CN116593701B

CN116593701B - Creatine kinase isoenzyme detection kit containing biological blocker

Info

Publication number: CN116593701B
Application number: CN202310885056.9A
Authority: CN
Inventors: 赵科; 李进; 黄本巧; 吴海波
Original assignee: Beijing Homa Biological Engineering Co ltd
Current assignee: Beijing Homa Biological Engineering Co ltd
Priority date: 2023-07-19
Filing date: 2023-07-19
Publication date: 2024-01-23
Anticipated expiration: 2043-07-19
Also published as: CN116593701A

Abstract

The invention discloses a creatine kinase isoenzyme detection kit containing a biological blocker, which comprises a first reagent and a second reagent. The biological blocking agent contains various animal immunity protein substances aiming at interference of a xenotropic antibody, an anti-animal antibody, a rheumatoid factor and the like, has good blocking effect on endogenous interference factors with conventional content, and can effectively reduce false positive results of detection caused by endogenous interference. By introducing the blocking agent to the detection reagent at 0.1% to 2% by v/v, the detection of antibodies to the opposite sex, antibodies to the animal, rheumatoid factors, RF interference and false positives are improved.

Description

Creatine kinase isoenzyme detection kit containing biological blocker

Technical Field

The invention belongs to the technical field of diagnostic reagents, and particularly relates to detection of creatine kinase isozymes by an immunoturbidimetry.

Background

CK was first studied in 1960 and thought to be useful for diagnosing ACS, but was inferior in specificity to 1970

And then replaced by creatine kinase isozyme (CKMB). Accordingly, CKMB is recommended as a diagnostic gold standard for the previous generation of myocardial infarction (AMI) due to its better specificity and sensitivity.

The latex enhanced immunoturbidimetry is a dynamic antigen-antibody binding assay method by measuring the antigen of interest

The corresponding antibody is coated on the latex microsphere to increase the volume of the antigen-antibody conjugate, and in the detection light path, the light is transmitted and

the intensity change of scattered light is more remarkable, thereby improving the sensitivity of detection. Latex microspheres with different average particle sizes can also be used

The combination of the two components further improves the sensitivity and the linear range of detection. However, the inventors found in the study that the latex was reinforced

When the immunoturbidimetry is applied to the detection of CK-MB in a blood sample, rheumatoid factor (rheometer) existing in the sample

factor, RF) can interfere with detection. In addition, the non-specific binding of components such as proteins in the sample to antibodies causes background signals and also interferes with detection.

In view of the foregoing, there remains a need in the art for a detection kit for CKMB that has high specificity, low false positives, is resistant to RF interference, and is inexpensive.

Disclosure of Invention

The invention provides a latex enhanced immunoturbidimetry kit for detecting creatine kinase isozymes CK-MB, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a buffer solution, a coagulant, a biological blocker, a surfactant, a protective agent and a preservative, and the R2 reagent comprises a buffer solution, CK-MB antibody coated nano-microspheres, a protective agent and a preservative;

wherein, the buffer solution in the R2 reagent is TAPS, and the biological blocker in the R1 comprises at least one of animal immune protein mouse IgG, mouse IgM, rabbit IgG, goat IgG, cow IgG and horse IgG; the CK-MB antibody of the R2 reagent coats the nano-microspheres in the nano-microspheres, and is a composite nano-microsphere prepared by mixing small-diameter microspheres with average diameters of 100-200nm and large-diameter microspheres with average diameters of 200-400nm according to a volume ratio of 1:20-20:1, wherein the concentration is 0.02% -0.5% (w/v).

Wherein the R1 buffer is at least one selected from HEPES buffer, TRIS-HCl buffer and glycine buffer.

In some embodiments, the R1 preservative is selected from at least one of sodium azide, thimerosal, proClin 300.

In some embodiments, the R1 surfactant is selected from at least one of Tween20, tween80, NP40, thesit.

In some embodiments, the R2 protectant is selected from at least one of bovine serum albumin, ovalbumin, skim milk, calf serum.

In some embodiments, the coagulant in R1 is polyethylene glycol, at least one selected from PEG6000, PEG8000, PEG12000, PEG20000, preferably PEG20000.

In some embodiments, the preservative in R2 is sodium azide.

In some embodiments, the R2 protective agent is bovine serum albumin, ovalbumin, skim milk, calf serum.

In some embodiments the biological blocker in R1 is rabbit IgG (1%) + goat IgG (1%).

According to some embodiments, there is provided the use of a blocking agent in improving false positives of detection results.

In some embodiments, the blocking agent is a biocomposite blocking agent. In some embodiments, the detection refers to creatine kinase isozyme latex enhanced turbidimetric immunoassay.

According to some embodiments, there is provided the use of a blocking agent to improve the detection of a xenotropic antibody, an anti-animal antibody, a rheumatoid factor, RF interference. In some embodiments, the blocking agent is a biocomposite blocking agent.

In some embodiments, the assay is a creatine kinase isozyme latex enhanced turbidimetric immunoassay. In some embodiments, the detection result false positive is improved by introducing the blocking agent to the detection reagent at 0.1% to 2% v/v.

Compared with the prior art, the invention has the beneficial effects that: the application discloses a creatine kinase isoenzyme detection kit containing a biological blocker, wherein the detection kit is used for improving a specific antibody, an anti-animal antibody, a rheumatoid factor, RF interference and false positive of a detection result in detection by introducing the blocker into the detection reagent in an amount of 0.1-2% in terms of v/v, and the biological blocker and the detection kit have a synergistic effect when being a combination of rabbit IgG and goat IgG, so that the detection effect can be better improved.

Detailed Description

The present invention is further illustrated by the following examples, which are provided to illustrate the invention and not to limit the scope of the invention.

Example 1

Preparation of the first reagent

1. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA and 15mL bio-blocker (5 mg/mL) are weighed, dissolved in 1.0L double distilled water, ph is regulated to 7.2, and the volume is fixed to 1.5L to obtain the reagent first reagent (1).

2. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA, 10mL blocker (5 mg/mL) were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and the volume was fixed to 1.5L to prepare the first reagent (2).

3. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA, 5mL blocker (5 mg/mL) were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and the volume was fixed to 1.5L to prepare the first reagent (3).

4. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, and 7.5g BSA were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and the volume was adjusted to 1.5L to prepare the first reagent (4).

5. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA and 15mL IIR blocker are weighed, dissolved in 1.0L double distilled water, ph is adjusted to 7.2, and the volume is fixed to 1.5L to prepare the first reagent (5).

6. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA and 15mL HBR blocking agent are weighed, dissolved in 1.0L double distilled water, ph is adjusted to 7.2, and the volume is fixed to 1.5L to prepare the first reagent (6).

7. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA, 15mL mouse IgG blocker were weighed, dissolved in 1.0L double distilled water, ph adjusted to 7.2, and the volume was fixed to 1.5L to prepare the first reagent (7).

8. 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA and 15mL RF blocker are weighed, dissolved in 1.0L double distilled water, ph is adjusted to 7.2, and the volume is fixed to 1.5L to prepare the first reagent (8).

Example 2

Preparation of antibody coated particle nano microsphere

1. The particles (80 nm, carboxyl) were diluted to 1% (w/v) with MES buffer; 2. diluting the antibody (mouse monoclonal antibody) to 0.5mg/ml with MES buffer; 3. adding EDAC water solution with concentration of 0.1-1% (w/v) into the granules in step 1, and reacting for 30min at 37 ℃ in a constant temperature shaking table; 4. after the reaction is finished, adding the antibody in the step 2, and reacting for 3 hours at 37 ℃ in a constant-temperature shaking table; 5. adding a sealing agent, and standing at normal temperature overnight; 6. and (3) adding the particles which are sealed overnight into a cleaning solution, cleaning and centrifuging for 3 times, and preserving for later use. The order of step 1 and step 2 is interchangeable.

Example 3

Preparation of the second reagent

400mL of double distilled water is added into the particles prepared in the embodiment 2, 4.87g of TAPS, 5g of BSA, 1g of sodium azide and 50g of sucrose are added, the mixture is stirred and mixed uniformly, ph is regulated to 7.5, the double distilled water is added to 500mL, and the second reagent (1) is obtained after the main wavelength absorbance is basically unchanged after ultrasound.

Example 4

Preparation of the kit the aforementioned reagents were assembled into kits according to the following table.

Assembly of kit first reagent second reagent

Kit 1 first reagent (1) second reagent (1)

Kit 2 first reagent (2) second reagent (1)

Kit 3 first reagent (3) second reagent (1)

Kit 4 first reagent (4) second reagent (1)

Kit 5 first reagent (5) second reagent (1)

Kit 6 first reagent (6) second reagent (1)

Kit 7 first reagent (7) second reagent (1)

Kit 8 first reagent (8) second reagent (1)

Test example 1

Sensitivity test 8 kits prepared in the above examples were tested using a fully automated biochemical analyzer (e.g., hitachi 7180). Measuring 660nm wavelength, sampling sample 10 μl, adding 150 μl of the first reagent, keeping the temperature at 37deg.C for 5min, adding 50 μl of the second reagent, reading absorbance A1 after 42 seconds, reading absorbance A2 after incubation for 4 min 18 seconds at 37deg.C, and reacting absorbance ΔA=A2-A1;

the performance of the 8 examples described above was verified as follows:

；

from the above measurement, it was found that the kits 1 to 8 each achieved good sensitivity.

Test example 2

The effect of blocking agent on false positive results comparative kits were formulated according to the kit preparation method, with the only difference that no blocking agent was included. Samples with or without the xenotropic antibody 5mg/ml, mouse IgG10mg/ml, and RF 500IU/ml were tested with each kit prepared. The results are as follows, showing that the blocker can significantly improve false positives of test results, avoiding RF interference.

；

Conclusion: the results of the comparison experiments show that the biological composite blocking agent has good effect, and can effectively control the interference of the anisotropic antibody, the anti-animal antibody and the RF in the sample, so that the kit is safer and more effective.

Test example 3

Taking the kit 1 as an example, a CK-MB sample containing 600IU/ml of RF and a CK-MB sample containing no 600IU/ml of RF are prepared by using a commercially available rheumatoid factor standard, the CK-MB content is measured as a test sample, and the recovery rate is calculated by the calculation formula: recovery = CMKB sample (500 IU/ml RF) measurement ++cmkb sample measurement (500 IU/ml RF absent), the results are shown in table 3 below.

；

Table 3 shows that when the kit does not contain a blocker, CK-MB samples containing 500IU/ml of RF and CK-MB samples containing no CKBB and 500IU/ml of RF have detected CK-MB contents of 140.36ng/ml and 75.92ng/ml, respectively, and recovery rates are as high as 184.88%, indicating that the detection of CK-MB by RF is severely disturbed. Whereas in the case of kits containing rabbit IgG antibodies or goat IgG antibodies, recovery rates were 121.19% and 117.92%, respectively, indicating that both can block RF interference with CKMB detection. While the experimenter surprisingly found that there was a synergistic effect between the combination of the blocking agents of rabbit IgG and goat IgG after trying the combination of the multiple blocking agent complexes, the recovery rate could be as low as 104.74%.

The above embodiments are preferred embodiments of the present invention, and besides, the present invention may be implemented in other ways, and any obvious substitution is within the scope of the present invention without departing from the concept of the present invention.

Claims

1. A latex enhanced immunoturbidimetry kit for detecting creatine kinase isozyme CK-MB comprises an R1 reagent and an R2 reagent, wherein the R1 reagent comprises a buffer solution, a coagulant, a biological blocker, a surfactant, a protective agent and a preservative, and the R2 reagent comprises a buffer solution, CK-MB antibody coated nano-microspheres, a protective agent and a preservative;

wherein the R1 reagent is HEPES, naCl, PEG20000, tween80, sodium azide, BSA and a biological blocker; wherein the biological blocker in R1 is rabbit IgG+goat IgG, and the ratio of the rabbit IgG to the goat IgG is 1:1, a step of; the R2 reagent is CK-MB antibody coated nano-microsphere, TAPS, BSA, sodium azide and sucrose, wherein the nano-microsphere in the CK-MB antibody coated nano-microsphere is a composite nano-microsphere prepared by mixing small-diameter microsphere with average diameter of 100-200nm and large-diameter microsphere with average diameter of 200-400nm according to the volume ratio of 1:20-20:1, and the concentration is 0.02% -0.5% (w/v).

2. The latex-enhanced immunoturbidimetry kit for detecting creatine kinase isozyme CK-MB according to claim 1, wherein the R1 reagent is specifically 4.766g HEPES, 5.844g NaCl, 12g PEG20000, 15g Tween80, 1.0g sodium azide, 7.5g BSA and 15mL biological blocker, and the R2 reagent is specifically 4.87g TAPS, 5g BSA, 1g sodium azide and 50g sucrose.

3. Use of a latex-enhanced immunoturbidimetry kit for the detection of the creatine kinase isozyme CK-MB according to claim 1 or 2 for improving the detection of allophilic antibodies, anti-animal antibodies, rheumatoid factors, RF interference.