CN111781357B - Kit for simultaneously detecting four tumor markers of lung cancer and application of kit - Google Patents

Kit for simultaneously detecting four tumor markers of lung cancer and application of kit Download PDF

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CN111781357B
CN111781357B CN202010664812.1A CN202010664812A CN111781357B CN 111781357 B CN111781357 B CN 111781357B CN 202010664812 A CN202010664812 A CN 202010664812A CN 111781357 B CN111781357 B CN 111781357B
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万东山
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/57423—Specifically defined cancers of lung

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Abstract

The invention belongs to the technical field of immunodetection, and particularly relates to a kit for simultaneously detecting four tumor markers of lung cancer and application thereof. The invention provides a kit for simultaneously detecting four tumor markers of lung cancer, which adopts a magnetic particle chemiluminescence method to simultaneously detect four tumor marker neuron specific enolase, cytokeratin fragments 19, gastrin release peptide precursors, squamous cell carcinoma antigens, has high sensitivity, high accuracy and good reliability, is not easily influenced by human operation factors, can simultaneously detect four tumor markers, is convenient and rapid to operate, is convenient to operate and standardized, and can be used for laboratory detection, field detection and the like.

Description

Kit for simultaneously detecting four tumor markers of lung cancer and application of kit
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a kit for simultaneously detecting four tumor markers of lung cancer and application thereof.
Background
The lung cancer prevention strategy is very important for reducing the death rate of lung cancer, enhancing the screening of high-risk groups and advocating the promotion of physical examination of healthy groups. Lung cancer is largely classified into small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). Small cell lung cancer is a tumor with high malignancy, bad biological behavior and strong prognosis. Compared with other lung cancer types, the small cell lung cancer has shorter symptom period and shorter survival time after diagnosis compared with the lung cancer types with the same spreading range, and one third of lung cancer patients belong to the lung cancer types. Non-small cell lung cancer, including squamous carcinoma, adenocarcinoma, large cell carcinoma, has slower growth division of cancer cells than small cell carcinoma, and relatively late diffusion metastasis. Small cell lung cancer is one of the basic types of lung cancer, belonging to the undifferentiated carcinoma, and its pathological types include oat cell type, intermediate cell type and complex oat cell type. The two lung cancer treatment methods are different, the key point of the lung cancer treatment is accurate clinical stage, and the method is the basis for preparing a scientific treatment scheme.
A neuron-specific enolase (NSE) is one of the enolases involved in the glycolytic pathway, and is present in neural tissue and neuroendocrine tissue. NSE has the highest activity in brain tissue cells, the central activity level of peripheral nerves and nerve secretory tissues, and the lowest value is found in non-nerve tissues, serum and spinal fluid. It was found to have excessive NSE expression in tumors associated with neuroendocrine tissue origin, particularly Small Cell Lung Cancer (SCLC), resulting in a significant increase in NSE in serum.
When Cytokeratin 19 fragment (Cytokeratin-19-fragment CYFRA 21-1) is an alveolar epithelial cell, the fragments of keratin contained in the cells are degraded to become soluble substances and enter the blood, so that the blood content is increased. Preferred markers for non-small cell lung cancer. Especially, the sensitivity of the currently preferred tumor marker of squamous cell carcinoma can reach 60 percent and the specificity can reach 95 percent. It has important significance for early diagnosis, curative effect monitoring and prognosis judgment of non-small cell lung cancer (NSCLC).
Gastrin-releasing peptide precursor (ProGRP) is an important regulatory molecule, and is involved in many physiological functions and pathological states of the human body. The gastrointestinal hormone is mammal homologous amphibian bombesin, is initially separated from pig gastric mucosa and is widely distributed in the nervous system, gastrointestinal tract and respiratory tract of mammal. As the signal peptide dissociates, its 148 amino acid prepro breaks down further to produce 27 amino acid gastrin-releasing peptide and 68 amino acid gastrin-releasing peptide precursor (ProGRP). Since the half-life of gastrin releasing peptide is short, only two minutes, and it is impossible to detect it in the blood, the detection of gastrin releasing peptide precursors, whose elevated levels are found in various neuroendocrine source tumors, includes: small cell lung cancer, carcinoid, undifferentiated large cell lung cancer with neuroendocrine function, medullary thyroid cancer, other neuroendocrine malignancies, and androgen-independent subgroups of prostate cancers with neuroendocrine function.
Squamous cell carcinoma antigen (squamous cell carcinoma antigen, SCC) is a subtype of the tumor-associated antigen TA-4 and is a glycoprotein. SCC is found in the cytoplasm of squamous cell carcinomas of the uterus, cervix, lung, head and neck, and the like, particularly in non-keratinized carcinoma cells, in greater abundance. Squamous cell carcinoma antigen (SCC) is a very specific and earliest tumor marker for diagnosis of squamous cell carcinoma. SCC inhibits apoptosis in normal squamous epithelial cells and is involved in differentiation of squamous epithelium, and in tumor cells is involved in tumor growth, which aids in diagnosis and monitoring of all squamous cell carcinoma of origin, for example: cervical cancer, lung cancer (non-small cell lung cancer), head and neck cancer, esophageal cancer, nasopharyngeal cancer, and squamous cell carcinoma of vulva. The auxiliary diagnosis of lung squamous carcinoma, the level of which is related to the tumor progression degree, and the combined detection of CYFRA21-1 and SCC can improve the diagnosis sensitivity of lung cancer patients.
In 18 cancer diagnosis and treatment specifications issued by the national Wei Jian commission, the detection of serum tumor markers is included in the category of auxiliary diagnosis of lung cancer, and particularly, the detection of multiple tumor markers such as Neuron Specific Enolase (NSE), cytokeratin fragment 19 (CYFRA 21-1), gastrin releasing peptide precursor (ProGRP), squamous cell carcinoma antigen (SCC) and the like are combined for use, so that the sensitivity and the specificity of the tumor markers in clinical application can be improved. Where NSE and ProGRP are ideal indicators for aiding in the diagnosis of SCLC. While an increased CEA, SCC, CYFRA21-1 level contributes to diagnosis of NSLCL. The traditional detection of four tumor markers is the independent detection of each tumor marker, for example, a chemiluminescent immunoassay kit for detecting cytokeratin 19 fragments, a quantitative detection kit for neuron-specific enolase of CN102914650B, a preparation method and application thereof and the like are disclosed in published Chinese application No. CN106483298B, and the method has the advantages of complex operation, high cost, low specificity, sensitivity and accuracy and limited clinical diagnosis value.
The combined detection of four tumor markers, neuron-specific enolase (NSE), cytokeratin fragment 19 (CYFRA 21-1), gastrin releasing peptide precursor (ProGRP), squamous cell carcinoma antigen (SCC), while theoretically being able to distinguish between small cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC) types, clearly increased numbers of markers during actual operation increase the difficulty of development.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide a kit for simultaneously detecting four tumor markers of lung cancer, which is based on a double-antibody sandwich method, can simultaneously detect four tumor markers of lung cancer, namely Neuron Specific Enolase (NSE), cytokeratin fragment 19 (CYFRA 21-1), gastrin releasing peptide precursor (ProGRP) and squamous cell carcinoma antigen (SCC), and has the advantages of high sensitivity, good accuracy, high reliability and convenient and rapid detection.
The invention also aims to provide application of the kit for simultaneously detecting four tumor markers of lung cancer.
The aim of the invention is achieved by the following technical scheme:
in a first aspect, the invention provides a kit for simultaneously detecting four tumor markers of lung cancer, comprising:
(1.1) a 1.1 container in which magnetic particles labeled with NSE antibodies are contained;
(1.2) a 1.2 container in which magnetic particles labeled with a CYFRA21-1 antibody are contained;
(1.3) a 1.3-th vessel in which magnetic particles labeled with ProGRP antibodies are contained;
(1.4) a 1.4 th container in which magnetic particles labeled with an SCC antibody are contained;
(2.1) a 2.1 container containing an acridinium ester conjugated to an NSE antibody;
(2.2) a 2.2 container containing an acridine ester coupled to a CYFRA21-1 antibody;
(2.3) a 2.3-th vessel containing an acridinium ester coupled to a ProGRP antibody;
(2.4) a 2.4 th vessel containing an acridinium ester coupled to an SCC antibody;
(3) A third container containing NSE antigen low value quality control;
(4) A fourth container in which the NSE antigen high-value quality control is contained;
(5) A fifth container containing CYFRA21-1 antigen low value quality control;
(6) A sixth container containing a CYFRA21-1 antigen high value quality control;
(7) A seventh container in which a ProGRP antigen low-value quality control substance is contained;
(8) An eighth container in which the ProGRP antigen high-value quality control material is contained;
(9) A seventh container in which the SCC antigen low value quality control is contained;
(10) An eighth container in which the SCC antigen high value quality control material is contained;
The kit can detect NSE antigen, CYFRA21-1 antigen, proGRP antigen and SCC antigen in a sample at the same time;
in a specific embodiment of the invention, the kit detects NSE antigen, CYFRA21-1 antigen, proGRP antigen and SCC antigen simultaneously, and the elevation of the antigens can assist diagnosis of lung cancer or other cancers to a certain extent;
it is noted that the assays of the present invention are used for the detection of ex vivo samples, the direct result of the detection being the presence or absence of antigen, and not the diagnostic result. Even if the detection method of the invention is used for detecting the antigen in the human blood sample, an experienced doctor is required to judge whether the detected quantity of the corresponding antigen can cause diseases according to the comprehensive conditions of physique, medical history, clinical symptoms and the like of the corresponding person, so that the disease cannot be directly judged according to the height of the antigen, the invention is not directly aimed at diagnosis, and the application of the kit of the invention is not a diagnosis method.
In this document, unless otherwise specified, ordinal numbers such as "1.1", "2.3", "third", "fifth", etc., are merely distinguishing between items or substances having the same or similar purpose and do not limit the structure, composition, nature, shape, etc. of the items or substances. For example, the fourth container and the fifth container may be containers of identical materials and shapes. The container may be a bottle, tube or cup such that the contents thereof are kept separately.
Preferably, in the kit of the first aspect of the present invention, the magnetic particles labeled with each antibody may be contained in a mixture in the same container, so that the simultaneous detection of four tumor markers is also facilitated without being affected, i.e. the 1.1 st container, the 1.2 nd container, the 1.3 rd container, and the 1.4 th container are the same container, e.g. the first container. Of course, in order to facilitate distinguishing the results of the synchronous detection, the 1.1 st container, the 1.2 nd container, the 1.3 rd container, and the 1.4 th container may be different containers.
It is also preferred that in the kit of the first aspect of the present invention, the acridinium esters coupled to each antibody may be contained in a mixture in the same container, so that simultaneous detection of tumor markers is also facilitated without being affected, i.e. the 2.1 nd container, the 2.2 nd container, the 2.3 nd container, the 2.4 nd container are the same container, e.g. the second container. Of course, in order to facilitate distinguishing the results of the synchronous detection, the 2.1 st container, the 2.2 nd container, the 2.3 rd container, and the 2.4 th container may be different containers.
Preferably, in the kit of the first aspect of the present invention, the magnetic particles are active epoxy group-containing magnetic particles having a particle diameter of 2.8. Mu.m. The present inventors have found that such magnetic particles have a high labeling rate on the antibody of the present invention, aggregate rapidly after application of magnetic force, and have good adsorption uniformity. In a specific embodiment of the present invention, the magnetic particles are magnetic particles of model M700.
More preferably, in the kit of the first aspect of the present invention, the method for preparing magnetic microparticles labeled with the corresponding antibody comprises the steps of:
(1) Washing the magnetic particles with PBS, discarding the supernatant, and suspending in the PBS again to obtain a magnetic particle diluent with the concentration of 5mg/ml; diluting the corresponding antibody by PBS, adding the diluted antibody into the magnetic particle diluent, and uniformly mixing to obtain a magnetic particle/antibody diluent;
(2) Adding PBS containing 3M ammonium sulfate into the magnetic particle/antibody diluent prepared in the step (1) until the final concentration of the ammonium sulfate in the system is 1M; then carrying out vibration treatment for 16-24 h at 37 ℃ for marking;
(3) After the labeling was completed, the supernatant was discarded, washed with PBS containing 0.1% (w/v) BSA, and then added to PBS containing 0.1% (w/v) BSA and 0.5% (v/v) Tween20 to obtain magnetic microparticles labeled with the corresponding antibodies, wherein the final concentration of the magnetic microparticles was 5mg/ml;
the number of times of washing in the step (1) is preferably 3 to 4 times;
the PBS concentration in the steps (1) and (2) is preferably 0.1M, and the pH is 7.2;
the dosage of the antibody in the step (1) is preferably that 20-30 mug of the antibody is added into 1mg of magnetic particles;
the PBS concentration in step (3) is preferably 0.01M and the pH is 7.2;
the number of times of washing in the step (3) is preferably 3 to 4 times;
Preferably in the kit of the first aspect of the invention, the acridine ester is an acridine succinimidyl ester. The inventor researches find that the acridinium ester does not substantially affect the antibody titer of the invention, and has good luminescence.
More preferably, in the kit of the first aspect of the present invention, the method for preparing an acridinium ester coupled to a corresponding antibody comprises the steps of:
dissolving acridine succinimidyl ester with Dimethylformamide (DMF) to obtain an acridine succinimidyl ester diluent; diluting the antibody by PB to obtain an antibody diluent; mixing the acridine succinimidyl ester diluent with the antibody diluent, stirring and reacting for 30min, then adding lysine for blocking, and finally dialyzing to obtain acridine ester coupled with the corresponding antibody;
the PB concentration is preferably 0.1M, and the pH is 8.0;
the concentration of the acridine succinimidyl ester in the acridine succinimidyl ester diluent is preferably 0.5mg/ml;
the concentration of the antibody in the antibody diluent is preferably 1mg/ml;
after the acridine succinimidyl ester diluent and the antibody diluent are mixed, the mass ratio of the acridine succinimidyl ester to the antibody is preferably 1:100;
the final concentration of lysine in the system is preferably 0.25% (w/v);
Preferably in the kit of the first aspect of the invention, the amino acid sequences of the NSE antigen, CYFRA21-1 antigen, proGRP antigen and SCC antigen are as set forth in SEQ ID NO:1 to 4;
preferably, in the kit of the first aspect of the present invention, the NSE antigen low-value quality control, NSE antigen high-value quality control, CYFRA21-1 antigen low-value quality control, CYFRA21-1 antigen high-value quality control, proGRP antigen low-value quality control, proGRP antigen high-value quality control, SCC antigen low-value quality control, SCC antigen high-value quality control are respectively formed by diluting synthetic tumor antigens, and the amino acid primary structure and secondary structure of the four tumor antigens are respectively compared with those of the corresponding natural tumor antigens in a human body, so that the coincidence rate of the amino acid primary structure and the secondary structure is more than 98%. Wherein the diluent was PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum.
In this context, "optional" has a dictionary meaning, i.e., covering the scope of either the optional or the non-optional cases. The kit of the first aspect of the invention optionally further comprises containers, either with or without these containers, for holding wash solutions, first excitation solution, second excitation solution, magnetic particles for diluting antibody labels or dilutions of antibody-coupled acridine esters, respectively. In a specific embodiment of the invention, the kit of the first aspect of the invention further comprises a container for containing a wash solution, a first excitation solution, a second excitation solution, a dilution solution for diluting the antibody-labeled magnetic particles or the antibody-conjugated acridine ester, respectively.
More preferably, in the kit of the first aspect of the present invention, the wash solution comprises Tween 20, and the first excitation solution comprises nitric acid and H 2 O 2 The second excitation solution comprises NaOH, the diluent for diluting the antibody-labeled magnetic particles comprises bovine serum albumin and tween 20, and/or the diluent for diluting the antibody-conjugated acridine ester comprises bovine serum albumin and tween 20, wherein the formulation of the diluent for diluting the antibody-labeled magnetic particles or antibody-conjugated acridine ester may be the same.
In addition, the kit of the first aspect of the invention may further comprise instructions describing its use (as in the second aspect of the invention). As another example, the kit of the first aspect of the invention may be sold in association with a magnetic plate, microplate and/or flash light instrument, etc., and thus the invention also provides a product comprising the kit of the first aspect of the invention and optionally an instrument.
In a second aspect, the invention provides the use of a kit of the first aspect of the invention in a method for simultaneous detection of NSE, CYFRA21-1, proGRP, SCC antigens in a sample; accordingly, the invention also provides the use of a kit of the first aspect of the invention in the preparation of a product for a method for simultaneous detection of NSE, CYFRA21-1, proGRP, SCC antigens in a sample.
Preferably in use according to the second aspect of the invention, the method of simultaneously detecting NSE, CYFRA21-1, proGRP, SCC antigens in a sample comprises, in order:
(1) Respectively adding a sample to be detected, NSE, CYFRA21-1, proGRP, SCC antigen low-value and high-value quality control products into the corresponding cup holes; wherein, the sample to be tested is provided with four treatments in parallel, and each quality control product is also provided with 4 treatments in parallel (NSE, CYFRA21-1, proGRP, SCC antigen low value and high value quality control products are used as standard products);
(2) Diluting the antibody-coupled acridine ester in the 2.1 th container, the 2.2 nd container, the 2.3 nd container and the 2.4 th container, respectively adding the diluted acridine ester into the four cup holes containing the sample to be tested in the step (1), and treating the quality control products in the same way;
(3) Mixing the magnetic particles in the 1.1 st container, the 1.2 nd container, the 1.3 rd container and the 1.4 th container (if the 1.1 st container, the 1.2 nd container, the 1.3 rd container and the 1.4 th container are the same container or are directly sampled), adding the mixture into four cup holes containing the sample to be tested in the step (1), incubating at 37 ℃ for 20min, and treating the quality control products in the same way;
(4) Applying magnetic force to attract the magnetic particles, discarding the liquid in the holes and washing;
(5) Simultaneously dripping a first excitation liquid and a second excitation liquid into each hole, and detecting the relative light-emitting units of each hole by using a flash light-emitting instrument;
(6) Respectively calibrating low-value and high-value quality control products for measuring four tumor markers, and quantifying a sample to be measured according to concentration values obtained by calibration;
in a third aspect, the invention provides a method of preparing a kit of the first aspect of the invention comprising the preparation of magnetic microparticles labeled with NSE antibody, the preparation of magnetic microparticles labeled with CYFRA21-1 antibody, the preparation of magnetic microparticles labeled with ProGRP antibody, the preparation of magnetic microparticles labeled with SCC antibody; preparation of acridine ester coupled to NSE antibody, preparation of acridine ester coupled to CYFRA21-1 antibody, preparation of acridine ester coupled to ProGRP antibody, preparation of acridine ester coupled to SCC antibody, and preparation of NSE, CYFRA21-1, proGRP, SCC antigen quality control; optionally further comprising the formulation of a wash solution, a first challenge solution, a second challenge solution, a dilution for diluting the antibody-labeled magnetic particles or antibody-conjugated acridine esters.
Compared with the prior art, the invention has the following advantages and effects:
(1) The kit for simultaneously detecting four tumor markers of lung cancer provided by the invention adopts a magnetic particle chemiluminescence method to simultaneously detect four tumor markers of lung cancer, namely Neuron Specific Enolase (NSE), cytokeratin fragment 19 (CYFRA 21-1), gastrin release peptide precursor (ProGRP) and squamous cell carcinoma antigen (SCC), has the advantages of high sensitivity, high accuracy, good reliability, difficult influence of human operation factors, convenience and rapidness in operation, convenience and standardization in operation, and can be used for laboratory detection, field detection and the like.
(2) The kit for simultaneously detecting the four tumor markers of the lung cancer and the application thereof provided by the invention can simultaneously detect the four tumor markers, and the detection result is rapid and accurate.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
Hereinafter, the invention will be described by means of specific examples. Unless otherwise indicated, the method may be carried out according to the methods listed in the laboratory manuals such as "molecular cloning laboratory Manual (third edition) (Cold Spring Harbor laboratory Press)," cell laboratory manual (scientific press, beijing, china, 2001), "RNA laboratory technical Manual (scientific press, beijing, china, 2004)," immunodetection technique "(scientific press, beijing, china, 1991), etc., which are familiar to those skilled in the art; the reagents are all commercially available, and in the examples, magnetic particles are selected from the M700 type magnetic particles available from Invitrogen corporation, and acridine esters are selected from the acridine succinimidyl esters available from Sigma-Aldrich.
EXAMPLE 1 preparation of magnetic microparticles labeled with NSE monoclonal antibody and acridinium ester conjugated with NSE monoclonal antibody
In the preliminary research process, monoclonal antibodies aiming at different sites of NSE antigens are adopted for preliminary screening and optimization, and according to the research of the invention, if four tumor markers are to be detected simultaneously, monoclonal antibody combinations (commercially available sigma, product numbers AMAB90556 and HPA 007007) aiming at different sites of NSE antigens are required, and through testing, the NSE monoclonal antibodies do not have cross reaction with CYFRA21-1, proGRP and SCC antibodies, and are not the monoclonal antibodies with highest titers in the test, but have the best accuracy in the simultaneous detection, so that NSE monoclonal antibodies with different sites are used for preparing labeled magnetic microparticles and acridine ester conjugates. The specific method comprises the following steps:
1.1 preparation of magnetic microparticles labeled with NSE monoclonal antibody
(1) Taking 5mg of magnetic particles, adding 2ml of PBS (0.1M, pH 7.2) for washing, repeating the washing for 3 times, and discarding the supernatant; then adding 1ml PBS (0.1M, pH 7.2) to resuspend the magnetic microbeads to obtain a magnetic microparticle diluent with the concentration of 5 mg/ml; diluting NSE monoclonal antibody (product number: AMAB90556, etc.) to 1mg/ml with PBS (0.1M, pH 7.2) to obtain NSE monoclonal antibody dilution; then adding 100 mu l NSE monoclonal antibody diluent into the magnetic particle diluent prepared in the step (1) according to the proportion of adding 20 mu g of antibody into 1mg of magnetic particles, and uniformly mixing to obtain magnetic particle/NSE monoclonal antibody diluent;
(2) Adding PBS (0.1M, pH 7.2) containing 3M ammonium sulfate into the magnetic particle/NSE monoclonal antibody diluent prepared in the step (1) until the final concentration of the ammonium sulfate in the system is 1M; then uniformly mixing and vibrating for 16-24 hours at 37 ℃ for marking;
(3) After completion of labeling, the supernatant was discarded, washed 4 times with PBS (0.01M, pH 7.2) containing 0.1% (w/v) BSA, and after discarding the supernatant, PBS (0.01M, pH 7.2) containing 0.1% (w/v) BSA and 0.5% (v/v) Tween20 was added to give a final concentration of 5mg/ml of magnetic microparticles, to give magnetic microparticles labeled with NSE monoclonal antibodies, and stored at 2 to 8 ℃.
1.2 preparation of acridinium esters conjugated with NSE monoclonal antibodies
Dissolving acridine succinimidyl ester with anhydrous dimethyl imide (DMF) to obtain acridine succinimidyl ester diluent with the concentration of 0.5 mg/ml; diluting NSE monoclonal antibody (product number: HPA007007, etc.) with PB (0.1M, pH 8.0) to 1mg/ml to obtain NSE monoclonal antibody dilution with antibody concentration of 1 mg/ml; 20. Mu.l of acridine succinimidyl ester diluent was added to 1ml of NSE monoclonal antibody diluent at a ratio of 10. Mu.g of acridine ester to 1mg of antibody, stirred at room temperature (25 ℃) for 30 minutes, PB (0.1M, pH 8.0) containing 1% (w/v) lysine was added to make the final concentration of lysine 0.25%, blocked at room temperature (25 ℃) for 30 minutes, dialysis was performed with PBS (0.01M, pH 6.3), and glycerol-20℃was added after dialysis to store, to obtain acridine ester coupled with NSE monoclonal antibody, wherein the molar concentration of acridine ester after glycerol addition was 0.36mM.
1.3 preparation of NSE antigen Low-value quality control product
NSE antigen (synthesized by biological engineering (Shanghai) Co., ltd. According to the amino acid sequence of SEQ ID NO: 1) was diluted to 18ng/ml with PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, mixed well and stored at 2-8deg.C.
The amino acid sequence of the NSE antigen (SEQ ID NO: 1) is shown below: MSIEKIWAREILDSRGNPTVEVDLHTAKGLFRAAVPSGASTGIYEALELRDGDKQRYLGKGVLKAVDHINTTIAPVLISSGLSVVEQEELDDLMLDLDGTENKSKFGANAILGVSLAVCKAGAAERELPLYRHIAQLAGNSDLILPVPAFNVINGGSHAGNKLAMQEFMILPVGAESFRDAMRLGAEVYHTLKGVIKDKYGKDATNVGDEGGFAPNILENSEALELVKEAIDKAGYTEKIVIGMDVAASEFYRDGKYDLDFKSPADPSRYITGDQLGALYQDFVRDYPVVSIEDPFDQDDWAAWSKFTANVGIQIVGDDLTVTNPKRIERAVEEKACNCLLLKVNQIGSVTEAIQACKLAQENGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCRSERLAKYNQHMRIEEELGDEARFAGHNFRNPSVL
1.4 preparation of NSE antigen high-value quality control product
NSE antigen (synthesized by biological engineering (Shanghai) Co., ltd. According to the amino acid sequence of SEQ ID NO: 1) was diluted to 250ng/ml with PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, mixed well and stored at 2-8deg.C.
1.5 experimental results
The method comprises the steps of respectively preparing NSE monoclonal antibody labeled magnetic particles from NSE monoclonal antibodies of different sources and acridinium esters coupled with the NSE monoclonal antibodies, combining the NSE antigen high-value quality control (250 ng/ml)/NSE antigen low-value quality control (18 ng/ml), CYFRA21-1 antigen high-value quality control (100 ng/ml)/ProGRP antigen high-value quality control (300 pg/ml)/SCC antigen high-value quality control (100 ng/ml), CYFRA21-1 monoclonal antibody high-value samples (CY 211-McAb1# +CY211-McAb2#) and other concentration mixtures, and screening out the CYFRA21-1 monoclonal antibody high-value samples (CY 211-McAb1#, total concentration is 1 mg/ml), the ProGRP monoclonal antibody high-McAb1+GRP-McAb2 and other concentration mixtures), the SCC monoclonal antibody high-value samples (BS-0040+BS-0041 and other concentration mixtures, wherein the total concentration is 1 mg/ml), and the monoclonal antibody high-value sample high in the NSE are detected, and the reagent high-sensitivity of the NSE antibody are satisfied. The results were as follows:
TABLE 1 NSE monoclonal antibody information
Name of the name Source Goods number Protein concentration
NSE monoclonal antibody 1 Sigma MAB324 3.5mg/ml
NSE monoclonal antibody 2 Sigma AMAB90556 4.3mg/ml
NSE monoclonal antibody 3 Sigma HPA007007 2.8mg/ml
NSE monoclonal antibody 4 Sigma SAB4200572 3.2mg/ml
NSE monoclonal antibody 5 Sigma SAB4500768 4.9mg/ml
TABLE 2 NSE monoclonal antibody screening results (including cross-reactions)
Analysis from the above results: NSE monoclonal antibody pairing is adopted to pair monoclonal antibody 2 and monoclonal antibody 3 (commercially available sigma, product numbers AMAB90556 and HPA 007007), the result meets the quality requirement, and the following experiment is carried out in the optimal combination.
EXAMPLE 2 preparation of magnetic microparticles labeled with CYFRA21-1 monoclonal antibody and acridinium ester conjugated with CYFRA21-1 monoclonal antibody
In the preliminary research process, monoclonal antibodies aiming at different sites of CYFRA21-1 antigen are adopted for preliminary screening and optimization, and if four tumor antigens are to be detected simultaneously, the monoclonal antibodies aiming at different sites of the CYFRA21-1 antigen are adopted for combination (purchased from the Phpeng biological Co., ltd., the product numbers are CY211-McAb1#, CY 211-McAb2#). According to tests, the CYFRA21-1 monoclonal antibodies and the NSE, proGRP, SCC antibodies have no cross reaction and have the best accuracy in simultaneous detection, so that the CYFRA21-1 monoclonal antibodies with different sites are used for preparing labeled magnetic particles and acridine ester conjugates. The specific method comprises the following steps:
1.1 preparation of magnetic microparticles labeled with CYFRA21-1 monoclonal antibody
(1) Taking 5mg of magnetic particles, adding 2ml of PBS (0.1M, pH 7.2) for washing, repeating the washing for 3 times, and discarding the supernatant; then adding 1ml PBS (0.1M, pH 7.2) to resuspend the magnetic microbeads to obtain a magnetic microparticle diluent with the concentration of 5 mg/ml; the CYFRA21-1 monoclonal antibody (product number: CY211-McAb1# and the like) is diluted to 1mg/ml by PBS (0.1M, pH 7.2) to obtain a CYFRA21-1 monoclonal antibody diluent; adding 125 mu l of CYFRA21-1 monoclonal antibody diluent into the magnetic particle diluent prepared in the step (1) according to the proportion of adding 25 mu g of antibody into 1mg of magnetic particles, and uniformly mixing to obtain the magnetic particle/CYFRA 21-1 monoclonal antibody diluent;
(2) Adding PBS (0.1M, pH 7.2) containing 3M ammonium sulfate into the magnetic particle/CYFRA 21-1 monoclonal antibody diluent prepared in the step (1) until the final concentration of the ammonium sulfate in the system is 1M; then mixing uniformly at 37 ℃ and vibrating for 16-24 hr for marking;
(3) After completion of labeling, the supernatant was discarded, washed 4 times with PBS (0.01M, pH 7.2) containing 0.1% (w/v) BSA, and after discarding the supernatant, 0.1% (w/v) BSA and 0.5% (v/v) Tween20 in PBS (0.01M, pH 7.2) were added to give a final concentration of 5mg/ml of magnetic particles, to give magnetic particles labeled with CYFRA21-1 monoclonal antibody, and stored at 2-8 ℃.
1.2 preparation of acridinium esters conjugated with CYFRA21-1 monoclonal antibodies
Dissolving acridine succinimidyl ester with anhydrous dimethyl imide (DMF) to obtain acridine succinimidyl ester diluent with the concentration of 0.5 mg/ml; taking CYFRA21-1 monoclonal antibody (product number: CY211-McAb2#, and the like), and diluting to 1mg/ml by PB (0.1M, pH8.0), thereby obtaining a CYFRA21-1 monoclonal antibody diluent with the antibody concentration of 1 mg/ml; 20. Mu.l of acridine succinimidyl ester diluent was added to 1ml of CYFRA21-1 monoclonal antibody diluent at a ratio of 10. Mu.g of acridine ester to 1mg of antibody, stirred at room temperature (25 ℃) for 30 minutes, PB (0.1M, pH 8.0) containing 1% (w/v) lysine was added to make the final concentration of lysine 0.25%, blocked at room temperature (25 ℃) for 30 minutes, dialyzed against PBS (0.01M, pH 6.3), and glycerol-20℃was added after dialysis to store, to obtain acridine ester coupled with CYFRA21-1 monoclonal antibody, wherein the molar concentration of acridine ester after glycerol addition was 0.36mM.
Preparation of 1.3CYFRA21-1 antigen low-value quality control product
The CYFRA21-1 antigen (synthesized by the division of biological engineering (Shanghai) according to the amino acid sequence of SEQ ID NO: 2) is diluted to 4ng/ml by PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, and is mixed uniformly and stored at 2-8 ℃.
The amino acid sequence of the CYFRA21-1 antigen (SEQ ID NO: 2) is shown below: MTSYSYRQSSATSSFGGLGGGSVRFGPGVAFRAPSIHGGSGGRGVSVSSARFVSSSSSGAYGGGYGGVLTASDGLLAGNEKLTMQNLNDRLASYLDKVRALEAANGELEVKIRDWYQKQGPGPSRDYSHYYTTIQDLRDKILGATIENSRIVLQIDNARLAADDFRTKFETEQALRMSVEADINGLRRVLDELTLARTDLEMQIEGLKEELAYLKKNHEEEISTLRGQVGGQVSVEVDSAPGTDLAKILSDMRSQYEVMAEQNRKDAEAWFTSRTEELNREVAGHTEQLQMSRSEVTDLRRTLQGLEIELQSQLSMKAALEDTLAETEARFGAQLAHIQALISGIEAQLGDVRADSERQNQEYQRLMDIKSRLEQEIATYRSLLEGQEDHYNNLSASKVL
1.4 Preparation of CYFRA21-1 antigen high-value quality control product
The CYFRA21-1 antigen (synthesized by the division of biological engineering (Shanghai) according to the amino acid sequence of SEQ ID NO: 2) is diluted to 100ng/ml by PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, and is mixed uniformly and stored at 2-8 ℃.
1.5 experimental results
And respectively preparing magnetic particles marked by CYFRA21-1 monoclonal antibodies from different sources and acridine esters coupled with the CYFRA21-1 monoclonal antibodies, combining the magnetic particles marked by the CYFRA21-1 monoclonal antibodies with the high-value quality control (100 ng/ml) of the CYFRA21-1 antigen, the low-value quality control (4 ng/ml) of the CYFRA21-1 antigen, the high-value quality control (250 ng/ml) of NSE antigen, the high-value quality control (300 pg/ml) of ProGRP antigen and the high-value quality control (100 ng/ml) of the SCC antigen, the high-value sample (AMAB 90556+HPA 007007) of NSE, the high-value sample (GRP-McAb1+GRP-Mc2 and other concentration mixtures, the total concentration of Ab is 1 mg/ml), the high-value sample (BS-MA 0040+BS-0041 and other concentration mixtures) of the NSE antigen prepared in examples 1, the total concentration of the NSE antigen is 1 mg/ml), detecting the high-value sample (AMAB 90556+HPA007007 and other concentration mixtures, and detecting the high-value sample (AMA and the high-quality control) of the high-quality control to be 1 mg/HPA and the high-quality control to obtain the monoclonal antibodies, and the monoclonal antibodies to meet the requirements of the monoclonal antibodies of the specific reagent and the monoclonal antibody and the test reagent and the test. The results were as follows:
TABLE 3 CYFRA21-1 mab information
Table 4 CYFRA21-1 mab screening results (including cross-reactions)
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Analysis from the above results: the following experiments were performed with optimal combinations using CYFRA21-1 mab pairing as mab 1 and mab 2 (available from Qingpeng biological Co., ltd.) and CY211-McAb1#, CY211-McAb2#, respectively.
EXAMPLE 3 preparation of magnetic microparticles labeled with ProGRP monoclonal antibody and acridinium ester conjugated with ProGRP monoclonal antibody
In the preliminary research process, monoclonal antibodies aiming at different sites of ProGRP antigen are adopted for preliminary screening and optimization, and according to the research of the invention, if four tumor markers are to be detected simultaneously, monoclonal antibody combinations (purchased from the Phpeng biological Co., ltd.) of different sites of ProGRP are adopted, and the product numbers are GRP-McAb1 and GRP-McAb2 respectively. Through tests, the GRP-McAb monoclonal antibody has no cross reaction with CYFRA21-1, NSE and SCC antibodies, and has the best accuracy in simultaneous detection, so the invention uses ProGRP monoclonal antibodies with different sites to prepare labeled magnetic particles and acridine ester conjugates. The specific method comprises the following steps:
1.1 preparation of magnetic microparticles labeled with ProGRP monoclonal antibody
(1) Taking 5mg of magnetic particles, adding 2ml of PBS (0.1M, pH 7.2) for washing, repeating the washing for 3 times, and discarding the supernatant; then adding 1ml PBS (0.1M, pH 7.2) to resuspend the magnetic microbeads to obtain a magnetic microparticle diluent with the concentration of 5 mg/ml; proGRP monoclonal antibody (product number: GRP-McAb1, etc.) was diluted to 1mg/ml with PBS (0.1M, pH 7.2) to give a diluted solution of ProGRP monoclonal antibody; adding 125 mu l of ProGRP monoclonal antibody diluent into the magnetic particle diluent prepared in the step (1) according to the proportion of adding 25 mu g of antibody into 1mg of magnetic particles, and uniformly mixing to obtain magnetic particle/ProGRP monoclonal antibody diluent;
(2) Adding PBS (0.1M, pH 7.2) containing 3M ammonium sulfate into the magnetic particle/ProGRP monoclonal antibody diluent prepared in the step (1) until the final concentration of the ammonium sulfate in the system is 1M; mixing uniformly at 37 ℃ and vibrating for 16-24 hr for marking;
(4) After completion of labeling, the supernatant was discarded, washed 4 times with PBS (0.01M, pH 7.2) containing 0.1% (w/v) BSA, and after discarding the supernatant, 0.1% (w/v) BSA and 0.5% (v/v) Tween20 in PBS (0.01M, pH 7.2) were added to give a final concentration of 5mg/ml of magnetic microparticles, to give magnetic microparticles labeled with ProGRP monoclonal antibodies, and stored at 2-8 ℃.
1.2 preparation of acridinium esters conjugated with ProGRP monoclonal antibodies
Dissolving acridine succinimidyl ester with anhydrous dimethyl imide (DMF) to obtain acridine succinimidyl ester diluent with the concentration of 0.5 mg/ml; taking ProGRP monoclonal antibody (product number: GRP-McAb2 and the like), diluting to 1mg/ml by PB (0.1M, pH8.0), and obtaining a ProGRP monoclonal antibody diluent with the antibody concentration of 1 mg/ml; 20. Mu.l of acridine succinimidyl ester diluent was added to 1ml of ProGRP monoclonal antibody diluent at a ratio of 10. Mu.g of acridine ester to 1mg of antibody, stirred at room temperature (25 ℃) for 30 minutes, PB (0.1M, pH 8.0) containing 1% (w/v) lysine was added to make the final concentration of lysine 0.25%, blocked at room temperature (25 ℃) for 30 minutes, dialyzed against PBS (0.01M, pH 6.3), and glycerol was added to store at-20℃after dialysis to give acridine ester coupled with ProGRP monoclonal antibody, wherein the molar concentration of acridine ester after glycerol addition was 0.36mM.
1.3 ProGRP antigen low-value quality control product preparation
ProGRP antigen (synthesized by biological engineering (Shanghai) Co., ltd. According to the amino acid sequence of SEQ ID NO: 3) was diluted to 75pg/ml with PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, mixed well and stored at 2-8deg.C.
The amino acid sequence of the PROGRP antigen (SEQ ID NO: 3) is as follows:
MRGRELPLVLLALVLCLAPRGRAVPLPAGGGTVLTKMYPRGNHWAVGHLMGKKSTGESSSVSERGSLKQQLREYIRWEEAARNLLGLIEAKENRNHQPPQPKALGNQQPSWDSEDSSNFKDLVDSLLQVLNVKEGTPS
1.4 Preparation of ProGRP antigen high-value quality control product
ProGRP antigen (synthesized by biological engineering (Shanghai) Co., ltd. According to the amino acid sequence of SEQ ID NO: 3) was diluted to 300pg/ml with PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, mixed well and stored at 2-8deg.C.
1.5 experimental results
The magnetic particles marked by the ProGRP monoclonal antibodies and acridine esters coupled with the ProGRP monoclonal antibodies are respectively prepared from different sources of ProGRP monoclonal antibodies and combined, the high-value quality control product (300 pg/ml) of the ProGRP antigen, the low-value quality control product (75 pg/ml) of the ProGRP antigen, the high-value quality control product (250 ng/ml) of the NSE antigen prepared in examples 1-2 and 4, the high-value quality control product (100 ng/ml) of the CYFRA21-1 antigen, the high-value quality control product (100 ng/ml) of the SCC antigen, the mixture of the NSE monoclonal antibody high-value sample (AMAB 90556+HPA 007007) and the like, the mixture of the high-value sample (CY 211-Mc1# +CY211-McAb2#) and the like, the mixture of the total concentration of the high-value sample (BS-0040+BS-0041) and the like, the total concentration of the high-value sample (1 mg/ml) of the SCC monoclonal antibody is 1 mg/ml), the high-value sample of the CYFRA21-1 monoclonal antibody is detected, and the monoclonal antibody is screened, and the monoclonal antibody, and the monoclonal antibody, the antigen, and the reagent, which have no requirement for cross-test are selected. The results were as follows:
TABLE 5ProGRP mab information
Name of the name Source Goods number Protein concentration
ProGRP monoclonal antibody 1 Feipeng biology Co.,Ltd. GRP-McAb1、 4.3mg/ml
ProGRP mab 2 Feipeng biology Co.,Ltd. GRP-McAb2 2.9mg/ml
ProGRP mab 3 Sigma SAB1409152 3.6mg/ml
ProGRP mab 4 Shenzhen heavy chain Biotechnology Co., ltd HA116-1M 3.7mg/ml
ProGRP mab 5 Shenzhen heavy chain Biotechnology Co., ltd HA116-2M 4.1mg/ml
TABLE 6 ProGRP monoclonal antibody screening results (including Cross-reactions)
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Analysis from the above results: the ProGRP monoclonal antibodies are matched with monoclonal antibody 1 and monoclonal antibody 2 (purchased from Qingpeng biological Co., ltd., the goods numbers are GRP-McAb1 and GRP-McAb 2) respectively, so that the quality requirements are met, and the following experiment is carried out in an optimal combination.
EXAMPLE 4 preparation of magnetic microparticles labeled with SCC monoclonal antibody and acridinium ester conjugated with SCC monoclonal antibody
In the preliminary research process, monoclonal antibodies aiming at different loci of SCC antigen are adopted for preliminary screening and optimization, and according to the research of the invention, if four tumor markers are to be detected simultaneously, monoclonal antibody combinations (available from Nanjing Bose biotechnology Co., ltd., product numbers are BS-MA0040 and BS-MA 0041) aiming at different loci of the SCC antigen are adopted. According to tests, the SCC monoclonal antibodies have no cross reaction with CYFRA21-1, NSE and GroRP antibodies, and the accuracy is the best in simultaneous detection, so that the SCC monoclonal antibodies with different sites are used for preparing labeled magnetic particles and acridine ester conjugates. The specific method comprises the following steps:
1.1 preparation of magnetic microparticles labeled with SCC monoclonal antibody
(1) Taking 5mg of magnetic particles, adding 2ml of PBS (0.1M, pH 7.2) for washing, repeating the washing for 3 times, and discarding the supernatant; then adding 1ml PBS (0.1M, pH 7.2) to resuspend the magnetic microbeads to obtain a magnetic microparticle diluent with the concentration of 5 mg/ml; SCC monoclonal antibody (product number: BS-MA0040, etc.) was diluted to 1mg/ml with PBS (0.1M, pH 7.2) to obtain SCC monoclonal antibody dilution; adding 100 μl of SCC monoclonal antibody diluent into the magnetic particle diluent prepared in the step (1) according to the proportion of adding 20 μg of antibody into 1mg of magnetic particles, and uniformly mixing to obtain magnetic particle/SCC monoclonal antibody diluent;
(3) Adding PBS (0.1M, pH 7.2) containing 3M ammonium sulfate into the magnetic particle/SCC monoclonal antibody diluent prepared in the step (2) until the final concentration of the ammonium sulfate in the system is 1M; then mixing uniformly at 37 ℃ and vibrating for 16-24 hr for marking;
(4) After completion of labeling, the supernatant was discarded, washed 4 times with PBS (0.01M, pH 7.2) containing 0.1% (w/v) BSA, and after discarding the supernatant, 0.1% (w/v) BSA and 0.5% (v/v) Tween20 in PBS (0.01M, pH 7.2) were added to give a final concentration of 5mg/ml of magnetic particles, to give magnetic particles labeled with SCC monoclonal antibodies, and stored at 2-8 ℃.
1.2 preparation of acridinium esters conjugated with SCC monoclonal antibodies
Dissolving acridine succinimidyl ester with anhydrous dimethyl imide (DMF) to obtain acridine succinimidyl ester diluent with the concentration of 0.5 mg/ml; diluting SCC monoclonal antibody (product number: BS-MA0041, etc.) to 1mg/ml with PB (0.1M, pH 8.0) to obtain SCC monoclonal antibody diluted solution with antibody concentration of 1 mg/ml; 20. Mu.l of acridine succinimidyl ester diluent was added to 1ml of SCC monoclonal antibody diluent at a ratio of 10. Mu.g of acridine ester to 1mg of antibody, stirred at room temperature (25 ℃) for 30 minutes, PB (0.1M, pH 8.0) containing 1% (w/v) lysine was added to make the final concentration of lysine 0.25%, blocked at room temperature (25 ℃) for 30 minutes, dialyzed against PBS (0.01M, pH 6.3), and glycerol-20℃was added after dialysis to store, to obtain acridine ester coupled with SCC monoclonal antibody, wherein the molar concentration of acridine ester after glycerol addition was 0.36mM.
1.3 Preparation of SCC low-value quality control product
SCC antigen (synthesized by biological engineering (Shanghai) Co., ltd. According to the amino acid sequence of SEQ ID NO: 4) was diluted to 2ng/ml with PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, mixed well and stored at 2-8deg.C.
The amino acid sequence of the SCC antigen (SEQ ID NO: 4) is shown below:
MRVRAGVKAFGGSICYVEFKEEKSALQALEMDRKSVEGRPMFVSPCVDKSKNPDFKVFRYSTSLEKHKLFISGLPFSCTKEELEEICKAHGTVKDLRLVTNRAGKPKGLAYVEYENSEQASQAVMKMDGMTIKENIIKVAISNPPQRKVPEKPETRKAPGGPMLLPQTYGARGKGRTQLSLLPRALQRPSAAAPQAENGPAAAPAVAAPAATEAPKMSNADFAKLFL
1.4 Preparation of SCC high-value quality control product
SCC antigen (synthesized by biological engineering (Shanghai) Co., ltd. According to the amino acid sequence of SEQ ID NO: 4) was diluted to 100ng/ml with PBS (0.01M, pH 7.2) containing 30% (v/v) calf serum, mixed well and stored at 2-8deg.C.
1.5 experimental results
The magnetic particles marked by the SCC monoclonal antibodies and the acridine ester coupled with the SCC monoclonal antibodies are respectively prepared from different sources of SCC monoclonal antibodies and combined, the SCC antigen high-value quality control (100 ng/ml) and the SCC antigen low-value quality control (2 ng/ml), NSE antigen high-value quality control (250 ng/ml)/CYFRA 21-1 antigen high-value quality control (100 ng/ml)/ProGRP antigen high-value quality control (300 pg/ml), NSE monoclonal antibody high-value samples (AMAB 90556+HPA007007 and other concentration mixtures, the total concentration is 1 mg/ml), CYFRA21-1 monoclonal antibody high-value samples (CY 211-Mc1# +CY211-Mc2# and other concentration mixtures), the ProGRP monoclonal antibody high-value samples (GRP-McAb 1+GRP-Mc2 and other concentration mixtures prepared in examples 1-3) are taken as the sample to be detected, and the monoclonal antibodies to be detected are screened, and the monoclonal antibodies, the NSF 21, the GRP and the specific reagents and the reagent are satisfied. The results were as follows:
TABLE 7 SCC mab information
Name of the name Source Goods number Protein concentration
SCC monoclonal antibody1 Nanjing Boston Biotechnology Co Ltd BS-MA0040 2.8mg/ml
SCC monoclonal antibody 2 Nanjing Boston Biotechnology Co Ltd BS-MA0041 3.1mg/ml
SCC monoclonal antibody 3 Sigma WH0029940M3 2.6mg/ml
SCC monoclonal antibody 4 Sigma SAB2500911 6.5mg/ml
Table 8 SCC mab screening results (including cross-reactions)
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Analysis from the above results: SCC monoclonal antibody pairs are adopted as monoclonal antibody 1 and monoclonal antibody 2 (Nanjing Bos gold biotechnology Co., ltd., the goods numbers are BS-MA0040 and BS-MA0041 respectively), and the result accords with the best; the SCC antibodies were paired with antibody 3 and antibody 4 (sigma, cat No. WH0029940M3 and SAB2500911, respectively) and both pairs met the quality requirements, and the following experiments were performed in the best combination.
Example 5 Simultaneous detection kit for four tumor markers
According to the best results obtained in examples 1 to 4, the synchronous detection kit of the present invention may comprise 16 containers, which are respectively filled with the labeled magnetic particles prepared in each of examples 1 to 4, the acridine ester conjugate prepared in each of examples 1 to 4, the low-value quality control product, and the high-value quality control product, and the synchronous detection kit not only can perform synchronous detection of four tumor markers, but also can confirm the concentration of the tumor markers according to the quality control product. If stability of concentration needs to be confirmed, specific tumor markers can be confirmed for detection using a part of the reagents therein.
Of course, if only for synchronous detection, the labeled magnetic particles prepared in each of examples 1 to 4 may be mixed and contained in one container, the acridine ester conjugates prepared in each of examples 1 to 4 are contained in different containers, and the high-value and low-value quality control substances prepared in each of examples 1 to 4 are contained in different containers, respectively, such a synchronous detection kit comprises 13 containers.
In addition, the detection kit of the present invention may further comprise containers each containing the following reagents: the washing solution (formula: phosphate buffer (0.01M, pH 7.4), 0.05% (v/v) Tween 20) and the first excitation solution (formula: 0.05M nitric acid solution, 0.15% (v/v) H 2 O 2 ) And a second excitation liquid (its formulation: 0.2M NaOH solution), and may further include a diluent for diluting the antibody-labeled magnetic particles (the formulation of which is: phosphate buffer (0.02M, pH 7.4), 1% (w/v, g/ml) bovine serum albumin, 0.1% (v/v) Tween 20) and a dilution for diluting the antibody-conjugated acridine ester (formulation: phosphate buffer (0.02M, pH 7.4), 1% (w/v, g/ml) bovine serum albumin, 0.1% (v/v) Tween 20.
EXAMPLE 7 synchronous detection of four tumor markers
1. The specific process of synchronous detection is as follows:
(1) Taking a sample to be measured, adding the sample to be measured into a cup hole (4 holes are formed, and four treatments are performed on the sample to be measured in parallel), wherein each hole is 50 mu l; meanwhile, NSE, CYFRA21-1, proGRP, SCC antigen low value and high value quality control substances are also added into the corresponding cup holes (4 holes of each quality control substance are provided with four parallel treatments) respectively, and 50 μl of each hole is obtained;
(2) 50. Mu.l of acridine ester conjugated with NSE monoclonal antibody (prepared in example 1, diluted to 0.02. Mu.M with diluent), 50. Mu.l of acridine ester conjugated with CYFRA21-1 monoclonal antibody (prepared in example 2, diluted to 0.03. Mu.M with diluent), 50. Mu.l of acridine ester conjugated with ProGRP monoclonal antibody (prepared in example 3, diluted to 0.025. Mu.M with diluent), 50. Mu.l of acridine ester conjugated with SCC monoclonal antibody (prepared in example 4, diluted to 0.018. Mu.M with diluent) were added to the four wells containing the sample to be tested in step (1), and the respective quality controls were treated similarly;
(3) Equal volume mixing of the magnetic particles labeled with each monoclonal antibody prepared in examples 1 to 4 (the total concentration of the magnetic particles is diluted to 0.5mg/ml by the magnetic particle diluent) to obtain magnetic particles labeled with the mixed monoclonal antibody; adding the magnetic particles marked by the mixed monoclonal antibodies into four cup holes containing the sample to be tested in the step (1), wherein 100 mu l of each hole is incubated for 20 minutes at 37 ℃, and all quality control products are treated similarly;
(3) Washing: applying magnetic force to absorb magnetic particles, discarding liquid in the holes, adding 300 mu l of washing liquid into each hole, removing magnetic force, vibrating and mixing uniformly, standing for 20 seconds, applying magnetic force, discarding liquid in the holes, and washing for 5 times; applying magnetic force to the bottom to attract the magnetic particles, and discarding the liquid in the hole;
(4) Each well was simultaneously filled with 100. Mu.l of each of the first and second excitation solutions, and the Relative Luminescence Units (RLU) of each well was measured with a flash luminometer (available from promage Corp.) at the same time of filling.
(5) Quantifying the sample according to the low-value quality control and the high-value quality control of each tumor marker;
2. the specific process of the individual detection is as follows:
step (1) and step (2) are synchronously detected;
(3) Mixing 25 μl of each monoclonal antibody labeled magnetic particle prepared in examples 1-4 with 75 μl of diluent, adding into the cup hole containing the sample to be tested in step (1), incubating at 37deg.C for 20 min, and treating the quality control product;
step (4) and step (5) synchronously detect;
3. sensitivity, specificity, and precision analysis
The four tumor markers are detected in a step and independently, and the sensitivity, specificity and precision samples are measured, wherein the performance of the four tumor markers is as follows:
a analytical sensitivity: performance comparison of simultaneous detection and analytical sensitivity of individual detection, parallel measurement of 10-well zero-value calibrator, calculation of Standard Deviation (SD) and Mean (Mean) of luminescence values, calculation of m=mean+2×sd, and confirmation of analytical sensitivity from the calculated luminescence values.
b precision: the accuracy of the synchronous detection and the accuracy of the independent detection are compared, quality control samples of 10-hole samples are respectively measured, the average concentration (Mean) and Standard Deviation (SD) of the measurement results are calculated, and the coefficient of variation (CV%) =SD/mean×100% is calculated.
c accuracy: and comparing the synchronous detection accuracy with the independent detection accuracy, respectively measuring low-value quality control products and high-value quality control products of all the items, and calculating the relative deviation of the low-value quality control products and the high-value quality control products.
d results
TABLE 1 sensitivity, specificity, precision results analysis
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Conclusion e: through research, each tumor marker is measured after magnetic particles are singly measured and mixed, and the sensitivity, the precision and the accuracy performance of the tumor marker meet the quality requirements.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (5)

1. Use of a kit for simultaneously detecting four tumor markers of lung cancer in the preparation of a product for a method for simultaneously detecting NSE, CYFRA21-1, proGRP, SCC antigens in a sample, the kit comprising:
(1.1) a 1.1 container in which magnetic particles labeled with NSE antibodies are contained; wherein NSE antibody was purchased from Sigma under the trade designation AMAB90556 and protein concentration was 4.3mg/ml;
(1.2) a 1.2 container in which magnetic particles labeled with a CYFRA21-1 antibody are contained; wherein the CYFRA21-1 antibody is purchased from the Phpeng biological Co., ltd, the product number is CY211-McAb1#, and the protein concentration is 5.4mg/ml;
(1.3) a 1.3-th vessel in which magnetic particles labeled with ProGRP antibodies are contained; wherein, proGRP antibody is purchased from Phpeng biological Co., ltd, the product number is GRP-McAb1, and the protein concentration is 4.3mg/ml;
(1.4) a 1.4 th container in which magnetic particles labeled with an SCC antibody are contained; wherein, SCC antibody is purchased from Nanjing Bos gold biotechnology Co., ltd, product number is BS-MA0040, and protein concentration is 2.8mg/ml;
(2.1) a 2.1 container containing an acridinium ester conjugated to an NSE antibody; wherein NSE antibody was purchased from Sigma under the accession number HPA007007 and protein concentration was 2.8mg/ml;
(2.2) a 2.2 container containing an acridine ester coupled to a CYFRA21-1 antibody; wherein CYFRA21-1
The antibodies were purchased from the Fipeng organism Co., ltd, cat# CY211-McAb2# with a protein concentration of 3.7mg/ml;
(2.3) a 2.3-th vessel containing an acridinium ester coupled to a ProGRP antibody; wherein, proGRP antibody is purchased from Phpeng biological Co., ltd, the product number is GRP-McAb2, and the protein concentration is 2.9mg/ml;
(2.4) a 2.4 th vessel containing an acridinium ester coupled to an SCC antibody; wherein, SCC antibody is purchased from Nanjing Bos gold biotechnology Co., ltd, product number is BS-MA0041, and protein concentration is 3.1mg/ml;
(3) A third container containing NSE antigen low value quality control;
(4) A fourth container in which the NSE antigen high-value quality control is contained;
(5) A fifth container containing CYFRA21-1 antigen low value quality control;
(6) A sixth container containing a CYFRA21-1 antigen high value quality control;
(7) A seventh container in which a ProGRP antigen low-value quality control substance is contained;
(8) An eighth container in which the ProGRP antigen high-value quality control material is contained;
(9) A seventh container in which the SCC antigen low value quality control is contained;
(10) An eighth container in which the SCC antigen high value quality control material is contained;
the magnetic particles are active epoxy group-containing magnetic particles with the particle diameter of 2.8 mu m;
the preparation method of the antibody-labeled magnetic particles comprises the following steps:
(1) Washing the magnetic particles with PBS, discarding the supernatant, and suspending in the PBS again to obtain a magnetic particle diluent with the concentration of 5 mg/ml; diluting the corresponding antibody by PBS, adding the diluted antibody into the magnetic particle diluent, and uniformly mixing to obtain a magnetic particle/antibody diluent;
(2) Adding PBS containing 3M ammonium sulfate into the magnetic particle/antibody diluent prepared in the step (1) until the final concentration of the ammonium sulfate in the system is 1M; then carrying out vibration treatment for 16-24 h at 37 ℃ for marking;
(3) After the labeling was completed, the supernatant was discarded, washed with PBS containing 0.1% (w/v) BSA, and then added to PBS containing 0.1% (w/v) BSA and 0.5% (v/v) Tween20 to obtain magnetic microparticles labeled with the corresponding antibodies, wherein the final concentration of the magnetic microparticles was 5mg/ml;
a method for preparing an acridine ester coupled to a corresponding antibody comprising the steps of:
dissolving acridine succinimidyl ester with dimethylformamide to obtain an acridine succinimidyl ester diluent; diluting the antibody with PBS to obtain an antibody diluent; mixing the acridine succinimidyl ester diluent with the antibody diluent, stirring and reacting for 30min, then adding lysine for blocking, and finally dialyzing to obtain acridine ester coupled with the corresponding antibody;
the method for simultaneously detecting NSE, CYFRA21-1, proGRP and SCC antigens in the sample sequentially comprises the following steps:
(1) Respectively adding a sample to be detected, NSE, CYFRA21-1, proGRP, SCC antigen low-value and high-value quality control products into the corresponding cup holes; wherein, the sample to be measured is provided with four treatments in parallel, and each quality control product is also provided with 4 treatments in parallel;
(2) Diluting the antibody-coupled acridine ester in the 2.1 th container, the 2.2 nd container, the 2.3 nd container and the 2.4 th container, respectively adding the diluted acridine ester into the four cup holes containing the sample to be tested in the step (1), and treating the quality control products in the same way;
(3) Mixing the magnetic particles in the 1.1 st container, the 1.2 nd container, the 1.3 st container and the 1.4 th container, adding the mixture into four cup holes containing the sample to be tested in the step (1), incubating at 37 ℃ for 20min, and treating all quality control products in the same way;
(4) Applying magnetic force to attract the magnetic particles, discarding the liquid in the holes and washing;
(5) Simultaneously dripping a first excitation liquid and a second excitation liquid into each hole, and detecting the relative light-emitting units of each hole by using a flash light-emitting instrument;
(6) And respectively calibrating the low-value quality control products and the high-value quality control products for measuring the four tumor markers, and quantifying the sample to be measured according to the concentration values obtained by calibration.
2. The use according to claim 1, characterized in that: the amount of the antibody in the step (1) is 20-30 mug of the antibody added to 1mg of the magnetic particles.
3. The use according to claim 1, characterized in that: the acridine ester is acridine succinimide ester.
4. The use according to claim 1, characterized in that: after the acridine succinimidyl ester diluent and the antibody diluent are mixed, the mass ratio of the acridine succinimidyl ester to the antibody is 1:100.
5. The use according to claim 1, characterized in that: the amino acid sequences of the NSE antigen, the CYFRA21-1 antigen, the ProGRP antigen and the SCC antigen are shown in SEQ ID NO:1 to 4.
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