CN114200137A - Ratio immunoassay method taking commercial magnetic beads as internal standard - Google Patents

Ratio immunoassay method taking commercial magnetic beads as internal standard Download PDF

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CN114200137A
CN114200137A CN202010971675.6A CN202010971675A CN114200137A CN 114200137 A CN114200137 A CN 114200137A CN 202010971675 A CN202010971675 A CN 202010971675A CN 114200137 A CN114200137 A CN 114200137A
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CN114200137B (en
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吕弋
王超群
刘睿
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Sichuan University
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Abstract

The invention discloses a ratio immunoassay method taking commercial magnetic beads as internal standards. The invention takes carcinoembryonic antigen (CEA) as a research model, magnetic beads as a carrier and gold nanoparticles as a report probe. Simultaneous acquisition of magnetic beads using high resolution Inductively Coupled Plasma Mass Spectrometry (ICPMS)57In Fe signalling and reporter probes197And Au signal.197Au/57The Fe signal ratio and the CEA concentration have a good linear relation in a certain range, so that the accurate quantitative analysis of the CEA is realized. The invention skillfully develops another function (internal standard) of the carrier magnetic bead, successfully avoids the magnetic bead loss in the magnetic separation process and the interference generated by the signal intensity fluctuation of the instrumentTherefore, the accuracy and the reliability of the immunoassay method based on the magnetic beads are improved. In view of the popularity of the use of commercial magnetic beads, the present invention may provide a new concept for magnetic bead-based bioanalytical methods.

Description

Ratio immunoassay method taking commercial magnetic beads as internal standard
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to the fields of immunoassay, ratio analysis method and stable isotope (Metal stable isotope) sensing.
Background
Magnetic beads (MPs) are usually made of gamma-Fe2O3Or Fe3O4The size of the microsphere can be from nanometerOn the meter to micron scale. The unique superparamagnetism avoids time-consuming and labor-consuming separation procedures such as centrifugation and chromatographic separation, and the magnetic microspheres are the most widely commercialized substrates in the world. Magnetic beads have enjoyed tremendous success in various biological applications such as cancer therapy, drug delivery, magnetic resonance imaging, blood purification, and molecular diagnostics. For example, near infrared MPs imaging tracers with photothermal and magnetocaloric properties were developed based on magnetic microspheres for tumor ablation in mice. And an MPs-based extracorporeal blood purification device for treating blood poisoning is created, which enables continuous removal of pathogens and toxins from blood without the need to identify the source of the infection. In addition, some bioanalytical methods based on magnetic beads, which have strong specificity, high sensitivity, and no radioactive contamination, have been widely used for the detection of bioactive substances. After these pioneering efforts, one important challenge is to achieve more accurate and reliable quantitative analysis.
Ratio analysis can help correct complex matrix problems and enable accurate and reliable analysis of biological samples. By adopting the Internal Standard (IS), the error in the sample pretreatment process can be eliminated, and the signal fluctuation in the instrument detection can be corrected. Ratio analysis quantitative analysis is performed by the ratio of two signals, which can significantly improve the accuracy of the method. Many researchers have also worked on the design of effective IS. Despite great success, the extensive use of SI in bioassays IS often hampered by complex IS labeling or synthesis procedures.
Disclosure of Invention
Technical problem to be solved
In order to realize accurate quantitative detection of the antigen, the invention provides a ratio immunoassay method taking commercial magnetic beads as internal standards.
The invention takes carcinoembryonic antigen (CEA) as a research model, magnetic beads as a carrier and gold nanoparticles as a report probe. Simultaneous acquisition of magnetic beads using high resolution Inductively Coupled Plasma Mass Spectrometry (ICPMS)57In Fe signalling and reporter probes197And Au signal. Study of197Au/57The relationship between the Fe signal ratio and the CEA concentration realizes the accurate quantification of the CEAAnd the accuracy and the reliability of the immunoassay method are improved.
(II) technical scheme
In order to solve the above problems, a first object of the present invention is to provide a ratio immunoassay method using commercial magnetic beads as internal standards to achieve accurate quantitative detection of CEA.
The invention develops a new function of the vector magnetic bead, provides a novel immunoassay method and realizes the quantitative detection of CEA. The principle of the invention is shown in figure 1, the invention takes carcinoembryonic antigen (CEA) as a research model, magnetic beads as a carrier and gold nanoparticles as a report probe. The magnetic beads and gold nanoparticles are first labeled with primary and secondary antibodies, respectively. And then immunoreacting with CEA with different concentrations. Finally, after magnetic separation and washing, simultaneously collecting the magnetic beads by adopting high-resolution Inductively Coupled Plasma Mass Spectrometry (ICPMS)57In Fe signalling and reporter probes197And Au signal.197Au/57The Fe signal ratio and the CEA concentration have a better linear relation in a certain range, so that the quantitative detection of target molecules can be realized, and the accuracy and the reliability of the immunoassay method are improved.
The detection method of the invention preferably comprises the following steps:
s1: synthesizing gold nanoparticles by a common sodium citrate reduction method;
s2: the gold nanoparticles were incubated with a quantity of antibody at room temperature for 40 minutes with rotation to form antibody-modified gold nanoparticles (Ab)2-AuNPs) and blocked with BSA;
s3: preparation of CEA antibody-modified magnetic microspheres (Ab)1-MMP）；
S4: adding the same volume of CEA and the Ab with different concentrations1-MMP mixing, reaction for a certain time at a certain temperature, magnetic separation and washing;
s5: adding Ab diluted by a certain time2-AuNPs, recognizing CEA antigen and forming a sandwich structure;
s6: after magnetic separation and washing, the solution is digested by aqua regia, and finally the solution is diluted to a certain volume;
s7: in solution for collecting inorganic element mass spectrum197Au and 57the signal intensity of Fe was investigated and the ratio was related to the concentration of CEA.
In the method, the dosage of the antibody S2 is 0-20 mug.
In the method of the present invention, the preparation method of S3: first, the purchased magnetic beads were placed on a vortex mixer and vortexed for 30 s to resuspend the beads in a vial. Subsequently, 100. mu.L of the streptavidin-coated magnetic bead solution of 10 mg/mL after washing for 2 times was added with 20. mu.L of biotin-modified CEA antibody of 1 mg/mL (0.01M PBS, pH 7.4), and incubated at 25 ℃ and 1500 rpm with shaking for 1 h.
In the method, the CEA concentration range of S4 is 0.6-300 ng/mL, the reaction temperature is 37 ℃, the reaction time is 30-90 minutes, and the reaction buffer is PBS pH 7.4.
In the method of the present invention, Ab is described in S52The optimal dilution of AuNPs is 5 times, the reaction temperature is 37 ℃, the reaction time is 30-90 minutes, and the buffer of the reaction is PBS pH 7.4.
In the method of the present invention, the reaction content of S6 is: after the reaction of the magnetic bead complex with 50. mu.L of aqua regia for 20 min, the solution was diluted to 8 mL with ultrapure water.
(III) advantageous effects
The invention has the beneficial effects that:
1. according to the invention, Fe element in the commercialized magnetic microsphere is used as an internal standard, so that the accuracy of the method is further improved, and inaccuracy caused by loss of magnetic beads can be avoided, thereby realizing accurate quantitative analysis of CEA.
2. The invention uses magnetic particles as a carrier, does not need to add an element or introduce another marker, and has simple method.
3. The method has universal applicability, and the invention can provide a new idea for a magnetic bead-based bioanalysis method.
Drawings
FIG. 1 is a schematic diagram of the experimental mechanism of the present invention.
Fig. 2 is a graph for exploring the optimal amount of the antibody for modifying gold nanoparticles according to the present invention.
FIG. 3 shows Ab in the present invention1-graphs of the results of the optimisation of the reaction time of the MPs with the antigen.
FIG. 4 shows Ab in the present invention2Results plot of the reaction time optimization of AuNPs with antigen.
FIG. 5 is a schematic diagram showing the analytical performance of the analytical method of the present invention for CEA detection.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The invention relates to a ratio immunoassay method using commercial magnetic beads as internal standards to realize accurate quantitative detection of CEA.
Further, the streptavidin-modified magnetic beads are combined with the biotin-modified antibody to form Ab1-MPs。
Further, the Ab synthesized2AuNPs were blocked with BSA.
The invention is used for CEA detection, and comprises the following steps:
s1: preparation of Ab1-MPs。
S2: preparation of Ab2-AuNPs。
S3: take 40. mu.L of diluted Ab1MMP and 100 mu L of CEA with different concentrations are mixed uniformly, and the mixture is incubated for 1 h under the conditions of 37 ℃ and 1500 rpm.
S4 the magnetic bead complexes were washed 2 times with 50. mu.L PBST solution. Then, 50. mu.L of diluted Ab was added to the sample2AuNPs, incubated at 37 ℃ and 1500 rpm for 1 h with shaking.
And S5, finally, reacting the washed magnetic bead complex with 50 mu L of aqua regia for 20 min, and diluting the solution to 8 mL by using ultrapure water. Finally, collecting the solution by high-resolution inductively coupled plasma mass spectrometry197Au and57the signal of Fe and the ratio of the two is calculated.
The embodiment of the invention comprises the following steps:
1. experimental apparatus and equipment used in embodiment of the invention
The instruments used in the experiment mainly include: constant temperature oscillator, ELEMENT XR high resolution inductively coupled plasma mass spectrometer.
2. Experimental reagent used in embodiment of the invention
Chlorauric acid tetrahydrate, trisodium citrate, boric acid, sodium tetraborate decahydrate, sodium dihydrogen phosphate (NaH 2PO 4), disodium hydrogen phosphate and Tween 20, hydrochloric acid and nitric acid, bovine serum albumin, streptavidin-modified magnetic microspheres, carcinoembryonic antigen, biotinylated mouse monoclonal CEA antibody (bio-Ab)1) Mouse monoclonal CEA antibody (Ab)2）。
Deionized Water (DIW, 18.2M. omega. cm) was obtained from Milli-Q Integral Water purification apparatus-1）。
Example 1:
s1: different amounts of CEA antibody were incubated with 1 mL of AuNPs solution for 40 minutes, respectively.
S2: and measuring the absorbance of the mixed solution.
S3: ab is prepared by2After the AuNPs were purified by centrifugation, 300. mu.L of 3M NaCl solution was added and mixed therewith.
And S4, observing the color and state of the solution.
The experimental results are as follows:
this experiment is optimized for the optimal amount of antibody that prefers the modified gold nanoparticles of the invention.
The reaction conditions in this experiment were as described in example 1.
The antibody dosage is respectively designed to be 0, 1, 3, 5, 7, 10, 13, 15, 17 and 20 mu g.
Ab when the amount of antibody added is greater than 10. mu.g2The absorbance of the AuNPs solution remained essentially unchanged. This indicates that when the amount of antibody is about 10. mu.g sufficient to complete the modification of AuNPs. And Ab when CEA antibody is added in an amount of more than 13. mu.g2The AuNPs solutions were almost identical in color and morphology to the original AuNPs solutions, indicating better stability under these conditions. Taking into account the high cost of the antibody and Ab2The anti-ion interference ability of AuNPs solution, we chose the antibody amount of the marker AuNPs to be 13. mu.g.
Example 2:
s1: preparation of Ab1-MPs。
S2: gold nanoparticles were incubated with 13 μ g antibody at room temperature for 40 min with rotationPreparation of Ab from Bell2AuNPs, and blocked with BSA.
S3: take 40. mu.L of diluted Ab1MMP and 100 mu L of CEA with fixed concentration are mixed uniformly, and the mixture is incubated for a certain time under the conditions of 37 ℃ and 1500 rpm.
S4 the magnetic bead complexes were washed 2 times with 50. mu.L PBST solution. Then, 50. mu.L of diluted Ab was added to the sample2AuNPs, incubated at 37 ℃ and 1500 rpm for 1 h with shaking.
And S5, finally, reacting the washed magnetic bead complex with 50 mu L of aqua regia for 20 min, and diluting the solution to 8 mL by using ultrapure water. Finally, collecting the solution by high-resolution inductively coupled plasma mass spectrometry197Au and57the signal of Fe and the ratio of the two is calculated.
The experimental results are as follows:
ab in the preferred invention1Optimal reaction time of MPs with antigen.
The amount of the modified gold nanobody in this experiment was 13. mu.g (as described in example 1), and the other reaction conditions were as described in example 2.
Ab1The reaction time of MPs and antigen is designed to be 30 min, 45 min, 60 min, 75 min, 90 min, respectively.
As shown in FIG. 3, the ordinate represents the S/N value, and the abscissa represents the reaction time. The reaction time increased to gradually increase the S/N value and to reach a plateau. We selected 60 min as the following experimental conditions.
Example 3:
s1: preparation of Ab1-MPs。
S2: ab preparation by gold nanoparticles and 13 ug antibody at room temperature with 40 min incubation2AuNPs, and blocked with BSA.
S3: take 40. mu.L of diluted Ab1MMP and 100 mu L of CEA with fixed concentration are mixed uniformly, and the mixture is incubated for 1 h under the conditions of 37 ℃ and 1500 rpm.
S4 the magnetic bead complexes were washed 2 times with 50. mu.L PBST solution. Then, 50. mu.L of diluted Ab was added to the sample2AuNPs, and shaking and incubating at 37 ℃ and 1500 rpm for a certain time.
And S5, finally, reacting the washed magnetic bead complex with 50 mu L of aqua regia for 20 min, and diluting the solution to 8 mL by using ultrapure water. Finally, collecting the solution by high-resolution inductively coupled plasma mass spectrometry197Au and57the signal of Fe and the ratio of the two is calculated.
The experimental results are as follows:
ab in the preferred invention2Optimal reaction time of AuNPs with antigen.
In this experiment, the amount of modified gold nanobody was 13. mu.g (as described in example 1), Ab1Reaction time of MPs with antigen was 60 min (as described in example 2), other reaction conditions were as described in example 3.
Ab2The reaction time of AuNPs and antigen is respectively designed to be 30 min, 45 min, 60 min, 75 min and 90 min.
As shown in FIG. 4, the ordinate represents the S/N value, and the abscissa represents the reaction time. The reaction time increases to gradually increase the S/N value, and the reaction time increases to slightly decrease the S/N value. We selected 60 min as the following experimental conditions.
Example 4:
s1: preparation of Ab1-MPs。
S2: ab preparation by gold nanoparticles and 13 ug antibody at room temperature with 40 min incubation2AuNPs, and blocked with BSA.
S3: take 40. mu.L of diluted Ab1MMP and 100 mu L of CEA with different concentrations are mixed uniformly, and the mixture is incubated for 1 h under the conditions of 37 ℃ and 1500 rpm.
S4 the magnetic bead complexes were washed 2 times with 50. mu.L PBST solution. Then, 50. mu.L of diluted Ab was added to the sample2AuNPs, incubated at 37 ℃ and 1500 rpm for 1 h with shaking.
And S5, finally, reacting the washed magnetic bead complex with 50 mu L of aqua regia for 20 min, and diluting the solution to 8 mL by using ultrapure water. Finally, collecting the solution by high-resolution inductively coupled plasma mass spectrometry197Au and57the signal of Fe and the ratio of the two is calculated.
The experimental results are as follows:
the experiment is to study the performance of the method for quantitatively detecting CEA.
In this experiment, the amount of modified gold nanobody was 13. mu.g (as described in example 1), Ab1Reaction time of MPs with antigen 60 min (as described in example 2), Ab2The reaction time of AuNPs with antigen was 60 min (as described in example 3), and other conditions were as described in example 4.
The concentration of the target CEA was designed to be 0.6 ng/mL, 6 ng/mL, 30 ng/mL, 60 ng/mL, 150 ng/mL, 300 ng/mL, respectively.
After the reaction is completed, high-resolution ICPMS detection is utilized197Au and 57the strength of Fe element and the ratio thereof are calculated, a standard curve is drawn according to the calculated ratio and the concentration of the target CEA, as shown in FIG. 5, wherein the abscissa is the CEA concentration and the ordinate is the CEA concentration197Au and 57the ratio of the Fe intensities; the standard curve is expressed asI Au/I Fe=5.874*10-3C +0.023, correlation coefficient R = 0.997; whereinIThe intensity value of ICPMS is shown, and C is the concentration of CEA. The linear range was 0.6 to 300 ng/mL and the limit of detection (LOD) was calculated to be about 0.09 ng/mL (calculated using a 3-fold SD divided by slope, SD is the standard deviation of 10 blank samples).

Claims (9)

1. A ratio immunoassay method taking commercial magnetic beads as internal standards is characterized in that the method can be used for the quantitative detection of antigens.
2. The ratiometric immunoassay method of claim 1, wherein: the ratio immunoassay method takes magnetic beads as a carrier and gold nanoparticles as a report probe.
3. The ratiometric immunoassay method of claim 1, wherein: the method is mainly the magnetic microsphere (Ab) modified by CEA antibody1MMP), CEA antigen, CEA antibody modified gold nanoparticles (Ab)2-AuNPs).
4. The ratiometric immunoassay method of claim 1, wherein: simultaneous acquisition of magnetic beads using high resolution Inductively Coupled Plasma Mass Spectrometry (ICPMS)57In Fe signalling and reporter probes197An Au signal;197Au/57and the Fe signal ratio realizes accurate quantitative analysis of the CEA.
5. The Ab of claim 31-MMPs, characterized in that: the specific combination of streptavidin and biotin is utilized to couple the antibody and the magnetic microspheres.
6. The Ab of claim 32-AuNPs, characterized in that: ab2Blocking with 1% BSA after AuNPs synthesis.
7. The ratiometric immunoassay method is used for antigen (CEA) detection, characterized in that: comprises the following steps
Ab prepared according to claim 51-MMP；
Ab prepared according to claim 62-AuNPs；
Adding the same volume of CEA and the Ab with different concentrations1-MMP mixing, reaction for a certain time at a certain temperature, magnetic separation and washing;
adding a certain fold dilution of the Ab2-AuNPs, recognizing CEA antigen and forming a sandwich structure;
after magnetic separation and washing, the solution is digested by aqua regia, and finally the solution is diluted to a certain volume;
in solution for collecting inorganic element mass spectrum197Au and 57the signal intensity of Fe was investigated and the ratio was related to the concentration of CEA.
8. The method for quantitatively detecting CEA according to claim 7, wherein: in step S3, the reaction temperature is 37 ℃ and the reaction time is 30-90 minutes.
9. The method of claim 7 for the quantitative detection of CEA,the method is characterized in that: in step S4, Ab2The AuNPs dilution factor is 0.5-10 times, the reaction temperature is 37 ℃, and the reaction time is 30-90 minutes.
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