A kit for detecting squamous cell carcinoma antigen

Abstract

The present invention discloses a kit for detecting squamous cell carcinoma antigens, the kit comprising reagent R1, reagent R2, calibrators and quality control products; the reagent R1 comprises: antibody-coated magnetic particles, buffer, electrolyte, surfactant, stabilizer, preservative; the reagent R2 comprises: acridinium ester compound or alkaline phosphatase labeled antibody, buffer, electrolyte, surfactant, stabilizer, preservative. The kit provided by the present invention has a wide linear range of 0.2ng / mL to 70ng / mL, within the linear range, the kit correlation coefficient r≥0.99, and has high precision and stability.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescent immunoassay, and in particular to a kit for detecting squamous cell carcinoma antigens. Background Art

[0002] Squamous cell carcinoma antigen (SCCA) is increasingly used in clinical oncology. SCCA can be used as a biomarker for non-small cell lung cancer, mainly for squamous cell carcinoma. Compared with other lung cancers, SCCA is closely related to smoking history. In addition, the most common tissue type of cervical cancer is SCC, and it has great value in predicting prognosis, monitoring, and identifying whether patients are at high risk of lymph node metastasis of squamous cell cervical cancer before treatment. SCCA in squamous cell carcinomas of other parts of the body have been studied, including the head and neck, esophagus, and skin. In general, the later the tumor stage, the higher the SCCA level. Continuous detection of SCCA can monitor disease recurrence, residual cancer tissue after treatment, and patient response to treatment.

[0003] At present, the kits for detecting squamous cell carcinoma antigens on the market are mainly electrochemiluminescence and chemiluminescent microparticle immunoassays. Both methods have high sensitivity, accuracy and specificity, but the reagents of imported manufacturers such as Roche and Abbott still occupy a large share in the market. In order to replace expensive imported reagents and reduce the medical burden, more and more domestic reagents have begun to be developed and flooded into the market.

[0004] CN201711288526.4 discloses a kit for determining the content of squamous cell carcinoma antigen and its test method, the kit includes a calibrator, a quality control product, an anti-reagent, a magnetic particle reagent, and a luminescent substrate; the anti-reagent is a squamous cell carcinoma antigen-coated antibody labeled with fluorescein isothiocyanate and a squamous cell carcinoma antigen-labeled antibody labeled with alkaline phosphatase; the magnetic particle reagent is a magnetic particle and a sheep anti-FITC linker. The kit disclosed in the patent uses a chemiluminescent enzyme-linked immunosorbent assay method in conjunction with a chemiluminescent measuring instrument to determine the SCC content in human serum, although it has a strong specificity. However, the kit disclosed in the patent has a low binding rate between magnetic beads and antibodies, and the precision and stability of the test need to be improved. In addition, when using the kit for sample detection, the operation is relatively cumbersome and requires manual sample addition, which greatly affects the accuracy of the test results.

[0005] CN202110216123.9 discloses a squamous cell carcinoma antigen detection kit, which includes a first reagent, a second reagent and a calibrator: the first reagent includes a glycine buffer, a dispersant, a coagulant, a surfactant, a preservative, BSA and a blocking agent; the second reagent includes a Hepes buffer, SCCA antibody-coated latex particles, trehalose, BSA, and a preservative. The kit disclosed in this patent has good accuracy, good anti-interference performance, high precision, linearity and prozone effect to meet clinical needs, and can be used on an automatic biochemical analyzer, but the detection principle of the kit is latex immunoturbidimetric detection, which has a poor sensitivity compared to the chemiluminescence detection method. Summary of the invention

[0006] The purpose of the present invention is to provide a kit for detecting squamous cell carcinoma antigens, so as to solve the problems of the current SCC kits, such as cumbersome operation, poor detection accuracy and sensitivity, and easy aggregation of magnetic beads during cross-linking with antibodies, resulting in poor precision.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A kit for detecting squamous cell carcinoma antigens, the kit comprising reagent R1, reagent R2, a calibrator and a quality control product;

[0009] The reagent R1 includes: antibody-coated magnetic particles, buffer, electrolytes, surfactants, stabilizers, and preservatives;

[0010] The reagent R2 includes: an acridinium ester compound or an alkaline phosphatase labeled antibody, a buffer, an electrolyte, a surfactant, a stabilizer, and a preservative.

[0011] Furthermore, the kit further comprises a reagent R3, wherein the reagent R3 comprises a buffer, an electrolyte, a surfactant, a stabilizer, and a preservative;

[0012] Preferably, reagent R3 comprises 100 mM PBS, 150 mM NaCl, 1.0% BSA, 0.05% Tween 20, 0.05% Proclin 300, and has a pH of 7.0-7.4.

[0013] Furthermore, the preparation method of the reagent R1 is as follows:

[0014] The antibody-coated magnetic particles are diluted with a magnetic particle diluent to obtain a reagent R1;

[0015] The magnetic particle diluent includes 20-100mM PBS, 10-200mM NaCl, 0.1-8% BSA, casein, one of γ-globulin, 0.05-2% Tween 20, Tween 80, Triton X405, one or two of surfactant S9, 0.05%-1% Proclin 300, Proclin 950, one or two of sodium azide, and the pH is 7.0-7.4. The capture antibody used in the present invention is suitable for storage in the magnetic particle diluent and the pH range. The ion strength contained in the magnetic particle diluent not only does not affect the affinity between the antibody and the antigen, but also allows the accuracy, precision and other performances to be optimized. In addition, the surfactant in the magnetic particle diluent can make the reagent more uniform and improve the detection precision; and the protein and preservative play a key role in the stability of the reagent. All substances in the magnetic particle diluent play their respective roles. When combined with the most suitable concentration, it can not only keep the antibody stable and active, but also facilitate the antibody to bind to the antigen, so that the reagent can play the best performance.

[0016] Furthermore, the preparation method of the antibody-coated magnetic particles is as follows:

[0017] After the magnetic microparticles are resuspended in a cross-linking buffer and activated with an activator, a cross-linking incubation solution containing a squamous cell carcinoma antigen capture antibody is added, and the suspension is reacted at 20 to 37° C. for 2 to 24 hours. After washing, a blocking agent is added, and the suspension is reacted at 20 to 37° C. for 6 to 24 hours for blocking. After washing with a washing solution, the supernatant is removed to obtain antibody-coated magnetic microparticles.

[0018] The cross-linking suspension reaction of magnetic particles and squamous cell carcinoma antigen capture antibody can achieve the same effect as 3 hours by suspending for 6 hours at room temperature (25°C); the blocking reaction in the antibody-coated magnetic beads experiment can achieve the blocking effect by suspending for 9 hours at room temperature (25°C), but extending the time to 18 hours or even longer will achieve better blocking effect, lower local noise and more stable reagents;

[0019] Furthermore, the mass ratio of the magnetic particles to the antibodies containing squamous cell carcinoma antigen capture is 1:(0.05-0.2); preferably, the mass ratio of the magnetic particles to the antibodies containing squamous cell carcinoma antigen capture is 1:(0.06-0.12); more preferably, the mass ratio of the magnetic particles to the antibodies containing squamous cell carcinoma antigen capture is 1:0.08.

[0020] Usually, the linear low-end sensitivity of squamous cell carcinoma antigen reaction is poor. If a suitable coupling process between antibodies and magnetic particles is explored, the steric hindrance between antibodies is reduced, and a certain distance between antibodies coated on magnetic particles is allowed to tightly grasp the antigen, and the antibodies that have been cross-linked on magnetic particles can be protected from falling off, the binding rate and uniformity with antigens can be effectively improved, solving the problem of poor low-end sensitivity. Too high a mass ratio of antibodies will cause overcrowding of antibodies on the surface of magnetic beads, and more antibodies will be non-specifically adsorbed on the surface of magnetic beads, resulting in uneven background measurement and a higher blank limit, poor low-end sensitivity, and will affect the precision and stability of the reagents; too low a mass ratio of antibodies will cause too much space on the surface of magnetic beads, and antibodies on the surface of magnetic beads will be adsorbed on the surfaces of other magnetic beads, causing the magnetic beads to agglomerate with each other, reducing the precision of the measurement, and too few antibodies cannot identify excessive antigens, affecting the linearity of the reagents.

[0021] Furthermore, the magnetic particles are carboxyl magnetic beads, the squamous cell carcinoma antigen is a human squamous cell carcinoma antigen, and the squamous cell carcinoma antigen capture antibody is a squamous cell carcinoma antigen monoclonal antibody;

[0022] Preferably, the particle size of the carboxyl magnetic beads is 1.5 μm-3 μm.

[0023] Furthermore, the activator is EDC, and the final concentration of the EDC added during the activation process is 0.5-5 mg / ml; the final concentration of the magnetic particle activator EDC is added to 0.5 mg / mL to achieve the effect of activating the carboxyl group, but increasing its concentration to 1 mg / mL has a better activation effect and a higher efficiency of coupling antibodies.

[0024] The cross-linking buffer comprises 20-100 mM MES, with a pH of 5.0-6.0;

[0025] The cross-linked incubation solution includes 20-100 mM MES, 0.5-10% BSA, casein, one of fish skin gelatin and pig skin gelatin, 0.05-0.2% Tween 20, Tween 80 and one of surfactant S17, and the pH is 5.0-6.0; SCC antibody is very prone to magnetic bead aggregation during the coupling process with magnetic beads, resulting in poor reagent precision and stability. Adding protein to the cross-linked incubation solution can protect the coupling structure of the antibody and magnetic beads; and adding surfactant can effectively prevent nonspecific adsorption between the antibody and the magnetic beads, and the surfactant can disperse the magnetic beads well to prevent them from agglomerating. Therefore, adding protein and surfactant to the cross-linked incubation solution can effectively improve the reagent precision and stability, and improve sensitivity.

[0026] The blocking agent includes 10-50mM Tris, 100-200mM NaCl, 0.05-0.2% Tween 20 or Tween 80, 0.5-10% BSA, casein, gamma globulin, glycine, and one of lysine, and the pH is 7.2-7.4; 2% BSA can be used as the protein in the blocking agent, but adding some small molecule amino acids (glycine) has a better effect, that is, 2% BSA+2% glycine, which not only has lower local noise, but also has better dispersion of cross-linked magnetic beads and lower detection imprecision.

[0027] The washing solution comprises 10-50 mM Tris, 100-200 mM NaCl, 0.05-0.2% Tween 20 or Tween 80, and the pH value is 7.2-7.4.

[0028] Furthermore, the preparation method of the reagent R2 is as follows:

[0029] The acridinium ester compound or alkaline phosphatase labeled antibody is diluted with a labeling diluent to obtain reagent R2;

[0030] The labeling diluent includes 50 mM MES, 150 mM NaCl, 0.5% BSA, 0.1% Proclin 300, and has a pH of 6.0-6.5.

[0031] Furthermore, the preparation method of the acridinium ester compound labeled antibody comprises: suspending the acridinium ester compound and the squamous cell carcinoma antigen detection antibody for reaction, removing the uncross-linked acridinium ester compound after the reaction is completed, and obtaining the acridinium ester compound labeled antibody;

[0032] The preparation method of the alkaline phosphatase labeled antibody comprises the following steps: mixing alkaline phosphatase and squamous cell carcinoma antigen detection antibody and then dialyzing, adding glutaraldehyde to react at room temperature during the dialysis process, continuing the dialysis, and diluting to a certain concentration after the dialysis is completed to obtain the alkaline phosphatase labeled antibody.

[0033] Furthermore, the calibrator and quality control product include human squamous cell carcinoma antigen, buffer, electrolyte, surfactant, stabilizer, preservative, mannitol and sugar;

[0034] The preparation method of the calibrator comprises: diluting the squamous cell carcinoma antigen with an antigen diluent to obtain the calibrator;

[0035] The preparation method of the quality control product comprises: diluting the squamous cell carcinoma antigen with an antigen diluent to obtain the quality control product;

[0036] The antigen diluent comprises 10-20mM PBS, one of 3-15mM EDTA or EDTA-2Na, one of 2-10% trehalose or sucrose, 2-10% mannitol, 10-200mM NaCl, one or two of 0.5-10% BSA, casein, gamma globulin, pig skin gelatin, fish skin gelatin, 0.05-2% Tween 20, Tween 80, Triton X405, one or two of surfactant S9, 0.05%-1% Proclin 300, Proclin 950, one or two of sodium azide, and pH is 7.0-7.5. Trehalose in the antigen diluent can also be replaced by sucrose. The calibration products and quality control products used in the present invention are all prepared with artificial matrix and contain antigen components derived from human cell lines. The buffer matrix PBS selected in this formula has similar properties to the serum matrix. Generally, proteases need the catalysis of metal ions to play a role in decomposing proteins, and EDTA can chelate with metal ions to inhibit protease decomposition. The phosphate buffer matrix and EDTA we selected can both play an antioxidant role, because both phosphate and EDTA can bind to metal ions. Common protein stabilizers include BSA, casein, γ-globulin, gelatin, etc. These substances can stabilize the structure of antigens without interfering with the determination. Sugars are also added as common protein stabilizing additives. Mannitol contains a polyhydroxy structure, which can reduce the oxidized part of the protein, and it plays an important role in the stability of squamous cell carcinoma antigens. Surfactants and preservatives have a certain auxiliary effect on protein stability. These substances can be reasonably matched to enable squamous cell carcinoma antigens to be stored at 2-8°C for more than one year. At the same time, they have good thermal stability and can be placed at 37°C for at least 7 days. They do not affect the affinity between antibodies and antigens and can be used as recovery standards and linear substances.

[0037] The beneficial effects of the present invention are:

[0038] 1. The kit for detecting squamous cell carcinoma antigen provided by the present invention, by determining the appropriate mass ratio of antibody to magnetic particles, avoids the problem that too high antibody mass ratio will make the antibodies on the surface of magnetic beads overcrowded, resulting in uneven background measurement values, high blank limit, and poor low-end sensitivity, and improves the sensitivity, so that the blank limit is not higher than 0.02ng / mL, the detection limit is not higher than 0.2ng / mL, and has a wide linear range of 0.2ng / mL to 70ng / mL. Within the linear range, the correlation coefficient of the kit r≥0.99.

[0039] 2. The kit for detecting squamous cell carcinoma antigen provided by the present invention can avoid the problem of SCC antibodies in the conventional cross-linking process being very easy to agglomerate magnetic beads during the cross-linking process of magnetic beads by using the cross-linking incubation solution of the present invention, and at the same time protect the coupling structure of the antibody and the magnetic beads, effectively prevent the non-specific adsorption between the antibody and the magnetic beads, and effectively improve the precision and stability of the reagent, with good precision, the intra-batch precision is not higher than 5%, and the inter-batch precision is not higher than 10%; the stability is strong, and the kit can be stored at 37°C for at least 7 days without affecting its performance, and can be stored at 2-8°C for at least one year.

[0040] 3. The kit for detecting squamous cell carcinoma antigen provided by the present invention has no effect on the test results when the potential interfering substances bilirubin ≤20mg / dL, hemoglobin ≤500mg / dL, triglyceride ≤3000mg / dL, and total protein ≤12g / dL. The specificity of this project was evaluated using samples containing other potential interfering substances (such as high RF800 and HAMA values), and the test recovery rate was between 85% and 115%, with no significant crossover.

[0041] 4. The kit for detecting squamous cell carcinoma antigens provided by the present invention has high accuracy. Using a recovery experiment, the recovery rate R is between 85% and 115%.

[0042] 5. The kit for detecting squamous cell carcinoma antigens provided by the present invention has a high cost-effectiveness. Compared with imported kits, the cost of the kit is low, and its performance and clinical use value are no less than those of imported kits. Moreover, the kit of the present invention can achieve good performance indicators on different detection instruments. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is a schematic diagram of the test results of the correlation between Example 1 of the present invention and the serum samples of the comparison kit. DETAILED DESCRIPTION

[0044] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for the terms in this document apply to the terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art would give them should be given based on the disclosure and context.

[0045] The reagents and equipment used in the present invention are all known products or obtained by purchasing commercially available products.

[0046] The present invention adopts a chemiluminescent immunoassay double antibody sandwich two-step method to develop a kit for quantitative detection of SCC. The sample and the magnetic microspheres cross-linked with anti-human squamous cell carcinoma antigen capture antibody are mixed (reagent R1), and the SCC in the sample is bound to the magnetic microspheres. After washing, the anti-human squamous cell carcinoma antigen detection antibody labeled with acridinium ester (reagent R2) is added to form a "capture antibody cross-linked magnetic microsphere-antigen-acridinium ester labeled detection antibody" complex. After washing again, an excitation liquid is added to the reaction mixture to excite the chemiluminescent reaction. The concentration of squamous cell carcinoma antigen in the sample is proportional to the relative luminescence value (RLU) detected by the system, and the concentration of squamous cell carcinoma antigen can be calculated through the calibration curve.

[0047] The present invention aims at the deficiencies of the prior art, overcomes the high background of SCC and the poor sensitivity of the low end of the linear antigen reaction, and explores a suitable coupling process of magnetic beads and antibodies by using a high-stability SCC calibration solution in combination, which can not only ensure that the antibodies coated on the magnetic beads are sufficient, but also minimize the steric hindrance between the antibodies on the surface of the magnetic beads, so that low-concentration squamous cell carcinoma antigens can be recognized and captured to the maximum extent, while high-concentration squamous cell carcinoma antigens can overcome the high-dose hook effect and expand the linear range. At the same time, a magnetic particle working solution is provided, which can provide favorable conditions for antibody recognition of antigens and improve antibody stability. And the invention can be applied to enzymatic chemiluminescence and electrochemiluminescence at the same time.

[0048] In order to more clearly illustrate the technical solution of the present invention, the specific implementation methods are described as follows:

[0049] Example 1

[0050] The kit of this embodiment includes reagent R1, reagent R2, calibrators and quality control products.

[0051] Reagent R1: Dilute the antibody coated on carboxyl magnetic beads (3 μm) to 0.8 mg / mL (based on magnetic particle concentration) with magnetic particle diluent (100 mM PBS, 150 mM NaCl, 1.0% BSA, 0.05% Tween 20, 0.05% Proclin 300, 0.09% sodium azide, pH 7.0-7.4) to obtain reagent R1.

[0052] The mass ratio of the magnetic particles to the antibodies is 1:0.08.

[0053] The specific preparation method of the antibody coated on the carboxyl magnetic beads is as follows:

[0054] (1) Take 10 mg (100 μL of fully suspended 100 mg / mL carboxyl magnetic beads) of carboxyl magnetic beads in a 2 mL centrifuge tube, remove the supernatant, wash three times with 1 mL of cross-linking buffer (100 mM MES, pH = 5.0), remove the supernatant, and finally resuspend with 1 mL of cross-linking buffer.

[0055] (2) Add EDC activator to a final concentration of 1 mg / mL and suspend and activate at 25°C for 30 minutes.

[0056] (3) Add 80 μg of squamous cell carcinoma antigen capture antibody, the cross-linking incubation solution is 100 mM MES, 0.5% porcine gelatin, 0.1% surfactant S17, pH 5.0, and the suspension reaction is carried out at 25° C. for 3 hours. After the cross-linking is completed, the supernatant is removed.

[0057] (4) Add 1 mL of blocking agent (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, 2% BSA, 2% glycine, pH 7.2-7.4), suspend and react at 25°C for 24 h for blocking. After blocking, remove the supernatant and wash three times with washing solution (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.2-7.4).

[0058] (5) Finally, add 1 mL of magnetic particle diluent to resuspend and obtain the antibody solution coated on the carboxyl magnetic beads. Then add 11.5 mL of magnetic particle diluent to dilute the antibody solution to 0.8 mg / mL (in terms of magnetic particle concentration) to obtain reagent R1, which is stored at 2-8°C.

[0059] Reagent R2: dilute the acridinium ester-labeled antibody to 0.1 ug / mL with labeling diluent (50 mM MES, 150 mM NaCl, 0.5% BSA, 0.1% Proclin 300, pH 6.0-6.5) to obtain reagent R2.

[0060] The preparation method of acridinium ester labeled antibody is as follows:

[0061] (1) Take 100 μg of squamous cell carcinoma antigen detection antibody, add 4 μg of acridinium ester, and suspend and react at 25°C for 20 minutes.

[0062] (2) Use an ultrafiltration tube or dialysis bag to remove the uncross-linked acridinium ester and retain only the acridinium ester-labeled antibody complex as much as possible.

[0063] (3) Dilute the labeled antibody complex to 0.1 ug / mL with labeling diluent to obtain reagent R2, which is stored at 2-8°C.

[0064] Calibrator: Squamous cell carcinoma antigen was prepared with antigen diluent (20mM PBS, 5mM EDTA, 1% trehalose, 5% mannitol, 2% BSA, 0.5% casein, 0.05% Tween 20, 0.05% Proclin 300, 0.05% Proclin 950, pH 7.0-7.5) to 0ng / mL (calibrator A), 1ng / mL (calibrator B), 5ng / mL (calibrator C), 20ng / mL (calibrator D), 40ng / mL (calibrator E) and 70ng / mL (calibrator F). It has been traced back to Abbott calibrators and the measured values ​​are consistent with those of Abbott calibrators.

[0065] Quality control: The squamous cell carcinoma antigen was prepared into 2 ng / mL (low value quality control) and 50 ng / mL (high value quality control) using antigen diluent (20 mM PBS, 5 mM EDTA, 1% trehalose, 5% mannitol, 2% BSA, 0.5% casein, 0.05% Tween 20, 0.05% Proclin 300, 0.05% Proclin 950, pH 7.0-7.5).

[0066] Embodiment 2:

[0067] The kit of this embodiment includes reagent R1, reagent R2, calibrator and quality control product. This embodiment is tested on a Beckman instrument. The reagent R1, calibrator and quality control product scheme of this embodiment is the same as that of Example 1.

[0068] Reagent R2: dilute the alkaline phosphatase labeled antibody to 0.1 ug / mL with labeling diluent (50 mM MES, 150 mM NaCl, 0.5% BSA, 0.1% Proclin 300, pH 6.0-6.5) to obtain reagent R2

[0069] Preparation of enzyme-labeled antibodies:

[0070] (1) Take 100 μg of squamous cell carcinoma antigen detection antibody, add 1 mg of alkaline phosphatase, and dialyze with 10Mm PBS at pH 7.2 for 18 hours, changing the solution three times during the process.

[0071] (2) Add 20 μl of 2.5% glutaraldehyde, react at room temperature for 2 hours, and dialyze overnight with 10 μm PBS (pH 7.2).

[0072] (3) The cell was dialyzed with 50 mM Tris buffer (pH 8.0) for 12 hours, during which the buffer was changed three times.

[0073] (4) Take out the labeled antibody and dilute it to 0.1 mg / mL with buffer (50 mM Tris, 1% BSA, pH 8.0).

[0074] Embodiment 3:

[0075] The kit of this embodiment includes reagent R1, reagent R2, reagent R3, calibrator and quality control product. This embodiment is tested on Roche instrument. The scheme of reagent R1, reagent R2, calibrator and quality control product of this embodiment is the same as that of embodiment 1.

[0076] Reagent R3 is an auxiliary reagent, and its components include 100 mM PBS, 150 mM NaCl, 1.0% BSA, 0.05% Tween 20, 0.05% Proclin 300, 0.09% sodium azide, and pH is 7.0-7.4.

[0077] Comparative Example 1:

[0078] The difference between the kit of comparative example 1 and example 1 is that the mass ratio of magnetic particles to antibodies is 1:0.05, and the rest is the same as example 1.

[0079] Comparative Example 2:

[0080] The kit of comparative example 2

[0081] The difference from Example 1 is that the mass ratio of magnetic particles to antibodies is 1:0.15, and the rest is the same as Example 1.

[0082] Comparative Example 3:

[0083] The difference between the kit of comparative example 3 and example 1 is that there is no pigskin gelatin in the cross-linking hatching solution, and the other aspects are the same as those of example 1.

[0084] Comparative Example 4:

[0085] The difference between the kit of comparative example 4 and example 1 is that there is no surfactant S17 in the cross-linking incubation solution, and the rest is the same as example 1.

[0086] Comparative Example 5:

[0087] The difference between the kit of Comparative Example 5 and Example 1 is that the 100 mM PBS in the magnetic particle diluent is changed to 100 mM Tris, and the rest is the same as Example 1.

[0088] Comparative Example 6:

[0089] The difference between the kit of Comparative Example 6 and Example 1 is that there is no Tween 20 in the magnetic particle diluent, and the other parts are the same as Example 1.

[0090] Comparative Example 7:

[0091] The difference between the kit of Comparative Example 7 and Example 1 is that the pH of the magnetic particle diluent is 6.5, and the rest is the same as Example 1.

[0092] Comparative Example 8:

[0093] The difference between the kit of Comparative Example 8 and Example 1 is that the pH of the magnetic particle diluent is 8.0, and the rest is the same as Example 1.

[0094] Comparative Example 9:

[0095] The difference between the kit of comparative example 9 and example 1 is that there is no mannitol in the antigen diluent, and the rest is the same as example 1.

[0096] The reagents prepared in Example 1 and Comparative Examples 1 to 9 were tested on a fully automatic chemiluminescence immunoassay analyzer, and the sensitivity, precision, linear range, serum recovery, uniformity and stability of calibrators and quality control products were examined for comprehensive evaluation.

[0097] 1. Sensitivity: Mainly investigate the blank limit and detection limit. Use Example 1 and Comparative Examples 1 to 7 to test the calibration substance A 20 times at the same time, obtain the RLU value of the 20 measurement results, and calculate the average value ( ) and standard deviation (SD), we get Will The corresponding RLU value is substituted into the linear equation obtained by two-point regression fitting of the concentration-RLU value of calibrator A and the adjacent low-concentration calibrator B to obtain the corresponding concentration value, which is the blank limit (LOB). The results are shown in Table 1. Then, 5 groups of low-analyte samples were tested 5 times each using Example 1 and Comparative Examples 1 to 9. The lowest concentration of detectable analytes measured at a probability of 95% is the detection limit (LoD). The results are shown in Table 2.

[0098] Table 1 Blank limit data of Example 1 and Comparative Examples 1 to 9

[0099]

[0100] As can be seen from the data in Table 1, the detection data CV of Example 1 is the smallest, more uniform, with a blank limit of 0.01ng / mL, and a higher detection sensitivity. The blank limits of Comparative Examples 1 and 2 are relatively high, indicating that the mass ratio of magnetic particles to antibodies is too low or too high, which will affect the detection blank limit. Too high an antibody mass ratio will cause the surface antibodies of the magnetic beads to be overcrowded, and antibodies that rely on surface adsorption rather than covalent coupling account for a large proportion, resulting in uneven background measurements and a higher blank limit, and poor low-end sensitivity; too low an antibody mass ratio will cause the surface space of the magnetic beads to be too large, and the surface antibodies of the magnetic beads will be adsorbed on the surfaces of other magnetic beads, causing the magnetic beads to agglomerate with each other, which will also cause uneven background measurements and a higher blank limit, and insufficient antibody will also cause the detection value to be low. The blank limits of Comparative Examples 3 and 4 are relatively high, indicating that the pigskin gelatin and surfactant S17 in the incubation process will have a certain impact on the sensitivity of reagent detection.

[0101] Table 2 Detection limit data of Example 1 and Comparative Examples 1 to 7

[0102]

[0103] From the data in Table 2, it can be seen that the detection limit of Example 1 is closer to 0.2 ng / mL. The detection limits of Comparative Examples 1 and 2 are relatively high, indicating that a too low or too high mass ratio of magnetic particles to antibodies will also significantly affect the detection limit.

[0104] From the data in Table 1 and Table 2, it can be seen that the blank limit of Example 1 is no higher than 0.02 ng / mL, and the detection limit reaches 0.2 ng / mL. The mass ratio of magnetic particles to antibodies will significantly affect the detection sensitivity, and the addition of protein to the cross-linked incubation solution will also affect the detection sensitivity to a certain extent. The mass ratio of magnetic particles to antibodies in Example 1 is 1:0.08, and the pH of the magnetic particle diluent is 7.0-7.4, which are both relatively suitable.

[0105] 2. Precision: In two time periods of one day, two concentration levels of quality control products and 0.2 ng / mL to 70 ng / mL serum samples were tested simultaneously using Example 1 and Comparative Examples 1 to 7, and the total CV of imprecision of the test concentration results was calculated. The results are shown in Table 3.

[0106] Table 3 Precision data of Example 1 and Comparative Examples 1 to 7

[0107]

[0108]

[0109] As can be seen from the data in Table 3, Example 1 has good precision, small CV, and accurate measurement value. Comparative Examples 1 and 2 have the worst precision and high CV, indicating that too low or too high mass ratio of magnetic particles to antibodies will affect the detection precision. Comparative Examples 3 and 4 are the second, indicating that adding pigskin gelatin and surfactant S17 to the cross-linked incubation solution is beneficial to the cross-linking of antibodies and magnetic particles, and improves the precision of the reagent.

[0110] 3. Linear range: The high-value serum samples of Example 1 and Comparative Examples 1 to 7 were simultaneously tested with samples gradiently diluted with zero-concentration serum, and the linear correlation coefficient was calculated. The results are shown in Table 4.

[0111] Table 4 Linear range data of Example 1 and Comparative Examples 1 to 7

[0112]

[0113] As can be seen from the data in Table 4, Example 1 has the best linearity. Comparative Example 1 is the worst, indicating that a too low antibody mass ratio will make the surface space of the magnetic beads too large, and the surface antibodies of the magnetic beads will be adsorbed on the surface of other magnetic beads, causing the magnetic beads to agglomerate with each other. The sites for recognizing antigens on the antibodies may be blocked by agglomeration, resulting in a low recognition rate and low sensitivity for low-concentration samples; at the same time, because the amount of antibodies is too small and cannot recognize excessive antigens, the measured value of high-concentration samples will be low, resulting in the linearity of the high-value segment becoming flat. In addition, the linearity of Comparative Examples 7 and 8 is relatively poor, indicating that the pH value of the magnetic particle diluent is too low or too high, which will affect the linear range of the kit.

[0114] 4. Recovery: Add 0.1 mL of standard solution to 0.9 mL of serum sample, and test the serum sample, standard solution, and analytical sample after adding standard solution to the serum sample three times using Example 1 and Comparative Examples 1 to 7, and take the average value. Calculate the recovery rate according to formula (1).

[0115]

[0116] Where: R-recovery rate; V-volume of standard solution added; V0-volume of human sample; C-detection concentration of human sample after adding standard solution; C0-detection concentration of human sample; Cs-concentration of standard solution. The results are shown in Table 5.

[0117] Table 5 Recovery data of Example 1 and Comparative Examples 1 to 7

[0118]

[0119] As can be seen from the data in Table 5, the recovery rate of Example 1 is more uniform, all close to 100%. The recovery rates of Comparative Example 1 are all on the high side, which may be related to the linear shape ("S-type") of Comparative Example 1. The recovery rates of Comparative Examples 5, 7 and 8 are also on the high side, indicating that the buffer components and pH value of the magnetic particle dilution solution will also affect the reagent recovery.

[0120] 5. Stability of calibration products and quality control products: 6 sets of calibration products and quality control products were extracted from Example 1 and Comparative Example 9, placed at 2-8°C, and 2 sets of test concentrations were extracted from each batch at 0, 12, and 15 months, respectively, and the concentration deviation from 0 months was calculated. The results are shown in Table 6. 4 sets of calibration products and quality control products were extracted from Example 1 and Comparative Example 9, respectively, placed at 37°C, and 2 sets of test concentrations were extracted from each batch at 0 and 7 days, respectively, and the concentration deviation from 0 days was calculated. The results are shown in Table 7

[0121] Table 6 2-8°C stability data of the calibrators and quality control products of Example 1 and Comparative Example 8

[0122]

[0123]

[0124] Table 7 37°C stability data of the calibrators and quality control products of Example 1 and Comparative Example 9

[0125]

[0126] It can be seen from the data in Table 6 that the antigen diluent in Example 1 allows the calibrators and quality control products to be stored at 2-8°C for at least one year, while the antigen diluent in Comparative Example 8 lacks mannitol, causing the calibrators and quality control products to drop by 20%-30% after being stored at 2-8°C for one year.

[0127] It can be seen from the data in Table 7 that the antigen diluent in Example 1 allows the calibrators and quality controls to be stored at 37°C for at least 7 days, while the antigen diluent in Comparative Example 8 lacks mannitol, causing the calibrators and quality controls to drop by about 30% after being stored at 37°C for 7 days.

[0128] 6. Specificity: No cross-reaction was observed when AFP (800 ng / mL), CEA (600 ng / mL), CA 125 (1200 U / mL), CA15-3 (800 U / mL), CA 19-9 (500 U / mL), and NSE (300 ng / mL) were tested using Example 1. The results are shown in Table 8. Potential interfering substances (bilirubin, hemoglobin, triglycerides, and total protein) were added to the serum samples respectively. The volume of the added potential interfering substances should not cause changes in the matrix. Physiological saline was used as a control. The concentrations of the above samples were tested using Example 1, and the concentration deviation from the control was calculated. The results are shown in Table 9. Samples containing other potential interfering substances (RF800, high HAMA values) were added with antigens at different concentrations, and a recovery test was performed using Example 1 to calculate the recovery rate. The results are shown in Table 10.

[0129] Table 8 Cross-reactivity data of the kit of Example 1 with other tumor markers

[0130] Tumor markers concentration Test concentration (ng / mL) AFP 800ng / mL 0.0 CEA 600ng / mL 0.0 CA 125 1200U / mL 0.0 CA 15-3 800U / mL 0.0 CA 19-9 500U / mL 0.0 NSE 300ng / mL 0.0

[0131] Table 9 Interference reaction data of the kit and potential interferents in Example 1

[0132]

[0133] It can be seen from the data in Table 9 that when the potential interfering substances bilirubin ≤20 mg / dL, hemoglobin ≤500 mg / dL, triglyceride ≤3000 mg / dL, and total protein ≤12 g / dL, there is no effect on the test results of Example 1.

[0134] Table 10 Interference reaction data of the kit in Example 1 and other potentially interfering clinical situations

[0135]

[0136] From the data in Table 10, it can be seen that in Example 1, samples containing other potential interferents (such as high RF800 and HAMA values) were used to evaluate the specificity of this project, and the test recovery rate was between 95% and 105%, with no significant crossover.

[0137] 7. Serum sample correlation: A total of 100 samples with different concentrations and covering the linear range were tested using Example 1, and compared with Abbott's squamous cell carcinoma antigen quantitative determination kit (chemiluminescent microparticle immunoassay) as a comparison kit. The linear regression equation was: Y = 0.9932x + 0.167, and the determination coefficient of linear regression was r2 = 0.9972. The results are as follows Figure 1 shown.

[0138] 8. Reagent stability: The kit of Example 1 was placed at 2-8°C, and 2 sets were extracted at 0, 12, and 15 months, respectively, and calibrated, and the detection limit, precision, and serum samples were tested, and compared with those at 0 months. The results are shown in Table 11. The kit of Example 1 was placed at 37°C, and 2 sets were extracted after 7 days, and the detection limit, precision, and serum samples were tested, and compared with the kit at 2-8°C. The results are shown in Table 12.

[0139] Table 11 Stability data of the test kit of Example 1 at 2-8°C

[0140]

[0141]

[0142] It can be seen from the data in Table 11 that the kit of Example 1 is stable at 2-8°C for at least 1 year without affecting its sensitivity, precision and serum sample correlation.

[0143] Table 12 Stability data of the kit of Example 1 at 37°C

[0144]

[0145]

[0146] It can be seen from the data in Table 12 that the kit of Example 1 is stable at 37°C for at least 7 days without affecting its sensitivity, precision and serum sample correlation.

[0147] In order to detect whether Example 2 and Example 3 also have high precision and sensitivity in the detection process, the blank limit data, detection limit data and precision data of Examples 1-3 were specifically tested. The specific detection results of the blank limit data are shown in Table 13 below, the detection results of the detection limit data are shown in Table 14 below, and the detection results of the precision data are shown in Table 15 below.

[0148] Table 13 Blank limit data of Examples 1 to 3

[0149]

[0150] Table 14 Detection limit data of Examples 1 to 3

[0151]

[0152] Table 15 Precision data of Examples 1 to 3

[0153]

[0154]

[0155]

[0156] It can be clearly concluded from the test results of blank limit, detection limit and precision of Examples 1-3 in Tables 13-15 above that the kit prepared in the present application has universal applicability in terms of detection equipment and can be used for different detection equipment respectively.

[0157] In summary, the present invention adopts a chemiluminescent immunoassay double antibody sandwich two-step method to develop a kit for quantitative detection of SCC. A suitable mass ratio of magnetic particles to antibodies is used to improve the sensitivity and accuracy of the reagent and expand the linear range. A cross-linked incubation solution is provided to effectively improve the precision and sensitivity of the reagent. At the same time, a magnetic particle working solution is provided to provide favorable conditions for antibodies to recognize antigens, so that all aspects of the performance of the kit are optimized. By using a highly stable antigen diluent in conjunction, the prepared calibrators and quality control products can be stable for at least 1 year at 2-8°C and at least 7 days at 37°C. In addition, the magnetic bead reagents produced by the invention can be applied to different luminescence platforms.

Claims

1. A kit for detecting squamous cell carcinoma antigens, characterized in that: The kit includes reagent R1, reagent R2, calibrators and quality control products; The reagent R1 includes: antibody-coated magnetic particles, buffer, electrolytes, surfactants, stabilizers, and preservatives; The reagent R2 includes: an acridinium ester compound or an alkaline phosphatase labeled antibody, a buffer, an electrolyte, a surfactant, a stabilizer, and a preservative; The kit further comprises a reagent R3, which comprises a buffer, an electrolyte, a surfactant, a stabilizer, and a preservative; The reagent R3 includes 100 mM PBS, 150 mM NaCl, 1.0% BSA, 0.05% Tween 20, 0.05% Proclin 300, 0.09% sodium azide, and the pH is 7.0-7.4; The preparation method of the reagent R1 is as follows: diluting the antibody-coated magnetic particles with a magnetic particle diluent to obtain the reagent R1; The magnetic particle diluent comprises 20-100 mM PBS, 10-200 mM NaCl, 0.1-8% BSA, casein, or γ-globulin, 0.05-2% Tween 20, Tween 80, Triton X405, or one or two of surfactant S9, and 0.05%-1% Proclin 300, Proclin 950, or one or two of sodium azide, with a pH of 7.0-7.4; The preparation method of the antibody-coated magnetic particles is as follows: After the magnetic particles are resuspended in a cross-linking buffer and activated by an activator, a cross-linking incubation solution containing a squamous cell carcinoma antigen capture antibody is added, the particles are suspended and reacted at 20-37°C for 2-24 hours, a blocking agent is added after washing, the particles are suspended and reacted at 20-37°C for 6-24 hours for blocking, a washing solution is added for washing, and the supernatant is removed to obtain antibody-coated magnetic particles; the cross-linking incubation solution comprises 100 mM MES, 0.5% pig skin gelatin, 0.1% surfactant S17, and a pH of 5.0; The mass ratio of the magnetic particles to the capture antibody containing squamous cell carcinoma antigen is 1: (0.06-0.12); The preparation method of the reagent R2 is as follows: diluting the acridinium ester compound or the alkaline phosphatase labeled antibody with a labeled diluent to obtain the reagent R2; The labeling diluent includes 50mM MES, 150mM NaCl, 0.5% BSA, 0.1% Proclin300, and pH is 6.0-6.5; The method for preparing the acridinium ester compound labeled antibody comprises: suspending the acridinium ester compound and the squamous cell carcinoma antigen detection antibody for reaction, removing the uncross-linked acridinium ester compound after the reaction is completed, and obtaining the acridinium ester compound labeled antibody; The preparation method of the alkaline phosphatase labeled antibody comprises the following steps: mixing alkaline phosphatase and squamous cell carcinoma antigen detection antibody and then dialyzing, adding glutaraldehyde to react at room temperature during the dialysis process, continuing the dialysis, and diluting to a certain concentration after the dialysis is completed to obtain the alkaline phosphatase labeled antibody.

2. The kit according to claim 1, characterized in that The mass ratio of the magnetic particles to the squamous cell carcinoma antigen-capturing antibody is 1:0.

08.

3. The kit according to claim 2, characterized in that The magnetic particles are carboxyl magnetic beads, the squamous cell carcinoma antigen is a human squamous cell carcinoma antigen, and the squamous cell carcinoma antigen capture antibody is a squamous cell carcinoma antigen monoclonal antibody; the particle size of the carboxyl magnetic beads is 1.5 μm-3 μm.

4. The kit according to claim 1, characterized in that The activator is EDC, and the final concentration of the EDC added during the activation process of the activator is 0.5-5 mg / ml; The cross-linking buffer comprises 20-100 mM MES, with a pH of 5.0-6.0; The blocking agent includes 10-50 mM Tris, 100-200 mM NaCl, 0.05-0.2% Tween 20 or Tween 80, 0.5-10% BSA, casein, gamma globulin, glycine, and lysine, and the pH is 7.2-7.4; The washing solution includes 10-50 mM Tris, 100-200 mM NaCl, 0.05-0.2% Tween 20 or Tween 80, and the pH value is 7.2-7.

4.

5. The kit according to claim 1, characterized in that The calibrators and quality control products include human squamous cell carcinoma antigen, buffer, electrolytes, surfactants, stabilizers, preservatives, mannitol and sugars; The preparation method of the calibrator comprises: diluting the squamous cell carcinoma antigen with an antigen diluent to obtain the calibrator; The preparation method of the quality control product comprises: diluting the squamous cell carcinoma antigen with an antigen diluent to obtain the quality control product; The antigen diluent comprises 10-20 mM PBS, one of 3-15 mM EDTA or EDTA-2Na, one of 2-10% trehalose or sucrose, 2-10% mannitol, 10-200 mM NaCl, one or two of 0.5-10% BSA, casein, γ-globulin, pig skin gelatin, and fish skin gelatin, 0.05-2% Tween 20, Tween 80, Triton X405, and surfactant S9, and one or two of 0.05%-1% Proclin 300, Proclin 950, and sodium azide, and the pH is 7.0-7.5.

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