride, potassium chloride, rubidium chloride, cesium chloride, etc., and one of these alone or these One or more of them can be mixed and used. Among the alkali metal ions exemplified, sodium chloride is particularly preferable in terms of ease of handling and price. On the other hand, rubidium chloride is the most powerful and particularly preferable for eliminating the influence of heparin.

The amount (concentration) of alkali metal ions to be coexisted may be determined according to the amount (concentration) of heparin in the biological sample to be measured. For example, “molar concentration of alkali metal ions to coexist” and “ The ratio of the molar concentration of heparin expected to be present in the sample to be analyzed is 1.0 × 10 ^{5 to} 1.2 × 10 ⁷ , more preferably 1.5 × 10 ^{6 to} 1.2 × 10 ^7. Especially preferably, it can be exemplified to be 2.0 × 10 ^{6 to} 8.6 × 10 ⁶ .

The alkali metal ion may be present when the anti-troponin I antibody and troponin I used for immunoassay of troponin I are immunoreacted. As a method of coexistence, an alkali metal ion is added to a biological sample before immunoassay. Add to the reagent for immunoassay, such as buffer solution and immune reaction reagent solution (anti-troponin I antibody solution, etc.) that add metal ions and allow alkali metal ions to coexist when immunoassay is performed later For example, it can be added so that alkali metal ions coexist when the immunoassay is performed later. The preferred reason for adding an alkali metal ion as part of an immunoassay reagent containing an anti-troponin I antibody is that the alkali metal ion is deeply involved in the reaction of the anti-troponin I antibody, and also from the viewpoint of long-term stability. This is because it is effective.
The immunoassay for measuring troponin I is not particularly limited as long as it utilizes an immune reaction between troponin I and an antibody or antigen, and various conventionally known techniques can be employed. For example, a sandwich method or a competitive method can be exemplified. Therefore, the reagent of the present invention is an anti-troponin I antibody (which may be a monoclonal antibody or a polyclonal antibody), a troponin I antigen or a troponin I antigen analog (an immunologically equivalent antibody reactivity to troponin I). (Any of which may be directly labeled with an enzyme such as alkaline phosphatase, a fluorescent substance, a radioisotope or a chemiluminescent substrate, or indirectly labeled with avidin or biotin, etc.) ) And any one of them may contain an alkali metal ion.

  According to the present invention, as will be apparent from Examples described later, it is possible to eliminate the influence that can be caused by heparin by an extremely simple configuration in which immunoassay is performed in the presence of an alkali metal. The alkali metal used in the present invention is inexpensive, and a technique for coexisting it is not particularly required to be skilled, such as adding to a biological sample, adding to an immunoassay reagent, and the like. Therefore, according to the present invention, it is possible to easily and inexpensively eliminate the influence that can be caused by heparin in the troponin I immunoassay.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this. In the following examples, a commercially available device AIA-1800 automatic immunoassay device (manufactured by Tosoh Corporation) was used as an immunoassay device, and cTnI was measured by a one-step sandwich method. Specifically, a water-insoluble carrier on which an anti-cTnI mouse monoclonal antibody is immobilized and an anti-cTnI mouse monoclonal antibody labeled with alkaline phosphatase (an antibody that binds to cTnI at a different site from the immobilized antibody) The solution containing the solution and the cTnI solution were added to the reaction cup, and then serum or heparin plasma was added, and the mixture was kept warm at 37 ° C. with stirring. Thereafter, unreacted substances were removed by B / F separation, alkaline phosphatase-labeled anti-cTnI mouse monoclonal antibody bound to a water-insoluble carrier via cTnI, 4-methylumbelliferyl phosphate was added, and unit time 4-methylumbelliferone production (nM / sec) per hit was quantified by fluorescence measurement. This is because the degree of formation is proportional to the amount of alkaline phosphatase, that is, the amount of cTnI that forms an immune complex on a water-insoluble carrier. In the sandwich immunoassay as described above, if a standard curve based on the fluorescence intensity and the cTnI concentration is prepared in advance using a standard product containing a known concentration of cTnI, the fluorescence intensity of the cTnI unknown sample can be measured. Thus, the cTnI concentration can be calculated from the standard curve.

  A solution containing an alkaline phosphatase-labeled anti-cTnI mouse monoclonal antibody (an antibody that binds to cTnI at a site different from the immobilized antibody) is added to a water-insoluble carrier to which an anti-cTnI mouse monoclonal antibody is bound; Lithium chloride, sodium chloride, potassium chloride, rubidium chloride or cesium chloride was added in an amount of 25 to 150 mM and lyophilized to prepare a cTnI measuring reagent. For control, a control reagent freeze-dried without adding an alkali metal was prepared.

  Using the reagent prepared as described above, the effects of the coexistence of various alkali metal salts were evaluated in the following manner.

Table 1 and FIG. 1 show that heparin-added serum was prepared by adding a certain amount (2.56 × 10 ⁻⁵ mM) of heparin sodium to healthy human serum (negative specimen), and cardiac troponin I was added to the prepared heparin-added serum. It is the result of adding and measuring a monomer. Whether the effect of heparin was avoided or not was calculated based on the calculated value ÷ theoretical value = recovery rate (%) after calculating the theoretical value of the amount of cardiac troponin I added to heparinized serum. did. When the alkali metal ion concentration is 150 mM (ratio of molar concentration of alkali metal ions / molar concentration of heparin present in the sample = 5.9 × 10 ⁶ ) or more, the recovery rate (Y axis) and [alkaline It was evaluated whether the influence of heparin was avoided by approximating the regression line from the figure of [Molar concentration of metal ion / molar concentration of heparin present in sample]] (X axis).

As shown in Table 1 and FIG. 1, the recovery rate was 53% when measured with a reagent to which no alkali metal ions were added, but the recovery rate was improved by adding alkali metal ions. The effect is recognized when the ratio of “molar concentration of alkali metal ions” to “molar concentration of heparin present in the sample” is in the range of 1.5 × 10 ^{6 to} 1.2 × 10 ⁷ , particularly 2.0 × It is remarkable in the range of 10 ^{6 to} 9.9 × 10 ⁶ .

Table 2 and FIG. 2 show the measurement results for cTnIC dimer in the same manner as described above. Also in the measurement of IC dimer, the recovery rate was 79% when measured with a reagent to which no alkali metal ions were added, but the recovery rate was improved by adding alkali metal ions. The effect of the ratio of “molar concentration of alkali metal ions” to “molar concentration of heparin present in the sample” is recognized to be in the range of 1.0 × 10 ^{5 to} 1.2 × 10 ⁷ , particularly 1.2 × 10 7. It is remarkable in the range of ^{6 to} 8.6 × 10 ⁶ .

Table 3 and FIG. 3 show the measurement results for cTnITC trimer in the same manner as described above. Even in the measurement of IC dimer, the recovery rate was 98% when measured with a reagent to which no alkali metal ions were added, but the recovery rate was almost 100% by adding alkali metal ions, particularly rubidium chloride or cesium chloride. improves. The effect is achieved by setting the ratio of “molar concentration of alkali metal ions” to “molar concentration of heparin present in the sample” to 8.8 × 10 ⁶ or less.

From the above results, in the immunoassay of troponin I existing in the form of monomer, IC dimer or ITC trimer, the measurement can be performed while eliminating the influence of heparin coexisting with all forms of troponin I. The ratio of “concentration” to “molar concentration of heparin present in the sample” is in the range of 1.5 × 10 ^{6 to} 1.2 × 10 ⁷ , preferably 2.0 × 10 ^{6 to} 8.6 × 10 ^6. It can be seen that it is preferable to coexist alkali metal ions.

It is a figure which shows the influence of the alkali metal ion in the immunoassay of cTnI monomer. In the figure, white circles indicate lithium chloride, white squares indicate sodium chloride, white triangles indicate potassium chloride, black circles indicate rubidium chloride, and black squares indicate the results when cesium chloride coexists. It is a figure which shows the influence of the alkali metal ion in the immunoassay of cTnIC dimer. In the figure, white circles indicate lithium chloride, white squares indicate sodium chloride, white triangles indicate potassium chloride, black circles indicate rubidium chloride, and black squares indicate the results when cesium chloride coexists. It is a figure which shows the influence of the alkali metal ion in the immunoassay of cTnITC trimer. In the figure, white circles indicate lithium chloride, white squares indicate sodium chloride, white triangles indicate potassium chloride, black circles indicate rubidium chloride, and black squares indicate the results when cesium chloride coexists.

Claims (1)

An immunological assay for troponin I in a biological sample, carried out in the presence of sodium chloride, from 2.9 × 10 ⁶ to the molar concentration of heparin present in said biological sample. Said measurement method wherein sodium chloride is present in a molar ratio of 3.9 × 10 ⁶ .

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