CN110887968A - Quantitative detection method of alpha-fetoprotein - Google Patents
Quantitative detection method of alpha-fetoprotein Download PDFInfo
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- CN110887968A CN110887968A CN201911261911.9A CN201911261911A CN110887968A CN 110887968 A CN110887968 A CN 110887968A CN 201911261911 A CN201911261911 A CN 201911261911A CN 110887968 A CN110887968 A CN 110887968A
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a quantitative detection method of alpha fetoprotein, which comprises the following steps: the kit comprises a biotin-labeled alpha fetoprotein antibody, a streptavidin magnetic particle suspension, an acridinium ester-labeled alpha fetoprotein antibody, a standard, a cleaning solution, a diluent, a chemiluminescent substrate solution A and a chemiluminescent substrate solution B; the streptavidin and the biotin have high-specificity binding capacity, and the streptavidin and the biotin-labeled high-purity antibody are specifically bound through non-covalent bonds, so that the streptavidin-labeled high-purity antibody has the effect of cascade amplification, and the reaction is highly specific; the invention combines the high specificity of the antibody-antigen reaction with the high sensitivity of the acridinium ester luminescence, utilizes the acridinium ester to capture photons generated by the reaction to detect the product concentration, and has the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to a quantitative detection method of alpha-fetoprotein.
Background
Alpha-fetoprotein (AFP) is a glycoprotein belonging to the albumin family, mainly synthesized by fetal liver cells and the yolk sac. The alpha-fetoprotein has higher concentration in fetal blood circulation, and is reduced after birth, and the alpha-fetoprotein is basically replaced by albumin after 2-3 months, so that the alpha-fetoprotein is difficult to detect in blood, and the content of the alpha-fetoprotein in serum of adults is extremely low. Alpha-fetoprotein has many important physiological functions including transport function, bidirectional regulatory function as a growth regulatory factor, immunosuppression, T-lymphocyte induced apoptosis, and the like. The alpha fetoprotein is closely related to the occurrence and development of liver cancer and various tumors, can show higher concentration in various tumors, and can be used as a positive detection index of various tumors. At present, the serum marker is mainly used as a serum marker of primary liver cancer clinically and is used for diagnosing and monitoring the curative effect of the primary liver cancer.
Therefore, clinical molecular diagnosis of AFP is important, especially for sensitive detection of early detection of tumors and treatment planning. Currently, there are many immunoassay methods for determining AFP, and the most common serum assay methods are: enzyme Immunoassay (EIA), Radioimmunoassay (RIA), chemiluminescence, and time-resolved immunofluorescence assay (TRFIA). The enzyme labeling method is a semi-quantitative reagent, has great limitations in accuracy and sensitivity, is very susceptible to labeling reaction, temperature, pH value, ion concentration in solution and other factors for enzyme activity, and has the defects of narrow linear range, time consumption and the like; the radioimmunoassay has the defects of radioactivity, short time for placing a marker, short effective period of a reagent and the like; the magnetic particle chemiluminescence technology is a novel labeling immunoassay technology which combines an immunomagnetic bead system, a chemiluminescence system and an immunoreaction system and is used for detecting trace antigens or antibodies. The reagent has the characteristics of high sensitivity of radioimmunity, simplicity, convenience and rapidness of enzyme-linked immunosorbent assay, easy automation operation, no use of harmful reagents in testing, long shelf life of the reagents, and application to biological, medical research and clinical experimental diagnosis.
Disclosure of Invention
The invention aims to provide a quantitative detection method of alpha fetoprotein, which has the advantages of high sensitivity, short reaction time, simple operation and high anti-interference performance.
In order to achieve the above object, the present invention provides a method for quantitatively detecting alpha-fetoprotein, comprising a biotin-labeled alpha-fetoprotein antibody, a streptavidin magnetic particle suspension, an acridinium ester-labeled alpha-fetoprotein antibody, a standard, a washing solution, a diluent, a chemiluminescent substrate solution a, and a chemiluminescent substrate solution B.
The streptavidin magnetic particle suspension is a streptavidin-coated magnetic particle solution.
The biotin-labeled alpha-fetoprotein antibody is a monoclonal antibody.
The diluent is PBS buffer solution containing a dissociation agent, and the dissociation agent is 2-methoxyestradiol.
The standard substance is PBS buffer solution containing alpha fetoprotein antigens with different concentrations.
The cleaning solution is Tris-HCl buffer solution with the concentration of 0.025mol/L, and the Tris-HCl buffer solution contains NaCl with the concentration of 0.15mol/L and 0.05% Tween-20.
The chemiluminescent substrate solution A is prepared from 1.5% of H by mass2O2And HNO with a concentration of 0.1mol/L3And (4) forming.
The chemiluminescence substrate solution B consists of 0.1mol/L Triton X-100 and 0.35mol/L NaOH mixed solution.
A detection method of an alpha-fetoprotein kit comprises the following steps:
1) adding 20ul of sample into a reaction tube, sequentially adding 30ul of biotin-labeled alpha-fetoprotein antibody and 30ul of acridinium ester-labeled alpha-fetoprotein antibody, incubating at 37 ℃ for 15min, finally adding 30ul of streptavidin magnetic particle suspension, and incubating at 37 ℃ for 15 min;
2) separating the reaction tube after incubation reaction on a magnetic separation frame for 2min, removing the supernatant, adding 500ul of cleaning solution, and cleaning on the magnetic separation frame for 1 min; removing supernatant, adding 300ul of cleaning solution, and cleaning on a magnetic separation frame for 1 min;
3) 100ul of chemiluminescent substrate solution A is added into a reaction tube, 100ul of chemiluminescent substrate solution B is added after 1min, and after incubation for 5min at 37 ℃ in the dark, the luminescence value is measured.
The invention has the following beneficial effects:
the kit for chemiluminescence detection of alpha fetoprotein provided by the invention is an acridinium ester chemiluminescence system, namely acridineThe ester luminescent system is simple, does not need a catalyst, and is placed in H2O2The solution can emit light without a catalytic process or a reinforcing agent, so that background luminescence is reduced, sensitivity is improved, and interference effect is small. The light release of the acridinium ester is fast and centralized, the light-emitting peak value is 0.4s, the half-life period is 0.8s, and the light-emitting efficiency and the intensity are high. And the acridinium ester is easy to be linked with protein, has small molecular weight, has small influence on the conformation of the linked antibody and has good labeling stability.
The streptavidin and the biotin have high-specificity binding capacity, and the streptavidin and the biotin-labeled high-purity antibody are specifically bound through non-covalent bonds, so that the streptavidin-labeled high-purity antibody has the effect of cascade amplification, and the reaction is highly specific. Therefore, the sensitivity is improved, non-specific interference is not increased, and the binding property is not affected by the high dilution of the reaction reagent, so that the non-specific action of the reaction reagent can be reduced to the maximum extent in practical application. The invention combines the high specificity of the antibody-antigen reaction with the high sensitivity of the acridinium ester luminescence, utilizes the acridinium ester to capture photons generated by the reaction to detect the product concentration, and has the characteristics of higher sensitivity, short reaction time, simple operation and high anti-interference performance.
Drawings
FIG. 1 is an image under an electron microscope of streptavidin magnetic particles prepared in example 2.
FIG. 2 is a standard curve diagram of the kit for detecting alpha-fetoprotein standards with different concentrations.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below.
Example 1
A kind of alpha fetoprotein magnetic particle chemiluminescence kit, including biotin-labeled alpha fetoprotein antibody, streptavidin magnetic particle suspension, acridinium ester-labeled alpha fetoprotein antibody, standard substance, cleaning solution, diluent, chemiluminescence substrate solution A, chemiluminescence substrate solution B;
the streptavidin magnetic particle suspension is a streptavidin-coated magnetic particle solution.
The biotin-labeled alpha-fetoprotein antibody is a monoclonal antibody.
The cleaning solution is Tris-HCl buffer solution with the concentration of 0.025mol/L, and the Tris-HCl buffer solution contains NaCl with the concentration of 0.15mol/L and 0.05% Tween-20.
The chemiluminescent substrate solution A is prepared from 1.5% of H by mass2O2And HNO with a concentration of 0.1mol/L3And (4) forming.
The chemiluminescence substrate solution B consists of 0.1mol/L Triton X-100 and 0.35mol/L NaOH mixed solution.
The standard substance is prepared by taking Tris-HCl buffer solution containing 0.5-5.0% BSA as a matrix and adding alpha-fetoprotein antigen, and the concentration gradient of the standard substance is as follows: 0ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml, 250 ng/ml.
The diluent is a buffer solution containing a dissociation agent, and the dissociation agent is 2-methoxyestradiol.
A detection method of an alpha fetoprotein magnetic particle chemiluminescence kit comprises the following steps:
1) adding 50ul of a sample or a standard substance into a reaction tube, sequentially adding 50ul of an alpha-fetoprotein antibody marked by biotin and 50ul of an alpha-fetoprotein antibody marked by acridinium ester to form an antibody-antigen-antibody sandwich compound, incubating for 15min at 37 ℃, finally adding 50ul of a streptavidin magnetic particle suspension, forming a solid phase by the interaction of streptavidin and biotin of the compound, and incubating for 10min at 37 ℃;
2) separating the reaction tube after the incubation reaction on a magnetic separation frame for 3min, removing the supernatant, adding 500ul of cleaning solution, and cleaning on the magnetic separation frame for 3 min; removing supernatant, adding 300ul of cleaning solution, and cleaning for 3min on a magnetic separation frame;
3) 100ul of chemiluminescent substrate solution A is added into a reaction tube, 100ul of chemiluminescent substrate solution B is added after 1min, and after incubation for 5min at 37 ℃ in the dark, the luminescence value is measured.
Example 2
The invention discloses a preparation method of streptavidin magnetic particles, which comprises the following specific steps:
a. system for makingPreparing nano magnetic particles: FeSO (ferric oxide) is added4.7H2O and FeCl3.6H2And mixing the O solution according to the mass ratio of 1:2, wherein the total concentration of ferric salt is 0.5mol/L, adding the mixture into a triangular flask, adding 200mL of distilled water, keeping the temperature at 30 ℃, introducing nitrogen, adjusting the pH to 10 by 0.25mol/LNaOH, rapidly stirring for 30min at 70 ℃, standing and crystallizing for 2h at 70 ℃, carrying out magnetic separation to obtain ferroferric oxide particles, washing for 3 times by using distilled water, and washing for 3 times by using absolute ethyl alcohol to obtain the ferroferric oxide nano magnetic particles.
b. Surface modification of magnetic particles: mixing ferroferric oxide nano magnetic particles with absolute ethyl alcohol according to the volume ratio of 1:8, adding an APTES reagent, stirring for 6-8 hours at room temperature, and carrying out magnetic separation to obtain silanized ferroferric oxide nano magnetic beads; washing the silanized ferroferric oxide nano magnetic beads for 2-3 times by using an absolute ethyl alcohol solution to obtain a silanized ferroferric oxide nano magnetic bead absolute ethyl alcohol mixed solution; washing the silanized ferroferric oxide nano magnetic bead absolute ethyl alcohol mixed solution for 2-3 times by PBS buffer solution to obtain magnetic particle mixed solution; adding glutaraldehyde solution into the magnetic particle mixed solution, oscillating and crosslinking for 1.5-2.5 hours at room temperature, performing magnetic separation, and removing supernatant to obtain aminated ferroferric oxide nano magnetic particle mixed solution
c. Preparation of streptavidin magnetic particles: coating the aminated ferroferric oxide nano magnetic particle mixed solution with streptavidin, oscillating and fixing for 6 hours at room temperature, carrying out magnetic separation to obtain the coated nano magnetic particle mixed solution, washing with distilled water and PBS buffer solution for 3-4 times respectively to obtain immune nano magnetic particles, finally dispersing the streptavidin magnetic particles in 5ml of PBS solution, and storing at 4 ℃ for later use.
The diameter of the prepared streptavidin magnetic particle is 40-60 nanometers, the nano magnetic particle has superparamagnetism, namely, the nano magnetic particle has stronger magnetic responsiveness in an external magnetic field, and after the magnetic field is removed, the magnetism of the magnetic particle disappears immediately, namely, the magnetic particle has no remanence and is uniformly dispersed in the solution again. Fig. 1 is an image of the streptavidin magnetic particle prepared in this example under an electron microscope, and it can be seen from fig. 1 that the streptavidin magnetic particle has uniform particle size, regular spherical shape, and uniform size.
Example 3
The kit is detected by using a standard substance, the detection result is shown in table 1, and the lowest sensitivity detection value is 2 ng/ml; the calibration curve is prepared based on the chemiluminescence values and the concentrations, as shown in FIG. 2, and the linear coefficient r20.9937, linear range 1 ~ 250 ng/ml.
TABLE 1
Example 3
The invention also carries out repeated tests to verify the accuracy of the kit.
A10 ng/ml alpha fetoprotein standard substance is taken to carry out 10 times of experiments, and a detection concentration value is calculated by using a linear equation y which is 10.187x + 19.141.
The detection results are shown in Table 3, the measured average values are 10.041ng/ml, the standard deviation is 0.122, the intra-batch variation coefficient is 1.2%, and the kit has high detection result accuracy and good linear relation and can be used as an important tool for auxiliary diagnosis of liver cancer.
TABLE 2
| Alpha fetoprotein standard (10ng/ml) | |
| For the first time | 9.87 |
| For the second time | 10.11 |
| The third time | 10.09 |
| Fourth time | 9.95 |
| Fifth time | 10.28 |
| The sixth time | 9.89 |
| The seventh time | 10.06 |
| The eighth time | 10.11 |
| The ninth time | 9.98 |
| The tenth time | 10.07 |
| Mean value of | 10.041 |
| Standard deviation of | 0.122 |
| Coefficient of variation within batch | 1.2％ |
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A quantitative detection method of alpha fetoprotein is characterized by comprising a biotin-labeled alpha fetoprotein antibody, a streptavidin magnetic particle suspension, an acridinium ester-labeled alpha fetoprotein antibody, a standard, a cleaning solution, a diluent, a chemiluminescent substrate solution A and a chemiluminescent substrate solution B.
2. The method for quantitatively detecting alpha-fetoprotein according to claim 1, wherein the streptavidin magnetic particle suspension is a streptavidin-coated magnetic particle solution.
3. The method of claim 1, wherein the biotin-labeled alpha-fetoprotein antibody is a monoclonal antibody.
4. The method for quantitatively detecting the alpha-fetoprotein as claimed in claim 1, wherein the diluent is a PBS buffer solution containing a dissociation agent, and the dissociation agent is 2-methoxyestradiol.
5. The method for quantitatively detecting the alpha-fetoprotein according to claim 1, wherein the standard substance is PBS buffer solution containing different concentrations of alpha-fetoprotein antigens.
6. The method of claim 1, wherein the washing solution is Tris-HCl buffer solution with concentration of 0.025mol/L, which contains NaCl with concentration of 0.15mol/L and 0.05% Tween-20.
7. The method for quantitatively detecting alpha-fetoprotein according to claim 1, wherein the chemiluminescent substrate solution A is prepared from 1.5% by mass of H2O2And HNO with a concentration of 0.1mol/L3And (4) forming.
8. The method for quantitative determination of alpha-fetoprotein as claimed in claim 1, wherein said chemiluminescent substrate solution B is a mixture of 0.1mol/L Triton X-100 and 0.35mol/L NaOH.
9. The detection method of the alpha fetoprotein kit is characterized by comprising the following steps:
1) adding 20ul of sample into a reaction tube, sequentially adding 30ul of biotin-labeled alpha-fetoprotein antibody and 30ul of acridinium ester-labeled alpha-fetoprotein antibody, incubating at 37 ℃ for 15min, finally adding 30ul of streptavidin magnetic particle suspension, and incubating at 37 ℃ for 15 min;
2) separating the reaction tube after incubation reaction on a magnetic separation frame for 2min, removing the supernatant, adding 500ul of cleaning solution, and cleaning on the magnetic separation frame for 1 min; removing supernatant, adding 300ul of cleaning solution, and cleaning on a magnetic separation frame for 1 min;
3) 100ul of chemiluminescent substrate solution A is added into a reaction tube, 100ul of chemiluminescent substrate solution B is added after 1min, and after incubation for 5min at 37 ℃ in the dark, the luminescence value is measured.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN108872594A (en) * | 2018-07-05 | 2018-11-23 | 潍坊市康华生物技术有限公司 | A kind of alpha-fetoprotein detection kit and preparation method thereof |
| CN110261626A (en) * | 2019-07-31 | 2019-09-20 | 宁波奥丞生物科技有限公司 | A kind of PLGF magnetic microparticle chemiluminescence kit and its detection method |
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- 2019-12-10 CN CN201911261911.9A patent/CN110887968A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
| CN108872594A (en) * | 2018-07-05 | 2018-11-23 | 潍坊市康华生物技术有限公司 | A kind of alpha-fetoprotein detection kit and preparation method thereof |
| CN110261626A (en) * | 2019-07-31 | 2019-09-20 | 宁波奥丞生物科技有限公司 | A kind of PLGF magnetic microparticle chemiluminescence kit and its detection method |
Non-Patent Citations (1)
| Title |
|---|
| 冯斌等: "3-氨丙基三乙氧基硅烷表面修饰的磁性Fe_3O_4纳米粒子合成与表征", 《化工新型材料》 * |
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