阅读说明：本技术 一种吖啶酯抗体标记方法及其应用 (Acridinium ester antibody labeling method and application thereof ) 是由 胡文波 于 2020-07-28 设计创作，主要内容包括：本发明公开了一种吖啶酯抗体标记方法及其应用；所述方法为：1)量取标记缓冲溶液于离心管中；2)加入髓过氧化物酶检测抗体,充分混匀；3)加入吖啶酯溶液,充分混匀,室温避光震荡反应；4)加入适量封闭液,室温避光振荡反应；5)加入适量的标记缓冲液定量,2～8℃密封保存。该方法可以得到灵敏度更高、线性范围广、也便于实现自动化的MPO检查产品。(The invention discloses an acridinium ester antibody labeling method and application thereof; the method comprises the following steps: 1) measuring a marking buffer solution in a centrifuge tube; 2) adding myeloperoxidase detection antibody, and mixing; 3) adding the acridinium ester solution, fully and uniformly mixing, and carrying out vibration reaction at room temperature in a dark place; 4) adding a proper amount of confining liquid, and carrying out shaking reaction at room temperature in a dark place; 5) adding a proper amount of a labeling buffer solution for quantification, and sealing and storing at 2-8 ℃. The method can obtain MPO inspection products with higher sensitivity, wide linear range and convenient realization of automation.)

1. A method for labeling an acridinium ester antibody, comprising the steps of:

1) measuring a marking buffer solution in a centrifuge tube;

2) adding myeloperoxidase detection antibody, and mixing;

3) adding the acridinium ester solution, fully and uniformly mixing, and carrying out vibration reaction at room temperature in a dark place;

4) adding a proper amount of confining liquid, and carrying out shaking reaction at room temperature in a dark place;

5) adding a proper amount of a labeling buffer solution for quantification, and sealing and storing at 2-8 ℃.

2. The method of claim 1, wherein: the buffer solution in the step 1) is PB buffer solution.

3. The method of claim 2, wherein: the marking buffer solution is 0.1mol/L PB buffer solution with pH7.6.

4. The method of claim 1, wherein: the ratio of the addition amount of the acridinium ester to the addition amount of the myeloperoxidase antibody is 2 (4-6).

5. The method of claim 4, wherein: the ratio of the addition amount of the acridinium ester to the addition amount of the myeloperoxidase antibody is 2: 5.

6. The method of claim 1, wherein: the time for the reaction of the antibody and the acridinium ester in the step 3) is 110-130 minutes.

7. The method of claim 1, wherein: the blocking solution of the step 4) is BSA solution with the concentration of 10%.

8. The method of claim 1, wherein: the reaction time of the step 4) is 25-35 minutes.

9. A method as claimed in claim 1, characterized in that the method comprises the following steps:

1) weighing 3mL of PB buffer solution with the concentration of 0.1mol/L and the pH value of 7.6 in a centrifuge tube;

2) adding 250 mu g of myeloperoxidase detection antibody, and fully and uniformly mixing;

3) adding 100 mu L of acridine ester solution (1mg/mL), fully and uniformly mixing, and shaking for 2 hours at room temperature in a dark place;

4) adding a proper amount of blocking solution until the concentration of BSA is 1%, and carrying out oscillation reaction for 30 minutes at room temperature in a dark place;

5) adding a proper amount of marking buffer solution to 6mL, and sealing and storing at 2-8 ℃.

10. Use of a labeled acridinium ester obtained by the method of any one of claims 1-9 in the preparation of a kit for detecting myeloperoxidase by chemiluminescence.

Technical Field

The invention relates to the field of biotechnology detection, in particular to a method for labeling an acridinium ester antibody in the preparation process of a chemiluminescence detection kit.

Background

Cardiovascular diseases are the first chronic diseases in China, the death accounts for more than 40% of the death of the diseases, the cardiovascular diseases are high in the first place, the disability rate is close to 40%, the life and health of individuals are seriously threatened, and huge economic burden and mental pain are brought to families and society. In recent years, the disease is becoming more and more younger, and sudden cardiac death in the third and forties years is rare. Such high lethality and disability rate are mainly caused by the acute onset and rapid progress, and many patients cannot be treated timely and effectively. Therefore, early discovery, early treatment is a powerful measure to avoid serious adverse consequences.

A large amount of clinical research data show that the level of Myeloperoxidase (MPO) is obviously increased several months before an adverse cardiovascular event occurs, the content level of MPO has obvious correlation with coronary artery lesion, the monitoring of the level of MPO in blood can evaluate and represent the risk of CAD occurrence of healthy people and the risk of cardiovascular accident recurrence of ACS patients, the missed diagnosis rate is only 9.3%, and the MPO detection of risk people or people with slight symptoms is beneficial to timely discovery of acute and severe cases, so that the possibility of avoiding the occurrence of malignant events is realized. However, most of the existing MPO detection methods are enzyme-linked immunosorbent assay, turbidimetric assay and colloidal gold assay, and due to the limitation of methodology, the sensitivity, stability, linear range and the like of detection are difficult to meet the clinical requirements, thereby influencing the function of MPO in the aspect of timely diagnosis and early treatment.

Therefore, there is an urgent need for a MPO detection method, such as chemiluminescence, which is convenient and rapid to detect and has more accurate results. The ChemiLuminescence (CL) method is a kind of molecular luminescence spectroscopy, and is a trace analysis method for determining the content of an analyte by detecting the ChemiLuminescence intensity of a system with an instrument according to the principle that the concentration of the analyte in a chemical detection system and the ChemiLuminescence intensity of the system are in a linear quantitative relationship under a certain condition. The chemiluminescence analysis method has the advantages of high sensitivity, simple equipment, convenient operation, wide linear range, fast analysis, convenient realization of automation and the like. The acridinium ester is a commonly used luminescent agent in a chemiluminescence method, and the process of labeling the antibody with the acridinium ester directly influences the luminous efficiency, namely influences the sensitivity and accuracy of a detection result.

Disclosure of Invention

The invention aims to solve the technical problem of providing an antibody labeling method of acridinium ester in the process of detecting myeloperoxidase by a chemiluminescence method so as to realize MPO detection with higher efficiency and more accurate result. The specific technical scheme is as follows:

the method for labeling the acridinium ester antibody comprises the following steps:

1) measuring a marking buffer solution in a centrifuge tube;

2) adding myeloperoxidase detection antibody, and mixing;

3) adding the acridinium ester solution, fully and uniformly mixing, and carrying out vibration reaction at room temperature in a dark place;

4) adding a proper amount of confining liquid, and carrying out shaking reaction at room temperature in a dark place;

5) adding a proper amount of a labeling buffer solution for quantification, and sealing and storing at 2-8 ℃.

The buffer solution in step 1) is a PB buffer solution (phosphate buffer). The link can use more buffers, such as PBS buffer (phosphate buffer), CB buffer (carbonate buffer) and the like, and different buffer systems and the concentration and pH of the buffer systems can influence the light reflection intensity of the acridinium ester. The labeling buffer of the present invention is preferably 0.1mol/L PB buffer, pH 7.6. The buffer solution of the step 5) is the same as that of the step 1).

The ratio of the addition amount of the acridinium ester to the myeloperoxidase antibody in the method is 2 (4-6), and the ratio is a weight ratio. The detection of different antibodies is realized, the dosage ratio of the acridinium ester to the antibodies is very different, and the conditions of weak luminescent signals or nonspecific combination and the like can occur in improper proportioning, so that the detection sensitivity is low or the result is inaccurate. Therefore, for the detection of MPO antibody, the relationship between the amount of acridinium ester and the amount of the antibody is obtained by creative labor.

In a preferred embodiment, the ratio of the addition of the acridinium ester to the myeloperoxidase antibody according to the invention is 2: 5.

The time for the reaction between the antibody in the step 3) and the acridinium ester under the condition of light shielding and shaking at room temperature is 110-130 minutes. The length of the reaction time generally affects the degree of antibody conjugation and the intensity of the luminescent signal, but the false positive phenomenon occurs with time, and therefore, it is necessary to examine the optimum binding site between the two.

The blocking solution in the step 4) is BSA (bovine serum albumin) solution with the concentration of 10%, and the solubility of the blocking solution is better. Further, the reaction time of the confining liquid is 25-35 minutes.

In another preferred embodiment, the method of the present invention comprises the steps of:

1) weighing 3mL of PB buffer solution with the concentration of 0.1mol/L and the pH value of 7.6 in a centrifuge tube;

2) adding 250 mu g of myeloperoxidase detection antibody, and fully and uniformly mixing;

3) adding 100 mu L of acridine ester solution (1mg/mL), fully and uniformly mixing, and shaking for 2 hours at room temperature in a dark place;

4) adding a proper amount of blocking solution until the concentration of BSA is 1%, and carrying out oscillation reaction for 30 minutes at room temperature in a dark place;

5) adding a proper amount of marking buffer solution to 6mL, and sealing and storing at 2-8 ℃.

Wherein, the step 4) is to add a proper amount of 10% BSA solution into the acridinium ester labeled antibody solution to reduce the concentration of BSA to 1%.

The invention also provides application of the marked acridinium ester obtained by the method in preparation of a kit for detecting myeloperoxidase by a chemiluminescence method. The MPO detection kit prepared by the method has strong detection specificity and luminescent signals and high sensitivity.

Due to the implementation of the technical scheme, compared with the prior art, the invention has the following advantages:

the method has the advantages of full coupling of the acridinium ester and the antibody, strong luminescent signal, no non-specific binding and false positive phenomena, simple labeling method, good repeatability and convenient large-scale application. The method can obtain MPO checking products with higher sensitivity, wide linear range and convenient realization of automation.

Detailed Description