CREATINE KINASE ISOENZYME ASSAY KIT

Abstract

A creatine kinase isoenzyme latex-enhanced immunoturbidimetric assay kit, comprising a first reagent and a second reagent. The first reagent comprises a buffer solution, an electrolyte, polyethylene glycol, a surfactant, a preservative, a blocking agent, and a protective agent. The second reagent comprises a buffer solution, polystyrene latex particles coated with a creatine kinase isoenzyme antibody, the creatine kinase isoenzyme antibody on the latex particles, a protective agent, a stabilizer, and a preservative.

Claims

1. A kit for detecting creatine kinase isoenzyme by latex-enhanced immunoturbidimetry, comprising a first reagent and a second reagent, wherein the first reagent comprises: TABLE-US-00019 10 mM-100 mM buffer, 5 g/L-50 g/L electrolyte, 1 g/L-10 g/L polyethylene glycol, 1 g/L-20 g/L surfactant, 0.5 g/L-1.5 g/L preservative, 0.1%-2%, v/v blocking agent, 1 g/L-20 g/L protective agent, pH 6.5-8.51; the second reagent comprises: TABLE-US-00020 10 mM-100 mM buffer, 0.1%-0.5%, w/v polystyrene latex particle coated with antibody against creatine kinase isoenzyme, 0.02 g/L-0.05 g/L antibody against creatine kinase isoenzyme on said latex particle, 1 g/L-20 g/L protective agent, 20 g/L-200 g/L stabilizer, 0.5 g/L-1.5 g/L preservative, pH 6.5-8.51; the buffer is selected from the group consisting of glycine buffer, TRIS-HCl buffer and HEPES buffer; the preservative is selected from the group consisting of sodium azide, thimerosal and ProClin300; the polyethylene glycol is selected from the group consisting of PEG 6000, PEG 8000, PEG 12000 and PEG 20000; the surfactant is selected from the group consisting of Tween 20, Tween 80, NP40 and thesit; the protective agent is selected from the group consisting of bovine serum albumin, ovalbumin, skim milk and calf serum; the blocking agent is goat anti-mouse IgG; the stabilizer is selected from the group consisting of sucrose, glycerol, trehalose and glucose; the surface functional group of the polystyrene latex particle is selected from the group consisting of amino group, carboxyl group, chloromethyl group and epoxy group; the polystyrene latex particle has an average particle size of from 200 nm to 500 nm; the antibody against creatine kinase isozyme is derived from mouse or goat; and the antibody against creatine kinase isozyme is monoclonal antibody.

2. The kit for detecting creatine kinase isoenzyme by latex-enhanced immunoturbidimetry according to claim 1, comprising a first reagent and a second reagent, wherein the first reagent comprises: TABLE-US-00021 50 mM HEPES buffer, 8.77 g/L NaCl, 9 g/L PEG20000, 10 g/L Tween 80, 1.0 g/L sodium azide, 1%, v/v blocking agent, 5 g/L BSA, pH 7.2; the second reagent comprises: TABLE-US-00022 50 mM glycine buffer, 0.1%-0.5%, w/v polystyrene latex particle coated with antibody against creatine kinase isoenzyme, 0.02 g/L-0.05 g/L antibody against creatine kinase isoenzyme on the latex particle, 5 g/L BSA, 1 g/L sodium azide, 100 g/L sucrose, pH 7.5; the antibody is murine monoclonal antibody; the average particle size of the latex particles is 300 nm; the latex particle is modified with carboxyl group; and the blocking agent is goat anti-mouse IgG.

3. The kit for detecting creatine kinase isoenzyme by latex-enhanced immunoturbidimetry according to claim 1, comprising a first reagent and a second reagent, wherein the first reagent comprises: TABLE-US-00023 50 mM HEPES buffer, 8.77 g/L NaCl, 9 g/L PEG20000, 10 g/L Tween 20, 1.0 g/L sodium azide, 1%, v/v blocking agent, 5 g/L BSA, pH 7.2; the second reagent comprises: TABLE-US-00024 50 mM glycine buffer, 0.1%-0.5%, w/v polystyrene latex particle coated with antibody against creatine kinase isoenzyme, 0.02 g/L-0.05 g/L antibody against creatine kinase isoenzyme on the latex particle, 5 g/L BSA, 1 g/L sodium azide, 100 g/L sucrose, pH 7.5; the antibody is murine monoclonal antibody; the average particle size of the latex particles is 300 nm; the latex particle is modified with carboxyl group; and the blocking agent is goat anti-mouse IgG.

4. The kit for detecting creatine kinase isoenzyme by latex-enhanced immunoturbidimetry according to claim 1, comprising a first reagent and a second reagent, wherein the first reagent comprises: TABLE-US-00025 50 mM glycine buffer, 8.77 g/L NaCl, 9 g/L PEG20000, 10 g/L Tween 80, 1.0 g/L sodium azide, 1%, v/v blocking agent, 5 g/L BSA, pH 7.2; the second reagent comprises: TABLE-US-00026 50 mM Tris buffer, 0.1%-0.5%, w/v polystyrene latex particle coated with antibody against creatine kinase isoenzyme, 0.02 g/L-0.05 g/L antibody against creatine kinase isoenzyme on the latex particle, 5 g/L BSA, 1 g/L sodium azide, 100 g/L sucrose, pH 7.5; the antibody is murine monoclonal antibody; the average particle size of the latex particles is 300 nm; the latex particle is modified with carboxyl group; and the blocking agent is goat anti-mouse IgG.

5. A method of improving false positive detection results, the method comprising using a blocking agent, wherein the blocking agent is goat anti-mouse IgG; and the detection is an assay for creatine kinase isoenzyme by latex-enhanced immunoturbidimetry.

6. A method of improving RF interference with detection, the method comprising using a blocking agent, wherein the blocking agent is goat anti-mouse IgG; and the detection is an assay for creatine kinase isoenzyme by latex-enhanced immunoturbidimetry.

7. The method according to claim 5, further comprising incorporating the blocking agent into a detection reagent at 0.1% to 2%, v/v.

Description

DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1 shows the calibration curve obtained by using the kit of the present application.

DETAILED DESCRIPTION OF THE DISCLOSURE

Example 1. Preparation of the First Reagent

[0052] 1. 17.87 g HEPES, 13.15g NaCl, 9 g PEG20000, 15 g Tween 80, 1.5 g sodium azide, 7.5 g BSA and 15 mL blocking agent (5 mg/m1) were weighed and dissolved in 1.0 L double distilled water, pH was adjusted to 7.2, and the total volume was adjusted to 1.5 L to obtain the first reagent (1):

TABLE-US-00009 50 mM HEPES buffer, 8.77 g/L NaCl, 9 g/L PEG20000, 10 g/L Tween 80, 1.0 g/L sodium azide, 1%, v/v blocking agent, 5 g/L BSA, pH 7.2.

[0053] 2. Alternatively, 9.09 g Tris, 13.15 g NaCl, 9 g PEG 20000, 15 g Tween 80, 1.5 g sodium azide, 7.5 g BSA and 10 mL blocking agent (the same as mentioned above) were weighed and dissolved in 1.0 L double distilled water, pH was adjusted to 7.2, and the total volume was adjusted to 1.5 L to obtain the first reagent (2):

TABLE-US-00010 50 mM Tris buffer, 8.77 g/L NaCl, 9 g/L PEG20000, 10 g/L Tween 80, 1.0 g/L sodium azide, 1%, v/v blocking agent, 5 g/L BSA, pH 7.2.

[0054] 3. Alternatively, 5.63 g glycine, 13.15 g NaCl, 9g PEG20000, 15 g Tween80, 1.5 g sodium azide, 7.5 g BSA, 10 mL blocking agent (the same as above) were weighed and dissolved in 1.0 L double distilled water, pH was adjusted to 7.2, and the total volume was adjusted to 1.5 L to obtain the first reagent (3):

TABLE-US-00011 50 mM glycine buffer, 8.77 g/L NaCl, 9 g/L PEG20000, 10 g/L Tween 80, 1.0 g/L sodium azide, 1%, v/v blocking agent, 5 g/L BSA, pH 7.2.

Example 2. Preparation of Particles Coated with Antibody 1. The particles (300 nm, carboxyl group) were diluted to 1% (w/v) with HEPES buffer;

[0055] 2. The antibody (mouse monoclonal antibody) was diluted to 0.5 mg/ml with HEPES buffer;

[0056] 3. The particles obtained from step 1 were added with 0.1% to 1% (w/v) of EDAC aqueous solution, and incubated at 37 C. for 30 min in a shaker at constant temperature;

[0057] 4. After the reaction was completed, the antibody obtained from step 2 was added, and reacted at 37 C. for 3 h in a constant temperature shaker;

[0058] 5. Then a blocking agent was added and incubated at room temperature overnight;

[0059] 6. The particles which have been blocked overnight were washed and centrifuged for 3 times with washing solution and then stored for use.

[0060] Steps 1 and 2 may be interchangeable.

Example 3. Preparation of the Second Reagent

[0061] 1. The particles prepared in Example 2 were added into 400 mL double distilled water, supplemented with 18.76 g glycine, 2.5 g BSA, 0.5 g sodium azide, 50 g sucrose, stirred and mixed, pH was adjusted to 7.5, supplemented with double distilled water to 500 mL, and ultrasonicated until the absorbance at the main wavelength was substantially stable. The second reagent (1) was obtained:

TABLE-US-00012 50 mM glycine buffer, 0.1%-0.5%, w/v polystyrene latex particle coated with antibody, 0.02 g/L-0.05 g/L antibody on the latex particle, 5 g/L BSA, 1 g/L sodium azide, 100 g/L sucrose, pH 7.5.

[0062] 2. Alternatively, the particles prepared in Example 2 were added into 400 mL double distilled water, supplemented with 5.96 g HEPES, 2.5 g BSA, 0.5 g sodium azide, 50 g sucrose, stirred and mixed, pH was adjusted to 7.5, supplemented with double distilled water to 500 mL, and ultrasonicated until the absorbance at the main wavelength was substantially stable. The second reagent (2) was obtained:

TABLE-US-00013 50 mM HEPES buffer, 0.1%-0.5%, w/v polystyrene latex particle coated with antibody, 0.02 g/L-0.05 g/L antibody on the latex particle, 5 g/L BSA, 1 g/L sodium azide, 100 g/L sucrose, pH 7.5.

[0063] 3. Alternatively, the particles prepared in Example 2 were added into 400 mL double distilled water, supplemented with 3.03 g Tris, 2.5 g BSA, 0.5 g sodium azide, 50 g sucrose, stirred and mixed, pH was adjusted to 7.5, supplemented with double distilled water to 500 mL, and ultrasonicated until the absorbance at the main wavelength was substantially stable. The second reagent (3) was obtained:

TABLE-US-00014 50 mM Tris buffer, 0.1%-0.5%, w/v polystyrene latex particle coated with antibody, 0.02 g/L-0.05 g/L antibody on the latex particle, 5 g/L BSA, 1 g/L sodium azide, 100 g/L sucrose, pH 7.5.

Example 4. Preparation of the Kit

[0064] The kits were assembled with the reagents mentioned above according to the following table.

TABLE-US-00015 TABLE 1 Assembly of the kit the first reagent the second reagent Kit 1 the first reagent (1) the second reagent (1) Kit 2 the first reagent (2) the second reagent (1) Kit 3 the first reagent (3) the second reagent (1) Kit 4 the first reagent (2) the second reagent (2) Kit 5 the first reagent (3) the second reagent (2) Kit 6 the first reagent (3) the second reagent (3)

TEST EXAMPLES

Test Example 1. Test for Sensitivity

[0065] The six kits prepared in the above examples were tested using an automatic biochemical analyzer (for example, Hitachi 7180).

[0066] The measurement wavelength was at 660 nm, and the sampling amount was 10 L. 150 L of the first reagent was added, incubated at constant temperature of 37 C. for 5 min, then 50 L of the second reagent was added, the absorbance A1 was read 42 seconds later, and the absorbance A2 was read after incubating at 37 C. for 4 minutes and 18 seconds. The reaction absorbance was calculated as A=A2A1. The performance of the above six examples was tested, and the results are shown as follows:

TABLE-US-00016 TABLE 2 Results of detection limit No. of Kit Performance 1 2 3 4 5 6 Detection limit (ng/ml) 1.10 1.32 1.56 1.78 1.65 1.35 Precision (%) 2.54 3.58 2.95 2.65 3.78 3.15 Low-value quality control

[0067] According to the above measurement, it was found that all the kits 1 to 6 achieved good sensitivity, among which kit 1 was optimal with sensitivity of 1 ng/ml.

Test Example 2. The Selection of Surfactant

[0068] The following kits were prepared according to the preparation method for Kit 1, except that the surfactant Tween 80 was replaced with Tween 20, NP40 and thesit, respectively, and the anti-interference effect against the triglyceride was tested.

TABLE-US-00017 TABLE 3 Screening of surfactant surfactant Tween 80 Tween 20 NP40 thesit Control 7.44 7.47 6.84 7.04 (without interference) 1000 mg/dL triglyceride 7.49 7.68 8.01 7.65 Relative deviation 0.67% 2.81% 17.11% 8.66%

[0069] It can be seen that Tween-80 has the strongest anti-chylosis tolerance.

Test Example 3. Effect of Blocking Agent on False Positive Results

[0070] The comparative kit was prepared according to the preparation method of Kit 1, except that the blocking agent was absent. Samples with or without 500 IU/ml RF were tested with each kit prepared. The results are shown as follows, showing that the blocking agent can significantly improve the detection results from being false positive and can avoid RF interference.

TABLE-US-00018 TABLE 4 Effect of blocking agent on false positive results Blank control with RF blocking agent without RF (500 IU/ml) Recovery rate Without blocking agent 47.20 ng/ml 200.2 ng/ml .sup.424% With blocking agent 47.45 ng/ml 48.23 ng/ml 101.6%