CN110780079A - Squamous cell carcinoma antigen detection reagent - Google Patents
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- CN110780079A CN110780079A CN201911189340.2A CN201911189340A CN110780079A CN 110780079 A CN110780079 A CN 110780079A CN 201911189340 A CN201911189340 A CN 201911189340A CN 110780079 A CN110780079 A CN 110780079A
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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Abstract
The invention provides a squamous cell carcinoma antigen detection reagent, which comprises magnetic microspheres coating a squamous cell carcinoma antigen antibody, a squamous cell carcinoma antigen antibody marked by alkaline phosphatase and a chemiluminescent substrate; wherein the magnetic microspheres are silicon dioxide magnetic composite particles. The squamous cell carcinoma antigen detection reagent provided by the invention has the advantages of simple components and high detection sensitivity, and can realize rapid and accurate detection in a short time.
Description
Technical Field
The invention belongs to the technical field of medical detection, and relates to a squamous cell carcinoma antigen detection reagent.
Background
Squamous Cell Carcinoma Antigen (SCCA) is a well-defined tumor marker that was the earliest diagnostic marker for cancer. It is a glycoprotein extracted from uterine tissue, belonging to the family of serine/cysteine protease inhibitors, consisting of highly homologous and biologically functionally distinct SCCA1 and SCCA2, each containing 390 amino acids and having a molecular weight of approximately 45 kDa. SCCA is widely present in normal tissues of different organs (in microscopic amounts) and in malignant diseased epithelial cells; the concentration of SCCA in serum is related to the differentiation degree of squamous cell carcinoma, and the concentration of SCCA in serum of normal human is less than 1.5 ng/mL. SCC elevation can also be seen in a few benign diseases, such as lung infections, dermatitis, renal failure and liver disease. The kit has higher diagnosis value, better sensitivity and specificity to cervical cancer, assists in diagnosing squamous carcinoma such as lung, esophagus and the like, has a level related to the development degree of tumor, and can improve the accuracy and sensitivity by combining with CYFRA21-1 for detection.
The currently known methods for measuring squamous cell carcinoma antigens include Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), latex-enhanced turbidimetry, and the like. However, ELISA methods are more limited, have poor specificity, low sensitivity and poor accuracy. Radioimmunotherapy is considered an accurate and reliable method, but the radiation effect of radioisotopes, the waste disposal requirement of specialized laboratories, the high cost and short half-life disadvantages have led to the gradual elimination of this method. The latex-enhanced immunoturbidimetry is simple and rapid to operate, but has low sensitivity and poor repeatability.
CN103454417A discloses a preparation method and application of a squamous cell carcinoma antigen biosensor, wherein a sandwich type electrochemical immunosensor is prepared based on nitrogen-doped graphene and a Pd-Au/C supported nano catalyst, and the sandwich type electrochemical immunosensor can realize rapid detection of squamous cell carcinoma antigens, but has low detection accuracy. CN104614526A discloses a squamous epithelial cell carcinoma SCC-TCT combined detection method; the method comprises the steps of sampling exfoliated cells at the cervix, and pretreating to obtain a sample solution; centrifuging saponin and sample solution, taking precipitate, adding phosphate buffer solution, mixing, and counting the total number of cells; centrifuging the counted cell suspension, adding a phosphate buffer solution, and performing freeze thawing for at least one time to obtain a sample solution after the SCC antigen is dissociated and released; then, carrying out enzyme labeling test analysis on the intracellular SCC antigen; judging and screening according to the determination result, screening out the samples higher than the critical value, preparing uniform smears of sample solutions corresponding to the samples on a liquid-based cytometer, fixing, staining by a papanicolaou staining method, analyzing the morphological of microscopic cells, and judging the morphology of tumor cells; the process is too complex.
Therefore, it is desired to provide a detection reagent having a simple preparation method and high detection sensitivity.
Disclosure of Invention
The squamous cell carcinoma antigen detection reagent provided by the invention has the advantages of simple components, high detection sensitivity and capability of realizing rapid and accurate detection in a short time.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a squamous cell carcinoma antigen detection reagent, which comprises magnetic microspheres coating squamous cell carcinoma antigen antibodies, squamous cell carcinoma antigen antibodies marked by alkaline phosphatase and chemiluminescent substrates;
wherein the magnetic microspheres are silicon dioxide magnetic composite particles.
Compared with other magnetic microspheres, the silica magnetic composite microsphere specifically selected by the invention has more binding sites and can bind more antibodies, so that the detection reagent provided by the invention has higher detection precision at lower detection concentration; and the coating performance is better, the elution is not easy, and the phenomenon of error increase caused by poor coating in the subsequent sample detection process can be reduced.
In the invention, the silicon dioxide magnetic composite particles comprise a silicon dioxide shell layer and a magnetic ferric oxide nanometer inner core.
The silica magnetic composite particles of the present invention are magnetic microspheres available in the prior art.
Preferably, the magnetic microspheres have a particle size of 5 to 10 μm, such as 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, etc., and more preferably 8 to 10 μm, such as 8.2 μm, 8.5 μm, 8.8 μm, 9.0 μm, 9.2 μm, 9.5 μm, 9.8 μm, etc.
The particle size of the magnetic microspheres is preferably in the range of 5-10 mu m, and if the particle size is too large, the coating amount of the squamous cell carcinoma antigen antibody is possibly too large, so that part of the squamous cell carcinoma antigen antibody is easy to fall off, and the measurement accuracy is further influenced; if the particle size is too small, the coating of the antigen-antibody of squamous cell carcinoma is not favorable.
In the present invention, the chemiluminescent substrate is 4-nitrophenyl phosphate disodium salt hexahydrate.
Compared with other luminescent substrates, the luminescent substrate has longer luminescent time, sensitive reaction and bright color.
Preferably, the preparation method of the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody comprises the following steps:
mixing the activated alkaline phosphatase solution and the activated squamous cell carcinoma antigen antibody solution, and then carrying out coupling reaction to obtain the squamous cell carcinoma antigen antibody marked by alkaline phosphatase.
Preferably, the magnetic microspheres are coupled with streptavidin.
Preferably, the squamous cell carcinoma antigen antibody is coated on the magnetic microsphere through streptavidin.
Preferably, the preparation method of the magnetic microsphere coated with the squamous cell carcinoma antigen antibody is as follows:
adding the squamous cell carcinoma antigen antibody solution into the magnetic microspheres, and incubating and coating to obtain the magnetic microspheres coated with the squamous cell carcinoma antigen antibody.
Preferably, the preparation method is as follows:
(1) adding a streptavidin solution into the magnetic microspheres for coupling reaction to obtain streptavidin-coupled magnetic microspheres;
(2) adding the squamous cell carcinoma antigen antibody solution into streptavidin coupled magnetic microspheres for incubation coating to obtain the magnetic microspheres coated with squamous cell carcinoma antigen antibody.
Preferably, in the squamous cell carcinoma antigen detection reagent, the concentration of the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody is 0.1-0.5. mu.g/mL, such as 0.2. mu.g/mL, 0.3. mu.g/mL, 0.4. mu.g/mL, or the like.
Preferably, in the squamous cell carcinoma antigen detection reagent, the concentration of the magnetic microspheres coating the squamous cell carcinoma antigen antibody is 0.1-0.5 μ g/mL, such as 0.2 μ g/mL, 0.3 μ g/mL, 0.4 μ g/mL, etc.
Compared with the prior art, the invention has the following beneficial effects:
(1) compared with other magnetic microspheres, the silica magnetic composite microsphere specifically selected by the invention has more binding sites and can bind more antibodies, so that the detection reagent provided by the invention has higher detection precision at lower detection concentration; the coating performance is good, elution is not easy, and the phenomenon of error increase caused by poor coating in the subsequent sample detection process can be reduced;
(2) the particle size of the magnetic microspheres is preferably in the range of 5-10 mu m, and if the particle size is too large, the coating amount of the squamous cell carcinoma antigen antibody is possibly too large, so that part of the squamous cell carcinoma antigen antibody is easy to fall off, and the measurement accuracy is further influenced; if the particle size is too small, the coating of the antigen-antibody of squamous cell carcinoma is not favorable.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
A squamous cell carcinoma antigen detection reagent comprises magnetic microspheres coating squamous cell carcinoma antigen antibodies, squamous cell carcinoma antigen antibodies marked by alkaline phosphatase and chemiluminescent substrates.
Wherein the concentration of the squamous cell carcinoma antigen antibody marked by alkaline phosphatase is 0.3 mug/mL, the concentration of the magnetic microsphere coating the squamous cell carcinoma antigen antibody is 0.3 mug/mL, and the chemiluminescent substrate is 4-nitrophenyl disodium phosphate hexahydrate.
The preparation method of the magnetic microsphere coated with the squamous cell carcinoma antigen antibody comprises the following steps:
(1) adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) into MES (2- (N-morpholino) ethanesulfonic acid) suspension of carboxylated nano magnetic microspheres (with the particle size of 8 mu m) for activation reaction, then adding streptavidin, suspending for 5h at 30 ℃, adding a magnetic field for separation, removing supernatant, and then resuspending by using Tris buffer solution to obtain suspension of magnetic microspheres coupled with the streptavidin, wherein the concentration of the suspension is 0.2 mu g/mL.
(2) Mixing the suspension of the magnetic microspheres coupled with streptavidin with the suspension of the squamous cell carcinoma antigen antibody (Tris buffer solution with the concentration of 4 mu g/mL), incubating for 1h at room temperature, adding a magnetic field for separation, removing supernatant, washing by using phosphate buffer solution with the pH value of 7, and resuspending to obtain the suspension of the magnetic microspheres coated with the squamous cell carcinoma antigen antibody coated with the double-stranded DNA antigen with the concentration of 0.3 mu g/mL.
The preparation method of the squamous cell carcinoma antigen antibody marked by alkaline phosphatase comprises the following steps:
(1) adding the squamous cell carcinoma antigen antibody into a coupling agent 2-iminothiolane hydrochloride, standing for 20min at 30 ℃, adding a glycine solution, standing for 5min again at 30 ℃, and then removing salt by using a G-25 gel column to obtain the activated squamous cell carcinoma antigen antibody with the concentration of 1 mug/mL.
(2) The alkaline phosphatase solution was added to a succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate solution, allowed to stand at 30 ℃ for 30 minutes, and then desalted by means of a G-25 gel column to give an activated alkaline phosphatase at a concentration of 1. mu.g/mL.
(3) The activated antibody and the activated alkaline phosphatase were mixed at 5 ℃ and left to stand for 20 hours, followed by purification using a Sephadex G250 column at a flow rate of 3mL/min to give the alkaline phosphatase-labeled antibody at a concentration of 0.3. mu.g/mL.
Examples 2 to 5
The difference from example 1 is that the silica magnetic composite fine particles used in the present example have particle diameters of 10 μm (example 2), 5 μm (example 3), 3 μm (example 4), and 12 μm (example 5).
Example 6
The difference from example 1 is that in this example the chemiluminescent substrate is 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt.
Example 7
A squamous cell carcinoma antigen detection reagent comprises magnetic microspheres coating squamous cell carcinoma antigen antibodies, squamous cell carcinoma antigen antibodies marked by alkaline phosphatase and chemiluminescent substrates.
Wherein the concentration of the squamous cell carcinoma antigen antibody marked by alkaline phosphatase is 0.1 mug/mL, the concentration of the magnetic microsphere coating the squamous cell carcinoma antigen antibody is 0.1 mug/mL, and the chemiluminescent substrate is 4-nitrophenyl disodium phosphate hexahydrate.
Preparation of magnetic microspheres coated with squamous cell carcinoma antigen antibody, squamous cell carcinoma antigen antibody labeled with alkaline phosphatase is referred to example 1.
Comparative example 1
The difference from example 1 is that the magnetic microspheres were replaced with an enzyme label plate.
Comparative example 2
CN109142753A example 1 provides a squamous cell carcinoma antigen chemiluminescence immunoassay kit
Performance testing
The detection reagents provided in examples 1-7 and comparative examples 1-2 were subjected to performance testing as follows:
(1) and (3) testing the sensitivity: the squamous cell carcinoma antigen is prepared into a standard substance with the concentration of 0.000ng/mL, 0.001ng/mL, 0.005ng/mL, 0.01ng/mL, 0.02ng/mL, 0.04ng/mL, 0.05ng/mL, 0.08ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL and 5ng/mL by using a standard substance buffer solution (0.066mol/L phosphate buffer solution with the pH value of 7.4), and the lowest detectable concentration (LOD) is recorded by using a detection reagent, namely the sensitivity of the sample.
(2) And (3) detecting errors: the squamous cell carcinoma antigen was prepared as a standard at a concentration of 5ng/mL with a standard buffer solution (0.066mol/L, phosphate buffer solution at pH 7.4), and then detected using the detection reagents provided in examples and comparative examples, the values of the detection thereof were recorded, and the percentage of detection error was calculated.
The test results are shown in table 1:
TABLE 1
As can be seen from the examples and performance tests, the detection reagent provided by the invention has the detection advantages of high sensitivity and small error value, wherein the LOD of the detection reagent is below 0.01ng/mL, and can be as low as below 0.005 ng/mL; the detection error is below 2 percent, and the lowest detection error can reach below 0.75 percent.
As can be seen from the comparison between examples 1 to 3 and examples 4 to 5, the magnetic microspheres have better detection sensitivity and lower detection error when the particle size is in the range of 8 to 10 μm. As can be seen from the comparison between example 1 and example 6, when the chemiluminescent substrate selected by the invention is 4-nitrophenyl phosphate disodium salt hexahydrate, the finally obtained detection reagent has good sensitivity and low detection error. As can be seen from the comparison between example 1 and comparative example 1, the invention adopts the magnetic microspheres as the solid phase carriers, which is more favorable for reducing the measurement error. As is clear from the comparison between example 1 and comparative example 2, the detection reagent of the present invention can achieve the object of detection as compared with the conventional detection reagents.
The applicant states that the present invention is illustrated by the above examples to the squamous cell carcinoma antigen detection reagent of the present invention, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be carried out. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. The squamous cell carcinoma antigen detection reagent is characterized by comprising magnetic microspheres coated with squamous cell carcinoma antigen antibodies, squamous cell carcinoma antigen antibodies marked by alkaline phosphatase and chemiluminescent substrates;
wherein the magnetic microspheres are silicon dioxide magnetic composite particles.
2. The squamous cell carcinoma antigen detection reagent of claim 1, wherein the silica magnetic composite particle comprises a silica shell and a magnetic ferric oxide nano-core.
3. The squamous cell carcinoma antigen detection reagent according to claim 1 or 2, wherein the particle size of the magnetic microsphere is 5-10 μm, further preferably 8-10 μm.
4. The squamous cell carcinoma antigen detection reagent according to any of claims 1-3, wherein the chemiluminescent substrate is 4-nitrophenyl phosphate disodium salt hexahydrate.
5. The squamous cell carcinoma antigen detection reagent according to any of claims 1-4, wherein the preparation method of the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody comprises the following steps:
mixing the activated alkaline phosphatase solution and the activated squamous cell carcinoma antigen antibody solution, and then carrying out coupling reaction to obtain the squamous cell carcinoma antigen antibody marked by alkaline phosphatase.
6. The squamous cell carcinoma antigen detection reagent according to any of claims 1-5, wherein the magnetic microspheres are coupled with streptavidin;
preferably, the squamous cell carcinoma antigen antibody is coated on the magnetic microsphere through streptavidin.
7. The squamous cell carcinoma antigen detection reagent according to any of claims 1-6, wherein the magnetic microsphere coated with squamous cell carcinoma antigen antibody is prepared by the following method:
adding the squamous cell carcinoma antigen antibody solution into the magnetic microspheres, and incubating and coating to obtain the magnetic microspheres coated with the squamous cell carcinoma antigen antibody.
8. The squamous cell carcinoma antigen detection reagent according to claim 7, wherein the preparation method is as follows:
(1) adding a streptavidin solution into the magnetic microspheres for coupling reaction to obtain streptavidin-coupled magnetic microspheres;
(2) adding the squamous cell carcinoma antigen antibody solution into streptavidin coupled magnetic microspheres for incubation coating to obtain the magnetic microspheres coated with squamous cell carcinoma antigen antibody.
9. The squamous cell carcinoma antigen detection reagent according to any of claims 1-8, wherein the concentration of the alkaline phosphatase-labeled squamous cell carcinoma antigen antibody in the squamous cell carcinoma antigen detection reagent is 0.1-0.5 μ g/mL.
10. The squamous cell carcinoma antigen detecting reagent according to any of claims 1-9, wherein the concentration of the magnetic microspheres coated with squamous cell carcinoma antigen antibody in the squamous cell carcinoma antigen detecting reagent is 0.1-0.5 μ g/mL.
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Cited By (2)
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| CN112129941A (en) * | 2020-09-22 | 2020-12-25 | 武汉生之源生物科技股份有限公司 | Chemiluminescence kit for detecting squamous cell carcinoma antigen |
| CN118150831A (en) * | 2024-03-18 | 2024-06-07 | 宁波瑞源生物科技有限公司 | A kit for detecting squamous cell carcinoma antigen |
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