CN113092414B - SARS-CoV-2 antibody detection method - Google Patents
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- CN113092414B CN113092414B CN202110366253.0A CN202110366253A CN113092414B CN 113092414 B CN113092414 B CN 113092414B CN 202110366253 A CN202110366253 A CN 202110366253A CN 113092414 B CN113092414 B CN 113092414B
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Abstract
The invention discloses an unmarked detection method of a novel coronavirus antibody. The method of the invention achieves the detection purpose by modifying an anti-SARS-CoV-2 antibody on the end face of the optical fiber, combining the SARS-CoV-2 antibody in a sample with the SARS-CoV-2 antibody on the end face of the optical fiber, increasing the refractive index at the end face of the optical fiber due to the specific combination of the two antibodies, and detecting the change of the light intensity in the optical fiber. The method has the advantages of extremely small required sample amount, simple operation and high accuracy, and can be used for specifically detecting two new crown antibodies.
Description
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a detection method of a SARS-CoV-2 antibody.
Background
The novel coronavirus (SARS-CoV-2) has high infectivity and pathogenicity, can be quickly spread after outbreak, causes global public health problems, and has important significance for timely and accurately detecting virus nucleic acid and antibody to epidemic situation control. At present, the common methods for detecting the novel coronavirus (SARS-CoV-2) mainly comprise: real-time polymerase chain reaction of virus nucleic acid, enzyme-linked immunoassay, chemiluminescence immunoassay, flow measurement immunoassay, etc.
The real-time polymerase chain reaction of viral nucleic acid is a common method for clinically diagnosing patients with new coronavirus, which is called as 'gold standard', but the test is carried out by adopting the method, and the test result often generates inevitable 'false negative' and 'false positive' due to the sampling process and the sample preservation problem. The serum detection of antibodies generated by the infection induction of the novel coronavirus, such as anti-SARS-CoV-2 immunoglobulin G (IgG) and immunoglobulin M (IgM), has high sensitivity, specificity and stability, and can be used as an effective complementary method for nucleic acid detection. The conventional assay kit for the novel coronavirus (SARS-CoV-2) antibody, such as enzyme-linked immunosorbent assay (ELISA), chemiluminescence enzyme immunoassay (CLIA) and Lateral Flow Immunoassay (LFIA) kit, can be used for specifically detecting SARS-CoV-2 antibody. The enzyme-linked immunosorbent assay (ELISA) detection has low requirements on equipment, large instruments and equipment are not needed, and quantitative detection on the new crown antibody can be realized, but the method has more complicated operation steps and longer detection period; chemiluminescence enzyme immunoassay (CLIA) can also realize quantitative detection of new corona antibody, but the method requires expensive and complicated instruments and professional operators; although the detection result can be obtained quickly by the flow immunoassay (LFIA), the method can only qualitatively or semi-quantitatively analyze the concentration of the antibody and cannot accurately detect the concentration of the antibody, so that the development of a quick, simple and sensitive serological detection technology for clinical diagnosis of the novel coronavirus pneumonia is urgently needed.
Disclosure of Invention
The invention aims to provide a novel immune mark detection method of coronavirus antibody, which is a serological immune quantitative detection method for realizing SARS-CoV-2 immune globulin M (IgM) and immune globulin G (IgG) more simply, conveniently, accurately and slightly, and aims to solve the defects of long time consumption, complex operation and the like of the existing chemiluminescence enzyme immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) for detecting the novel coronavirus antibody. The method has the advantages of extremely small required sample amount, simple operation and high accuracy, and can be used for specifically detecting two new crown antibodies.
In a first aspect, the present invention provides a functionalized fiber optic probe.
The functionalized optical fiber probe provided by the invention is prepared by the method comprising the following steps:
a) Removing a coating layer on the surface of the optical fiber by using the multimode optical fiber as a detection probe, and grinding and polishing two ends of the optical fiber;
b) Placing the fiber end face at V (H) 2 SO 4 ):V(H 2 O 2 ) =3:1, carrying out reaction in the piranha solution, and cleaning and drying the optical fiber by using ultrapure water after the reaction is finished;
c) Placing the end face of one end of the optical fiber in the step b) in a toluene solution of MTS (methyl p-toluenesulfonate) for soaking, and cleaning and blow-drying after the reaction is finished;
d) And c), placing the end face of the optical fiber treated in the step c) in a GMBS (4-maleimidobutyrate-N-succinimidyl ester) ethanol solution for soaking, and cleaning the optical fiber for later use after the reaction is finished.
e) Soaking the end face of the optical fiber treated in the step d) in an anti-SARS-CoV-2 antibody (namely a second antibody which is a specific antibody aiming at SARS-CoV-2 antibody) solution, reacting in a refrigerator at 4 ℃ overnight, and cleaning the optical fiber by using ultrapure water after the reaction is finished;
f) Blocking unreacted site on the fiber end face with BSA solution for 2 hr to prepare SARS-CoV-2IgG functional detecting fiber or SARS-CoV-2IgM functional detecting fiber.
In the step a), the diameter of the multimode optical fiber can be 400-600 microns, and the numerical aperture of the optical fiber is 0.18-0.22. The length of the optical fiber may be 3-5 cm.
In step b), the reaction conditions are as follows: heating at 70 deg.C for 20-40 min.
In the step c), the mass concentration of MTS in the MTS toluene solution is 1-5% (specifically 2%); the soaking time may be 1-3 hours.
In step d), the concentration of GMBS in the GMBS (4-maleimidobutyrate-N-succinimidyl ester) ethanol solution can be 10-50 mM (specifically, 20 mM); the soaking time may be 0.5 to 2 hours.
In step e) of the above method, the concentration of the anti-SARS-CoV-2 antibody in the anti-SARS-CoV-2 antibody solution may be from 100. Mu.g/mL to 1000. Mu.g/mL. The anti-SARS-CoV-2 antibody can be anti-SARS-CoV-2 IgG antibody or anti-SARS-CoV-2 IgM antibody.
In step f) of the method, the mass concentration of the BSA solution is 1-5%. The sealing time is 2-3 hours.
The functionalized optical fiber probe prepared by the method also belongs to the protection content of the invention.
In a second aspect, the invention also protects the application of the functionalized optical fiber probe.
The application of the functional optical fiber probe provided by the invention is the application of the functional optical fiber probe in the preparation of a kit for detecting SARS-CoV-2 antibody.
The SARS-CoV-2 antibody can be a SARS-CoV-2IgG antibody and/or a SARS-CoV-2IgM antibody.
In the third aspect, the invention also protects the application of the functionalized fiber probe and the SARS-CoV-2 antibody standard product.
The invention provides the application of the functional fiber probe and the SARS-CoV-2 antibody standard substance in the preparation of the reagent kit for detecting SARS-CoV-2 antibody.
The SARS-CoV-2 antibody can be a SARS-CoV-2IgG antibody and/or a SARS-CoV-2IgM antibody.
In a fourth aspect, the invention also provides a kit for detecting SARS-CoV-2 antibody.
The kit for detecting SARS-CoV-2 antibody provided by the invention comprises the functional optical fiber probe provided by the invention.
Furthermore, the kit for detecting the SARS-CoV-2 antibody also comprises a SARS-CoV-2 antibody standard product.
Furthermore, the kit for detecting SARS-CoV-2 antibody further comprises a carrier, and the carrier describes the following method for detecting SARS-CoV-2 antibody.
In a fifth aspect, the invention also provides a method for detecting the content of SARS-CoV-2 antibody.
The method for detecting the content of the SARS-CoV-2 antibody provided by the invention comprises the following steps:
c1 Introducing PBS buffer solution on the surface of the prepared functionalized optical fiber probe for 1-2 minutes to stabilize optical signals and obtain a reflected light intensity signal value of 1;
c2 Introducing SARS-CoV-2 antibody standard substances with different concentrations into the fiber end face of the functionalized fiber probe, reacting for 5-7 minutes to obtain a reflected light intensity signal value of 2;
c3 Then introducing a regenerating solution to the surface of the optical fiber for 3-5 minutes to ensure that the SARS-CoV-2IgG antibody is dissociated from the anti-SARS-CoV-2 IgG antibody on the surface of the functionalized optical fiber probe, and finally introducing a PBS buffer solution to the surface of the optical fiber for 1-5 minutes to obtain a detection result of a signal value 1-a signal value 2;
c4 Repeating the steps c 1) to c 3), measuring the reflected light intensity signal value 2 corresponding to the SARS-CoV-2 antibody standard product with the series concentration, obtaining the detection result corresponding to the SARS-CoV-2 antibody standard product with the series concentration, and performing Logistic fitting to draw a standard curve graph by taking the SARS-CoV-2 antibody standard product concentration as a horizontal coordinate and the detection result signal value corresponding to the SARS-CoV-2 antibody standard product concentration as a vertical coordinate, so as to obtain a standard curve equation;
c5 Detecting a sample to be detected according to the method in c 1) to c 3) to obtain a detection result signal value corresponding to the sample to be detected, substituting the detection result signal value into the standard curve equation obtained in c 4), and calculating to obtain the concentration of the SARS-CoV-2 antibody in the sample to be detected.
The above method can be used for detecting SARS-CoV-2IgG antibody or SARS-CoV-2IgM antibody.
When detecting SARS-CoV-2IgG antibody, the fiber end face of the functionalized fiber probe has anti-SARS-CoV-2 IgG antibody;
when detecting SARS-CoV-2IgM antibody, the optical fiber end face of the functionalized optical fiber probe has anti-SARS-CoV-2 IgM antibody.
In the above method, the regenerating solution is an SDS aqueous solution with a mass concentration of 0.5%.
The sample to be detected can be serum to be detected. Before detection, the serum to be detected needs to be diluted by 20-50 times by PBS buffer solution and placed at 4 ℃ for later use.
Due to the adoption of the technical scheme, the invention has the following advantages: 1. the invention relates to a novel coronavirus (SARS-CoV-2) antibody detection method, which is characterized in that an anti-novel coronavirus (SARS-CoV-2) antibody is modified on the end surface of an optical fiber, the novel coronavirus (SARS-CoV-2) antibody in a sample is combined with the novel coronavirus (SARS-CoV-2) antibody on the end surface of the optical fiber, the refractive index of the end surface of the optical fiber is increased due to the specific combination of the two antibodies, and the detection aim is achieved by detecting the change of light intensity in the optical fiber. 2. The novel coronavirus (SARS-CoV-2) antibody detection method of the invention has the advantages of high detection speed and capability of specifically and quantitatively detecting the content of the novel coronavirus.
Drawings
FIG. 1 is a typical graph of the SARS-CoV-2IgG assay for the novel coronavirus;
FIG. 2 is a graph of standard curve for SARS-CoV-2IgG assay.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to specific embodiments so that those skilled in the art can better understand the present invention and can implement the present invention, but the examples are not intended to limit the present invention.
This example is the case of the spiking detection of novel coronavirus IgG antibodies in human serum samples.
The SARS-CoV-2IgG antibody standard used in the following examples was purchased from BF02020, a Beijing Boolong immuno-technology Co., ltd.
The anti-SARS-CoV-2 IgG antibody used in the following examples was purchased from BF03082-S, beijing Boolong Immunol technology Co.
The multimode optical fibers used in the following examples were obtained from RMDX-23, first technologies, inc. of Beijing.
Examples
The invention provides a method for detecting a novel coronavirus (SARS-CoV-2) IgG antibody, which comprises the following steps:
(1) Manufacture of functional optical fiber probe
a1 A multimode optical fiber with the diameter of 600 microns is used as a detection probe, the numerical aperture of the optical fiber is 0.22, a coating layer on the surface of the optical fiber is removed, the length of the removed coating layer is 5 cm, the total length of the optical fiber probe is 5 cm, and two ends of the optical fiber are ground flat and polished.
b1 The end face of the optical fiber is placed at V (H) 2 SO 4 ):V(H 2 O 2 ) =3:1, heating at 70 ℃ for 30 minutes, and after the reaction is finished, cleaning and drying the optical fiber by using ultrapure water.
c1 Placing the end face of one end of the optical fiber in the step b 1) in MTS (methyl p-toluenesulfonate) toluene solution with the mass concentration of 2 percent to be soaked for 2 hours, and cleaning and blow-drying after the reaction is finished.
d1 Placing the end face of the optical fiber treated in the step c) in a 20mM ethanol solution of GMBS (4-maleimidobutyric acid-N-succinimidyl ester) for soaking for 1 hour, and cleaning the optical fiber for standby after the reaction is finished.
e1 The end face of the optical fiber treated in the step d) was immersed in an anti-SARS-CoV-2 IgG antibody solution at a concentration of 100. Mu.g/mL in a refrigerator at 4 ℃ for reaction overnight, and after the reaction was completed, the optical fiber was washed with ultrapure water.
f1 BSA solution with the mass concentration of 2%) is used to block the unreacted sites on the end face of the optical fiber for 2 hours, so as to manufacture the SARS-CoV-2IgG functional detection optical fiber.
(2) Establishment of a Standard Curve
Taking SARS-CoV-2IgG antibody standard substance with gradient concentration of 10, 20, 50, 100, 500, 1000ng/mL for detection, wherein the detection process specifically comprises the following steps: and (2) introducing PBS buffer solution to the surface of the anti-SARS-CoV-2 IgG antibody functional detection optical fiber prepared in the step (1) for 60 seconds to stabilize an optical signal and obtain a reflected light intensity signal value of 1, introducing a novel coronavirus IgG antibody standard product (namely SARS-CoV-2IgG antibody standard product) to the end face of the optical fiber, and reacting for 300 seconds to obtain a reflected light intensity signal value of 2. Then, a regenerating solution (SDS aqueous solution with a mass concentration of 0.5%; SDS) was passed through the surface of the optical fiber for 300 seconds to dissociate SARS-CoV-2IgG antibody from the secondary antibody on the surface of the optical fiber (the secondary antibody is a specific antibody against SARS-CoV-2IgG antibody, namely, the aforementioned anti-SARS-CoV-2 IgG antibody), and finally PBS buffer was passed through the surface of the optical fiber for 75 seconds to clean the surface of the optical fiber. The detection result is signal value 1-signal value 2, and the real-time typical curve of the detection is shown in figure 1. The SARS-CoV-2IgG antibody standard with different concentrations is sequentially introduced to obtain a SARS-CoV-2IgG antibody detection standard curve as shown in FIG. 2.
(3) Sample detection
Respectively adding novel coronavirus IgG antibodies with the concentrations of 25, 50 and 100ng/mL into serum of 6 different healthy people for detection, wherein the specific detection steps are as follows:
a2 The test serum was diluted 20-fold with PBS buffer and left at 4 ℃ until use.
b2 PBS buffer solution was introduced into the surface of the optical fiber for 60 seconds to stabilize the optical signal and obtain a signal value of 1, diluted serum to be measured was introduced into the end surface of the optical fiber and reacted for 300 seconds to obtain a signal value of 2, a regenerating solution (0.5% sds aqueous solution by mass concentration) was introduced into the surface of the optical fiber and washed for 300 seconds, and finally PBS buffer solution was introduced into the surface of the optical fiber for 75 seconds to restore the optical signal to the original value.
c2 The detection result is 'signal value 1 minus signal value 2', the detection result is compared with the established SARS-CoV-2IgG detection standard curve to obtain the concentration of SARS-CoV-2IgG in the serum to be detected, and the results are as follows:
the results show that the recovery rate of the SARS-CoV-2IgG antibody by adding standard is 95.2-105.2%, the relative standard deviation is less than 10%, and the detection result is reliable.
The detection limit is defined as the lowest concentration detected, the linear range of detection is obtained by the established standard curve, the detection limit of the standard curve shown in the figure is 5ng/mL, and the linear range is 10.025-109.393 ng/mL.
The detection principle of the invention is as follows:
the light is transmitted in the optical fiber, the end face of the optical fiber is smooth and flat enough, the Fresnel reflection of the light occurs at the end face of the optical fiber due to the different refractive indexes inside and outside the optical fiber, the light reflected back to the optical fiber can be detected, the light intensity reflected back to the optical fiber is related to the refractive index at the end face of the optical fiber, and the larger the refractive index at the end face of the optical fiber is, the smaller the light intensity reflected back to the optical fiber is. The new crown virus antibody is modified at the end face of the optical fiber, the new crown antibody contained in the sample to be detected can be specifically combined with the new crown virus antibody at the end face of the optical fiber, the refractive index at the end face of the optical fiber is increased due to the specific combination of the two antibodies, the reflected light is reduced, the refractive index changes caused by the antibodies with different concentrations are different, the reflected light intensity at the end face of the optical fiber is also different, and the purpose of quantitatively detecting the concentration of the new crown virus antibody in the serum sample can be achieved by detecting the change of the light intensity.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes performed by the present invention or directly or indirectly applied to other related fields are also included in the scope of the present invention.
Claims (10)
1. A method for preparing a functionalized optical fiber probe comprises the following steps:
a) Removing a coating layer on the surface of the optical fiber by using the multimode optical fiber as a detection probe, and grinding and polishing two ends of the optical fiber;
b) Placing the end face of the optical fiber in the piranha solution for reaction, and cleaning and drying the optical fiber by using ultrapure water after the reaction is finished;
c) Placing the end face of one end of the optical fiber in the step b) in a toluene solution of methyl p-toluenesulfonate for soaking, and cleaning and drying after the reaction is finished;
d) Placing the end face of the optical fiber treated in the step c) in an ethanol solution of 4-maleimidobutyric acid-N-succinimidyl ester for soaking, and cleaning the optical fiber for later use after the reaction is finished;
e) Soaking the end face of the optical fiber treated in the step d) in an anti-SARS-CoV-2 antibody solution, reacting in a refrigerator at 4 ℃ overnight, and cleaning the optical fiber by using ultrapure water after the reaction is finished;
f) And blocking unreacted sites on the end face of the optical fiber by using BSA solution to prepare the functionalized optical fiber probe.
2. The method of claim 1, wherein: in the step a), the diameter of the multimode optical fiber is 400-600 micrometers, and the numerical aperture of the optical fiber is 0.18-0.22; the length of the optical fiber is 3-5 cm;
in the step b), the piranha solution is V (H) 2 SO 4 ):V(H 2 O 2 ) =3: 1;
in the step b), the reaction conditions are as follows: heating at 70 deg.C for 20-40 min;
in the step c), the mass concentration of the methyl p-toluenesulfonate in the toluene solution of the methyl p-toluenesulfonate is 1% -5%; the soaking time is 1-3 hours;
in the step d), the concentration of the 4-maleimidobutyric acid-N-succinimide ester in the ethanol solution of the 4-maleimidobutyric acid-N-succinimide ester is 10 to 50mM; the soaking time is 0.5-2 hours;
in the step e), the concentration of the anti-SARS-CoV-2 antibody in the anti-SARS-CoV-2 antibody solution is 100 μ g/mL-1000 μ g/mL; the anti-SARS-CoV-2 antibody is anti-SARS-CoV-2 IgG antibody or anti-SARS-CoV-2 IgM antibody;
in the step f), the mass concentration of the BSA solution is 1-5%; the sealing time is 2-3 hours.
3. A functionalized optical fiber probe prepared according to the method of claim 1 or 2.
4. Use of the functionalized fiber-optic probe of claim 3 for the preparation of a kit for detecting SARS-CoV-2 antibody.
5. The use of the functionalized fiber-optic probe and the SARS-CoV-2 antibody standard substance of claim 3 in the preparation of a kit for detecting SARS-CoV-2 antibody.
6. Use according to claim 4 or 5, characterized in that: the SARS-CoV-2 antibody is SARS-CoV-2IgG antibody and/or SARS-CoV-2IgM antibody.
7. A kit for detecting SARS-CoV-2 antibodies comprising the functionalized fiber optic probe of claim 3.
8. The kit of claim 7, wherein: the kit for detecting the SARS-CoV-2 antibody also comprises a SARS-CoV-2 antibody standard substance;
further, the kit for detecting SARS-CoV-2 antibody further comprises a carrier, wherein the carrier is recorded with the method for detecting SARS-CoV-2 antibody content according to claim 9 or 10.
9. A method for detecting the content of SARS-CoV-2 antibody for non-diagnostic purposes, comprising the steps of:
c1 Introducing PBS buffer solution on the surface of the functionalized optical fiber probe of claim 3 for 1-2 minutes to stabilize the optical signal and obtain a reflected light intensity signal value of 1;
c2 Introducing SARS-CoV-2 antibody standard substances with different concentrations into the fiber end face of the functionalized fiber probe, reacting for 5-7 minutes to obtain a reflected light intensity signal value of 2;
c3 Then introducing a regeneration liquid to the surface of the optical fiber for 3-5 minutes to ensure that the SARS-CoV-2IgG antibody is dissociated from the anti-SARS-CoV-2 IgG antibody on the surface of the functionalized optical fiber probe, and finally introducing a PBS buffer solution to the surface of the optical fiber for 1-5 minutes, wherein the detection result is a signal value 1-a signal value 2;
c4 Repeating the steps c 1) to c 3), measuring the reflected light intensity signal value 2 corresponding to the SARS-CoV-2 antibody standard product with the series concentration, obtaining the detection result corresponding to the SARS-CoV-2 antibody standard product with the series concentration, and performing Logistic fitting to draw a standard curve graph by taking the SARS-CoV-2 antibody standard product concentration as a horizontal coordinate and the detection result signal value corresponding to the SARS-CoV-2 antibody standard product concentration as a vertical coordinate, so as to obtain a standard curve equation;
c5 Detecting the serum sample to be detected according to the method in c 1) -c 3) to obtain a detection result signal value corresponding to the serum sample to be detected, substituting the detection result signal value into the standard curve equation obtained in c 4), and calculating to obtain the concentration of the SARS-CoV-2 antibody in the serum sample to be detected.
10. The method of claim 9, wherein: the method is used for detecting SARS-CoV-2IgG antibody or SARS-CoV-2IgM antibody;
when detecting SARS-CoV-2IgG antibody, the fiber end face of the functionalized fiber probe has anti-SARS-CoV-2 IgG antibody;
when detecting SARS-CoV-2IgM antibody, the optical fiber end face of the functionalized optical fiber probe is provided with anti-SARS-CoV-2 IgM antibody;
the regeneration solution is an SDS aqueous solution with the mass concentration of 0.5 percent;
before detection, the serum to be detected needs to be diluted by 20-50 times by PBS buffer solution and placed at 4 ℃ for later use.
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