CN111879758A - Acridinium ester antibody labeling method and application thereof - Google Patents
Acridinium ester antibody labeling method and application thereof Download PDFInfo
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- CN111879758A CN111879758A CN202010735336.8A CN202010735336A CN111879758A CN 111879758 A CN111879758 A CN 111879758A CN 202010735336 A CN202010735336 A CN 202010735336A CN 111879758 A CN111879758 A CN 111879758A
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
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.
Description
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
Example 1 (50 tests/batch, the same applies hereinafter)
A method for labeling an acridinium ester antibody, comprising 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 345 microliter of 10% BSA solution, mixing uniformly, 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 ℃.
Example 2
A method for labeling an acridinium ester antibody, comprising the steps of:
1) weighing 3mL of PB buffer solution with the concentration of 0.1mol/L and the pH value of 7.4 in a centrifuge tube;
2) adding 200 mug 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 110 minutes at room temperature in a dark place;
4) adding 345 mu L of 10% BSA solution, and carrying out oscillation reaction for 30 minutes at room temperature in the dark;
5) adding a proper amount of marking buffer solution to 6mL, and sealing and storing at 2-8 ℃.
Example 3
A method for labeling an acridinium ester antibody, comprising 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 200 mug 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 130 minutes at room temperature in a dark place;
4) adding 345 mu L of 10% BSA solution, and carrying out oscillation reaction at room temperature in the dark for 25 minutes;
5) adding a proper amount of marking buffer solution to 6mL, and sealing and storing at 2-8 ℃.
Example 4
A method for labeling an acridinium ester antibody, comprising 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 300 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 130 minutes at room temperature in a dark place;
4) adding 345 mu L of 10% BSA solution, and carrying out oscillation reaction at room temperature in the dark for 25 minutes;
5) adding a proper amount of marking buffer solution to 6mL, and sealing and storing at 2-8 ℃.
Example 5
A method for labeling an acridinium ester antibody, comprising the steps of:
1) weighing 3mL of PB buffer solution with the concentration of 0.1mol/L and the pH value of 7.4 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 130 minutes at room temperature in a dark place;
4) adding 345 mu L of 10% BSA solution, and carrying out oscillation reaction for 35 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 ℃.
Example 6
A method for labeling an acridinium ester antibody, comprising the steps of:
1) weighing 3mL of PB buffer solution with the concentration of 0.1mol/L and the pH value of 7.4 in a centrifuge tube;
2) adding 300 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 120 minutes at room temperature in a dark place;
4) adding 345 mu L of 10% BSA solution, and carrying out oscillation reaction at room temperature in the dark for 25 minutes;
5) adding a proper amount of marking buffer solution to 6mL, and sealing and storing at 2-8 ℃.
The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.
Claims (10)
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.
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