CN113252906A - Detection method of paraneoplastic syndrome related marker antibody - Google Patents
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Abstract
The invention discloses a detection method of a marker antibody related to paraneoplastic syndrome, which combines microspheres with a flow analysis technology to jointly detect 14 marker antibodies related to paraneoplastic syndrome, such as Hu, Yo, Ri, CV2, Amphiphysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like, wherein the detection method comprises the following steps: activating 14 kinds of microspheres; carrying out coupling reaction on the microspheres and the capture antibody; sealing the microspheres; mixing the microspheres; adding a serum sample into a mixed reaction system to perform antigen-antibody immune binding reaction; adding excessive FITC labeled goat anti-mouse IgG for immunofluorescence reaction; the detection method greatly shortens the operation time required by detection, improves the detection efficiency of the related antibodies, further improves the sensitivity and specificity of the detection method for detecting the related marker antibodies, and can realize multi-parameter analysis of the indexes of the detected samples.
Description
Technical Field
The invention belongs to the technical field of clinical medical detection, and particularly relates to a method for detecting a paraneoplastic syndrome related marker antibody.
Background
The secondary tumor syndrome refers to the pathological changes of endocrine, nerve, digestion, hematopoiesis, bone joint, kidney, skin and other systems caused by abnormal immune reaction of tumor products or other unknown reasons, and corresponding clinical manifestations appear, wherein the manifestations are not directly caused by the primary tumor or the part where a metastasis is located, but indirectly caused by the above way. Paraneoplastic syndromes are diseases that occur in certain malignant patients and cause dysfunction without the appearance of metastasis, i.e., that have affected distant self-organs. The distant self-organ affected is as in the nervous system, also known as the nervous system paraneoplastic syndrome. It is not a group of symptoms resulting from the direct invasion of the tissue or organ by the tumor, but the distant effects of systemic cancer, such as lung cancer, ovarian cancer, etc., can manifest as distant effects of gray matter inflammation and neurodegeneration in the central nervous system.
The paraneoplastic syndrome generally occurs a lot before the occurrence of tumors of patients, about 80% of patients with the paraneoplastic syndrome (PNS) have obvious lesion expression before the occurrence of the tumors according to statistics, which provides a chance for discovering the existence of malignant tumors as soon as possible, and can remarkably improve the survival time of the patients if the cancers seriously threaten the life and health of human beings can be discovered and diagnosed and treated as soon as possible.
At present, the detection of related marker antibodies clinically used for diagnosing the paraneoplastic syndrome disease mainly comprises 14 antibodies such as Hu, Yo, Ri, CV2, Amphihysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like, and the detection of the 14 antibodies is mainly suitable for auxiliary diagnosis or screening inspection of the paraneoplastic syndrome, differential diagnosis of paraneoplastic encephalitis and autoimmune encephalitis, differential diagnosis of demyelinating myelitis of the central nervous system and paraneoplastic related myelitis, differential diagnosis of cerebellar ataxia, differential diagnosis of autoimmune peripheral neuropathy and paraneoplastic related peripheral neuropathy and the like.
At present, the clinical detection of 14 related marked antibodies of the paraneoplastic syndrome is mainly determined by an immunoblotting method, an enzyme-linked immunosorbent assay method and the like, the immunoblotting method is a determination method of separating antigens by polyacrylamide gel electrophoresis, transferring the antigens to a membrane support, and then incubating the membrane support with serum, and the method has the defects that the time for detecting the 14 related marked antibodies of the paraneoplastic syndrome is long, an X-ray negative film or a chemiluminescence imaging instrument is required to display results, and the problems of more influencing factors, standardization and the like exist when the enzyme-linked immunosorbent assay method is used for detecting the 14 related marked antibodies of the paraneoplastic syndrome.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art that 14 marker antibodies of the paraneoplastic syndrome are determined by the prior determination method, the invention provides the method for detecting the 14 marker antibodies of the paraneoplastic syndrome, which combines a microsphere analysis technology and a flow analysis technology and performs combined detection on the 14 marker antibodies of Hu, Yo, Ri, CV2, Amphihysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like related to the paraneoplastic syndrome, so that the operation time required by detection is greatly shortened, the sensitivity and the specificity of the detection method for the related marker antibodies are improved, the multi-parameter analysis of the indexes of a detection sample can be realized, and compared with the one-to-one determination method of enzyme-linked immunosorbent assay, the detection efficiency of the related antibodies and the accuracy of the detection results are greatly improved.
The technical scheme adopted by the invention is as follows: a detection method of a marker antibody related to paraneoplastic syndrome combines microspheres with a flow analysis technology, and performs combined detection on 14 marker antibodies related to paraneoplastic syndrome, such as Hu, Yo, Ri, CV2, Amphiphysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like, wherein the detection method specifically comprises the following steps:
1) preparing 14 kinds of microspheres, and respectively activating the 14 kinds of microspheres;
2) preparing 14 capture antibodies corresponding to the 14 marker antibodies related to the paraneoplastic syndrome, wherein the capture antibodies correspond to antigens and are used for carrying out antigen-antibody immune binding reaction with the marker antibodies to be detected in serum;
3) respectively carrying out coupling reaction on the microspheres activated in the step 1) and the capture antibodies prepared in the step 2);
4) sealing the microspheres after the coupling reaction in the step 3) is finished, and sealing the sites which are not combined with the capture antibody on the microspheres by using a sealing liquid;
5) mixing the 14 microspheres subjected to the sealing treatment respectively to form a mixed reaction system containing 14 microspheres coupled with different capture antibodies, wherein the mixed reaction system can be used for detecting the to-be-detected marker antibody in serum simultaneously;
6) adding a serum sample into the mixed reaction system in the step 5), wherein the capture antibody coupled on the microspheres and the to-be-detected marker antibody in the serum generate an antigen-antibody immunological binding reaction, and after the binding reaction is finished, an antigen-antibody complex combining the to-be-detected marker antibody and the capture antibody is formed on the microspheres;
7) step 6), after the reaction is finished, washing out the unreacted serum protein, adding excessive FITC labeled goat anti-mouse IgG for immunofluorescence reaction, and performing fluorescence quantitative analysis on the to-be-detected marker antibody in the serum sample;
8) detecting and analyzing the microspheres obtained after the immunofluorescence reaction in the step 7) by using a flow cytometer.
The microsphere in the step 1) is a polystyrene microsphere with carboxyl on the surface and embedded organic fluorescent molecules, the particle size is uniform, the polystyrene microsphere can be directly purchased from related biological product companies, and the specification of the microsphere is about 1.25 multiplied by 107each/mL, 14 kinds of the microspheres have 14 different colors, namely microsphere 1, microsphere 2, microsphere 3, microsphere 4, microsphere 5, microsphere 6, microsphere 7, microsphere 8, microsphere 9, microsphere 10, microsphere 11, microsphere 12, microsphere 13 and microsphere 14, and the fluorescence color of each kind of the microspheres is different.
In the step 1), microspheres are activated by a microsphere activation buffer solution, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide salt and N-hydroxysuccinimide sulfide, wherein the concentrations of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide salt and the N-hydroxysuccinimide sulfide are both 50mg/mL, and the microspheres can be directly purchased from the market.
Specifically, the activation method for the microspheres in step 1) specifically comprises the following steps: taking microsphere stock solution, centrifuging to remove supernatant, adding microsphere washing buffer solution for washing, centrifuging to obtain supernatant, adding microsphere activation buffer solution, resuspending, mixing uniformly, adding 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide salt and vulcanized N-hydroxysuccinimide, shaking at room temperature in a dark place, adding 1 XPBS buffer solution, mixing uniformly, centrifuging to remove supernatant, adding 1 XPBS buffer solution, resuspending, mixing uniformly to obtain activated microspheres, mixing the microsphere washing buffer solution used in activating the microspheres by mixing 1 XPBS with 95% pH value of 7.4 and 5% Tween-20, and mixing the microsphere activation buffer solution used is 0.1mol/L NaH with pH value of 6.22PO4。
The capture antibodies in step 2) are respectively: the monoclonal antibody can be a mouse anti-human Hu monoclonal antibody, a mouse anti-human Yo monoclonal antibody, a mouse anti-human Ri monoclonal antibody, a mouse anti-human CV2 monoclonal antibody, a mouse anti-human Amphihysin monoclonal antibody, a mouse anti-human Ma1 monoclonal antibody, a mouse anti-human Ma2 monoclonal antibody, a mouse anti-human SOX1 monoclonal antibody, a mouse anti-human Tr monoclonal antibody, a mouse anti-human Zic4 monoclonal antibody, a mouse anti-human Titin monoclonal antibody, a mouse anti-human Recoverin monoclonal antibody, a mouse anti-human PKC gamma monoclonal antibody, a mouse anti-human GAD65 monoclonal antibody, and the capture antibodies can be directly purchased from related biological product companies.
The blocking solution used for blocking the microspheres in the step 4) is prepared from 1 XPBS buffer solution, bovine serum albumin and sodium azide, the preparation concentration between the bovine serum albumin and the 1 XPBS buffer solution is 10g/l, the preparation concentration between the sodium azide and the 1 XPBS buffer solution is 0.5g/l, and corresponding bovine serum albumin and sodium azide are respectively added into the 1 XPBS with the pH value of 7.4, so that the blocking solution is obtained.
The detection principle of the detection method of the paraneoplastic syndrome related marker antibody provided by the invention is as follows: the detection method aims at 14 side-effect syndrome related marker antibodies such as Hu, Yo, Ri, CV2, Amphiphysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like, and carries out one-time combined detection through 14 microspheres marked by different fluorescent dyes, the activated 14 microspheres are respectively and correspondingly coupled with 14 capture antibodies, after the coupling reaction is finished, each microsphere is coupled with a corresponding capture antibody, the capture antibodies coupled on the microspheres are respectively and correspondingly subjected to antigen-antibody immune combination reaction with the marker antibody to be detected of a serum sample, the antibody to be detected in the serum sample is combined on the corresponding microspheres coupled with the capture antibodies to form a microsphere-capture antibody-antibody system to be detected, subsequently added FITC marked anti-goat IgG is combined with an antigen-antibody compound formed on the microspheres, and a flow cytometer detects the serum sample after the reaction is finished, each microsphere corresponds to a cell, and sequentially passes through a detection zone of a flow cytometer at high speed, the fluorescence and scattered light of the microspheres (FL2, FL3) and FITC (FL1) can be detected and recorded by a corresponding detection system, the microspheres with various colors can be separated according to the intensities of FL2 and FL3, and the concentration of a corresponding marker antibody to be detected can be obtained according to the average intensity of FL 1.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a detection method of a marker antibody related to paraneoplastic syndrome, which combines microspheres with a flow analysis technology for the first time and realizes combined detection aiming at 14 marker antibodies related to paraneoplastic syndrome, such as Hu, Yo, Ri, CV2, Amphihysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like;
(2) the detection method is utilized to detect 14 paraneoplastic syndrome related marker antibodies such as Hu, Yo, Ri, CV2, Amphipsin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like, so that the detection method has the advantages of greatly shortening the operation time required by detection, improving the sensitivity and specificity of the detection method for the related marker antibodies, realizing multi-parameter analysis of detection sample indexes, and greatly improving the detection efficiency of the related antibodies and the accuracy of detection results;
(3) in the detection method, 14 microspheres with different fluorescent colors are placed in the same reaction system for detection, so that various physiological and pathological indexes can be detected at one time, and the detection method is greatly different from the traditional one-by-one detection mode in efficiency for detecting various antibodies at one time.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent.
A detection method of a marker antibody related to paraneoplastic syndrome combines microspheres with a flow analysis technology, and performs combined detection on 14 marker antibodies related to paraneoplastic syndrome, such as Hu, Yo, Ri, CV2, Amphiphysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma, GAD65 and the like, wherein the detection method specifically comprises the following steps:
1) directly purchasing 14 microspheres with different colors from the market, namely microsphere 1, microsphere 2, microsphere 3, microsphere 4, microsphere 5, microsphere 6, microsphere 7 and microsphere8. Microsphere 9, microsphere 10, microsphere 11, microsphere 12, microsphere 13 and microsphere 14, the specification of the purchased microsphere stock solution is about 1.25 multiplied by 107/mL, 14 types of microspheres are respectively activated, and the activation operation method is as follows: 1) taking 2500 mu L of one purchased microsphere stock solution, placing the microsphere stock solution in a centrifuge tube, centrifuging at 10000r/min for 10min, and discarding the supernatant; 2) adding 2500 mu L of microsphere washing buffer solution into a centrifuge tube with the supernatant discarded, wherein the microsphere washing buffer solution is prepared by mixing 95% of 1 XPBS with the pH value of 7.4 and 5% of Tween-20, performing vortex oscillation for 10 seconds, performing ultrasonic cleaning for 10 seconds, centrifuging for 10 minutes at 10000r/min after washing, and discarding the supernatant; 3) adding 2500. mu.L of microsphere activation buffer solution into the centrifuge tube with the supernatant discarded, wherein the microsphere activation buffer solution is 0.1mol/L NaH with the pH value of 6.22PO4Adding 1-ethyl-3- (3-dimethylaminopropyl) and 250 mul of sulfuration N-hydroxysuccinimide, wherein the concentration of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide salt and the concentration of sulfuration N-hydroxysuccinimide are both 50mg/mL, reacting for 8-10min, 4) adding 2500 mul of 1 XPBS buffer solution into a centrifuge tube after the activation reaction of carboxyl on the microsphere is finished, shaking for 15 seconds, centrifuging for 10min at 10000r/min, and discarding the supernatant; 5) adding 2500 mu L of 1 XPBS buffer solution into a centrifuge tube, shaking and cleaning for 20 seconds at a speed of 10000r/min, centrifuging for 10min, discarding supernatant, and then suspending the microspheres in 2500 mu L of 1 XPBS buffer solution to obtain activated microspheres, wherein the activation methods of the rest 13 microsphere stock solutions are all activated by adopting the methods, and finally 14 activated microspheres are respectively obtained;
2) 14 capture antibodies corresponding to the 14 marker antibodies related to the paraneoplastic syndrome are directly purchased from the market, and the 14 capture antibodies are respectively: mouse anti-human Hu monoclonal antibody, mouse anti-human Yo monoclonal antibody, mouse anti-human Ri monoclonal antibody, mouse anti-human CV2 monoclonal antibody, mouse anti-human Amphihysin monoclonal antibody, mouse anti-human Ma1 monoclonal antibody, mouse anti-human Ma2 monoclonal antibody, mouse anti-human SOX1 monoclonal antibody, mouse anti-human Tr monoclonal antibody, mouse anti-human Zic4 monoclonal antibody, mouse anti-human Titin monoclonal antibody, mouse anti-human Recoverin monoclonal antibody, mouse anti-human PKC gamma monoclonal antibody, mouse anti-human GAD65 monoclonal antibody, the initial concentration of the purchased capture antibodies is 0.2mg/ml,
3) adding 100 mu L of purchased capture antibody stock solution into 200 mu L of activated microspheres, adjusting the final volume to 800 mu L by using 1 multiplied by PBS, carrying out coupling reaction for 2h, 15000r/min, centrifuging for 5min, removing supernatant, and suspending the coupled microspheres in 800 mu L of 1 multiplied by PBS buffer solution, thus completing the coupling process of the microspheres and the capture antibodies, wherein 14 microspheres and 14 capture antibodies are subjected to coupling reaction by adopting the method;
4) sealing the microspheres after the coupling capture antibody is completed, and sealing the sites which are not combined with the capture antibody on the microspheres by using sealing liquid: 1) preparation of a sealing liquid: respectively adding 100g of bovine serum albumin and 5g of sodium azide into 1 XPBS with the value of 10LpH being 7.4 to obtain 10L of confining liquid; the sealing method comprises the following steps: and (3) centrifuging the coupled microsphere buffer solution at 15000 rpm for 5min, discarding the supernatant, adding 1500 mu L of microsphere sealing buffer solution into the centrifuged microspheres, centrifuging at 15000 rpm for 5min, removing the supernatant, and then suspending the microspheres in 800 mu L of microsphere sealing buffer solution to complete the sealing process, thus obtaining the final product: microsphere 1-mouse anti-human Hu monoclonal antibody, microsphere 2-mouse anti-human Yo monoclonal antibody, microsphere 3-mouse anti-human Ri monoclonal antibody, microsphere 4-mouse anti-human CV2 monoclonal antibody, microsphere 5-mouse anti-human Amphihysin monoclonal antibody, microsphere 6-mouse anti-human Ma1 monoclonal antibody, microsphere 7-mouse anti-human Ma2 monoclonal antibody, microsphere 8-mouse anti-human SOX1 monoclonal antibody, microsphere 9-mouse anti-human Tr monoclonal antibody, microsphere 10-mouse anti-human Zic4 monoclonal antibody, microsphere 11-mouse anti-human Titin monoclonal antibody, microsphere 12-mouse anti-human Recoverin monoclonal antibody, microsphere 13-mouse anti-human PKC gamma monoclonal antibody, microsphere 14-mouse anti-human GAD65 monoclonal antibody 14 respectively coupled with microspheres for capturing antibodies;
5) mixing 200 mu L of each of the 14 microspheres obtained in the step 4) to form a mixed reaction system containing 14 microspheres coupled with different capture antibodies, wherein the mixed reaction system is 2800 mu L and can simultaneously detect the marker antibody to be detected in serum;
6) adding 200 mu L of a prepared serum sample into the mixed reaction system in the step 5), carrying out antigen-antibody immune binding reaction on the capture antibody coupled on the microsphere and the marker antibody to be detected in the serum, and forming an antigen-antibody complex combining the marker antibody to be detected and the capture antibody on the microsphere after the binding reaction is finished, thus finally obtaining: microsphere 1-mouse anti-human Hu monoclonal antibody-Hu antibody, microsphere 2-mouse anti-human Yo monoclonal antibody-Yo antibody, microsphere 3-mouse anti-human Ri monoclonal antibody-Ri antibody, microsphere 4-mouse anti-human CV2 monoclonal antibody-CV 2 antibody, microsphere 5-mouse anti-human Amphihysin monoclonal antibody-Amphihysin antibody, microsphere 6-mouse anti-human Ma1 monoclonal antibody-Ma 1 antibody, microsphere 7-mouse anti-human Ma2 monoclonal antibody-Ma 2 antibody, microsphere 8-mouse anti-human SOX1 monoclonal antibody-SOX 1 antibody, microsphere 9-mouse anti-human Tr monoclonal antibody-Tr antibody, microsphere 10-mouse anti-human Zic4 monoclonal antibody-Zic 4 antibody, microsphere 11-mouse anti-human Titin monoclonal antibody-Titin antibody, microsphere 12-mouse anti-human Recoverin monoclonal antibody-Recoverin antibody, Microsphere 13-mouse anti-human PKC γ monoclonal antibody-PKC γ antibody, microsphere 14-mouse anti-human GAD65 monoclonal antibody-GAD 65 antibody;
in order to perform quantitative analysis on the marker antibody to be detected subsequently, a series of control experiments on 14 standard substances of the marker antibody to be detected are carried out by using the same reaction method of the microspheres and a serum sample, and the 14 standard substances corresponding to the marker antibody to be detected can be directly purchased in the market;
7) step 6), after the reaction is finished, washing away the unreacted serum protein, wherein the washing method used in the step can adopt a washing method in a microsphere activation process, firstly, centrifuging the reaction solution after the antigen-antibody immune binding reaction is finished, then, suspending the centrifuged microspheres in a microsphere washing buffer solution, after washing, suspending the microspheres in 2500 mu L of 1 XPBS buffer solution, adding excessive FITC labeled goat anti-mouse IgG for immunofluorescence reaction, and carrying out fluorescence quantitative analysis on the marker antibody to be detected in the serum sample;
8) detecting and analyzing the microspheres obtained after the immunofluorescence reaction in the step 7) by a flow cytometer: when the detection is carried out by the flow cytometer, each microsphere corresponds to a cell, the cell sequentially passes through a detection area of the flow cytometer at high speed, the fluorescent light and the scattered light of the microsphere (FL2, FL3) and the FITC marker (FL1) are detected and recorded by a corresponding detection system, the microspheres with different colors can be separated according to the intensities of FL2 and FL3, and the concentration of the corresponding marker antibody to be detected can be obtained according to the average intensity of FL 1.
The combined detection method of the 14 paraneoplastic syndrome associated marker antibodies in one serum sample can be specifically implemented according to the number of the serum samples.
Effects of the embodiment
In order to further verify the feasibility and effectiveness of the combined detection of the 14 marker antibodies Hu, Yo, Ri, CV2, amphihysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC γ, and GAD65 related to the paraneoplastic syndrome provided by the present invention, the results of the combined detection of the 14 marker antibodies related to the paraneoplastic syndrome according to the implementation procedures of the above embodiment are compared with the results of the currently clinically used elisa method, which is performed by using 20 serum samples and expressed in ± s (pg/mL), and the results are shown in the following table:
TABLE 1 comparison table of the results of the joint detection method and Elisa determination method provided by the present invention
The detection results show that compared with the conventional enzyme-linked immunosorbent assay method for detecting the related antibodies, the combined detection method for 14 marker antibodies related to the paraneoplastic syndrome, provided by the invention, has no significant difference from the results, and shows that the technical scheme provided by the invention has effectiveness and feasibility; secondly, from the fluctuation among the measured result numbers, the fluctuation among the measured data pre-data of the detection method provided by the invention is smaller, the repeatability of the result is better, and the technical scheme provided by the invention is further explained to have higher precision and sensitivity for carrying out combined detection on the 14 side-effect syndrome related marker antibodies.
The above-mentioned embodiments only represent the specific embodiments of the present application, and it is obvious to those skilled in the art that several variations and modifications can be made without departing from the technical solution concept of the present application, and these embodiments are all within the protection scope of the present application.
Claims (10)
1. A detection method of a paraneoplastic syndrome related marker antibody is characterized in that microspheres are combined with a flow analysis technology, and the detection method is used for carrying out combined detection on 14 paraneoplastic syndrome related marker antibodies including Hu, Yo, Ri, CV2, Amphihysin, Ma1, Ma2, SOX1, Tr, Zic4, Titin, Recoverin, PKC gamma and GAD 65.
2. The method for detecting the paraneoplastic syndrome-associated marker antibody of claim 1, wherein the method comprises the following steps:
1) preparing 14 kinds of microspheres, and respectively activating the 14 kinds of microspheres;
2) preparing 14 capture antibodies corresponding to the 14 marker antibodies related to the paraneoplastic syndrome, wherein the capture antibodies correspond to antigens and are used for carrying out antigen-antibody immune binding reaction with the marker antibodies to be detected in serum;
3) respectively carrying out coupling reaction on the microspheres activated in the step 1) and the capture antibodies prepared in the step 2);
4) sealing the microspheres after the coupling reaction in the step 3) is finished, and sealing the sites which are not combined with the capture antibody on the microspheres by using a sealing liquid;
5) mixing the 14 microspheres subjected to the sealing treatment respectively to form a mixed reaction system containing 14 microspheres coupled with different capture antibodies, wherein the mixed reaction system can be used for detecting the to-be-detected marker antibody in serum simultaneously;
6) adding a serum sample into the mixed reaction system in the step 5), wherein the capture antibody coupled on the microspheres and the to-be-detected marker antibody in the serum generate an antigen-antibody immunological binding reaction, and after the binding reaction is finished, an antigen-antibody complex combining the to-be-detected marker antibody and the capture antibody is formed on the microspheres;
7) step 6), after the reaction is finished, washing out the unreacted serum protein, adding excessive FITC labeled goat anti-mouse IgG for immunofluorescence reaction, and performing fluorescence quantitative analysis on the to-be-detected marker antibody in the serum sample;
8) detecting and analyzing the microspheres obtained after the immunofluorescence reaction in the step 7) by using a flow cytometer.
3. The method of claim 2, wherein the microsphere in step 1) is a polystyrene microsphere with carboxyl groups on the surface and embedded organic fluorescent molecules, and the particle size is uniform.
4. The method of claim 3, wherein the size of the microsphere is 1.25X 107 microspheres/mL.
5. The method of claim 2, wherein 14 of the microspheres have 14 different colors, namely microsphere 1, microsphere 2, microsphere 3, microsphere 4, microsphere 5, microsphere 6, microsphere 7, microsphere 8, microsphere 9, microsphere 10, microsphere 11, microsphere 12, microsphere 13 and microsphere 14, and each of the microspheres has different fluorescence colors.
6. The method for detecting the paraneoplastic syndrome-associated marker antibody of claim 2, wherein the microspheres are activated in step 1) by a microsphere activation buffer, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide salt and sulfurized N-hydroxysuccinimide.
7. The method for detecting the paraneoplastic syndrome-associated marker antibody of claim 6, wherein the concentration of each of the 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide salt and the sulfurized N-hydroxysuccinimide is 50 mg/mL.
8. The method for detecting the paraneoplastic syndrome-associated marker antibody of claim 2, wherein the capture antibodies of step 2) are each selected from the group consisting of: mouse anti-human Hu monoclonal antibody, mouse anti-human Yo monoclonal antibody, mouse anti-human Ri monoclonal antibody, mouse anti-human CV2 monoclonal antibody, mouse anti-human Amphihysin monoclonal antibody, mouse anti-human Ma1 monoclonal antibody, mouse anti-human Ma2 monoclonal antibody, mouse anti-human SOX1 monoclonal antibody, mouse anti-human Tr monoclonal antibody, mouse anti-human Zic4 monoclonal antibody, mouse anti-human Titin monoclonal antibody, mouse anti-human Recoverin monoclonal antibody, mouse anti-human PKC gamma monoclonal antibody, and mouse anti-human GAD65 monoclonal antibody.
9. The method for detecting the paraneoplastic syndrome-associated marker antibody of claim 2, wherein the blocking solution used for blocking the microspheres in step 4) is: adding corresponding bovine serum albumin and sodium azide into 1 XPBS with the pH value of 7.4 respectively to prepare the product.
10. The method of claim 9, wherein the concentration of bovine serum albumin in 1 XPBS buffer is 10g/l and the concentration of sodium azide in 1 XPBS buffer is 0.5 g/l.
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