Human extracellular matrix metalloproteinase inducible factor magnetic particle chemiluminescence immunoassay quantitative detection kit and detection method thereof

[0032] Example 1. Preparation of the kit

[0033]The magnetic particles of the present invention are used as the solid phase of the immune reaction, and the chemiluminescence immunoassay method is used in conjunction with a chemiluminescence analyzer to measure the content of human extracellular matrix metalloproteinase-inducing factors in human body samples. The kit includes streptavidin-coated magnetic particle suspension, alkaline phosphatase-labeled human extracellular matrix metalloproteinase-inducing factor (CD147) monoclonal antibody (referred to as enzyme-labeled monoclonal antibody), and biotin-labeled human cells Extracellular matrix metalloproteinase inducible factor (CD147) monoclonal antibody (referred to as biotin-labeled monoclonal antibody), human extracellular matrix metalloproteinase inducible factor (CD147) calibrator, etc. The kit according to the invention uses magnetic particles as the solid phase of the immune reaction, and uses the chemiluminescence immunoassay method to cooperate with a chemiluminescence analyzer, which is used for the determination of human extracellular matrix metalloproteinase inducible factor (CD147) in human serum/plasma. content. The technical principle of the reaction is as follows: figure 1 As shown in the figure, the antigen in the human extracellular matrix metalloproteinase-inducing factor (CD147) of the test sample or calibrator is combined with biotin-labeled monoclonal antibody and alkali-labeled monoclonal antibody to form a complex, and the complex is a biotin-labeled monoclonal antibody -Antigen-enzyme-labeled monoclonal antibody complex; subsequently, streptavidin-coated magnetic particles are added to the complex, and the antigen-antibody complex is linked to the magnetic particles through the combination of streptavidin and biotin, Direct precipitation in an applied magnetic field separates the immunoreactive complexes from unbound other substances. Wash the precipitated complex, add the enzymatic chemiluminescence substrate, the substrate is catalytically cleaved under the action of the enzyme to form an unstable excited state intermediate, and when the excited state intermediate returns to the ground state, it emits photons to form a luminescence reaction, The luminescence intensity of the reaction was measured using a luminometer. Within the measurement range, the luminescence intensity is proportional to the concentration of human extracellular matrix metalloproteinase-inducing factor (CD147) in the sample. Using the improved four-parameter Logistic equation fitting, the induced human extracellular matrix metalloproteinase in the sample can be quantitatively calculated. factor (CD147) concentration.

[0034] The CD147 antibody of the present application is a recombinant protein, the protein amount is 46.3kD, and the protein sequence (SEQ ID NO.1) is as follows:

[0035] MASMTDQQAEARAFLSEEMIAEFKAAFDMWDADGGGDISTKELGTVMRMLGQNP TKEELDAIIEEVDEDGSGTIDFEEFLVMMVRQMKEDAKGKSEEELANCFRIFDKNADGFI DIEELGEILRATGEHVIEEDIEDLMKDSDKNNDGRIDFDEFLKMMEGELGEAHEAEEVHE EAHHEEAHHAEAHHEEAHAHAEEVHEPEEAPEEEEKPRINGSEPGTVFTTVEDLGSKILL TCSLNDSATEVTGHRWLKGGVVLKEDALPGQKTEFKVDSDDQWGEYSCVFLPEPMGTANIQLHGPPRVKAVKSSEHINEGETAMLVCKSESVPPVTDWAWYKITDSEDKALMNGSESR FFVSSSQGRSELHIENLNMEADPGQYRCNGTSSKGSDQAIITLRVRSHLAKLAAALEHHH HHH

[0036] The CD147 protein sequence in the present invention is expressed by Escherichia coli, and detected by agarose gel electrophoresis after the expression, and the detection result is as follows: figure 2 As shown, the CD147 protein band in the figure is compared with the protein Marker, and it can be seen that the size of the protein fragment is correct.

[0037] 1. The preparation process of the kit is as follows:

[0038] (1) Preparation of enzyme-labeled magnetic particle buffer:

[0039] materials Dosage Tris(hydroxymethylaminomethane) 2.42g Sodium chloride 9.00g Tween-20 0.50g bovine serum albumin 50.00g Proclin300 1.00g

[0040] The above materials were added to 1000mL of deionized water, fully stirred to dissolve, and the pH was adjusted to 8.00±0.10.

[0041] (2) Preparation of enzyme marker buffer:

[0042] materials Dosage Tris(hydroxymethylaminomethane) 2.42g Sodium chloride 18.00g Tween-20 1.00g bovine serum albumin 100.00g Proclin300 1.00g

[0043] The above materials were added to 1000mL of deionized water, fully stirred to dissolve, and the pH was adjusted to 8.00±0.10.

[0044] (3) Preparation of biotin label buffer:

[0045] materials Dosage Tris(hydroxymethylaminomethane) 2.42g Sodium chloride 9.00g Tween-20 1.00g bovine serum albumin 50.00g Proclin300 1.00g

[0046] The above materials were added to 1000mL of deionized water, fully stirred to dissolve, and the pH was adjusted to 8.00±0.10.

[0047] (4) Calibrator buffer preparation:

[0048] materials Dosage Tris(hydroxymethylaminomethane) 2.42g Sodium chloride 36.00g Tween-20 1.00g fetal bovine serum 100mL Proclin300 1.00g

[0049] Add the above materials into 1000mL deionized water, stir and dissolve fully, and adjust the pH to 7.40±0.10.

[0050] (5) Detection method of streptavidin-coated magnetic particle suspension:

[0051] The streptavidin-coated magnetic particle stock solution (purchased from Nanjing Pangu Gene Nanotechnology Co., Ltd.) was diluted with enzyme-labeled magnetic particle buffer to a concentration of 0.6 mg/mL.

[0052] (6) Detection method of human extracellular matrix metalloproteinase-inducing factor (CD147) monoclonal antibody labeled with alkaline phosphatase:

[0053] Add 200ug of alkaline phosphatase to 1mL of 10mM sodium carbonate buffer (pH8.0), add 20ug of human extracellular matrix metalloproteinase-inducing factor (CD147) monoclonal antibody (purchased from Shenzhen Yawei Company), and react at 37°C for 2 hours , Purify the enzyme-labeled antibody with ProteinG affinity column (GE Company) to obtain the enzyme-labeled human extracellular matrix metalloproteinase-inducing factor (CD147) monoclonal antibody. Dilute in enzyme marker buffer at a dilution ratio of 1:400.

[0054] (7) Detection method of biotin-labeled human extracellular matrix metalloproteinase-inducing factor (CD147) monoclonal antibody:

[0055] Add 20ug human extracellular matrix metalloproteinase inducible factor (CD147) monoclonal antibody (mouse, monoclonal, purchased from Abcam company) into 1mL 10mM sodium carbonate buffer (pH8.0), add 20ug biotin derivative, 37 ℃ After 2 hours of reaction, the biotin-labeled antibody was purified with a ProteinG affinity column (GE Company) to obtain a biotin-labeled human extracellular matrix metalloproteinase inducer (CD147) monoclonal antibody. Dilute in biotin labeling buffer at a dilution ratio of 1:800.

[0056] (8) Detection method of human extracellular matrix metalloproteinase inducible factor (CD147) calibrator:

[0057] Human extracellular matrix metalloproteinase-inducible factor (CD147) antigen was diluted with calibrator buffer to working concentrations of 0, 0.50, 1.00, 2.50, 5.00, 10.00 ng/mL, respectively.

[0058] (9) Assembling: Assemble the above reagent components into a box and store at 2-8°C.

[0059] Embodiment 2, the test method of test kit

[0060] (1) Sample addition and incubation process: Pipette 50uL of human extracellular matrix metalloproteinase-inducing factor (CD147) calibrator or fresh patient sample into the reaction tube, and then add alkaline phosphatase-labeled human extracellular matrix metalloproteinase-inducing factor ( CD147) monoclonal antibody 50uL and biotin-labeled human extracellular matrix metalloproteinase-inducing factor (CD147) monoclonal antibody 50uL, incubate at 37°C for 10 minutes; then add streptavidin-coated magnetic particle suspension 50uL, Incubate the reaction at 37°C for 10 minutes;

[0061] (2) Magnetic separation and cleaning process: put the reaction tube after the incubation reaction is completed on the magnetic separation rack for 1 minute, and remove the supernatant; add 300uL of magnetic bead coating buffer for the first time, and place it on the magnetic separation rack Let stand for 1 minute, remove the supernatant; add 300uL of magnetic bead coating buffer for the second time, place it on the magnetic separation rack for 1 minute, remove the supernatant; add magnetic bead coating buffer for the third time 300uL of liquid, put it on a magnetic separation rack for 1 minute, and remove the supernatant;

[0062] (3) Luminescence process: 200uL of Lumi-Phos 530 substrate solution (purchased from Lumigen, USA) was added, and after incubation at 37°C for 5 minutes in the dark, the luminescence value was measured with a Hamamatsu 9507 semi-automatic chemiluminescence instrument.

[0063] Example 3. Performance test results of the kit.

[0064] Kit performance evaluation indicators: The accuracy, linearity, precision and stability of the kit prepared by this method were determined. The standard curve is drawn after detection with protein calibrator. The standard curve is as follows: image 3 As shown, the standard curve formula y=1.0606x-0.1840, and the linearity of the test results is evaluated at the same time. The test results are shown in the following table:

[0065] 1. Accuracy test results

[0066]

[0067] 2. Linearity test results

[0068]

[0069] 3. Precision test results

[0070] repeat times low value samples 1 0.49 2 0.50 3 0.50 4 0.51 5 0.52 6 0.51 7 0.52 8 0.52 9 0.49 10 0.51 average value 0.51 Coefficient of Variation CV 2.38％

[0071] 4. Stability test results

[0072]

[0073] 5. Sensitivity test results

[0074]

[0075] It can be seen from the above detection results and analysis that the detection results of the kit of the present invention have wide linearity, high sensitivity and good repeatability, and the results are all statistically significant.

[0076] The detection kit of the present invention is compared with commercially available ELISA kits, and the measurement results are as follows:

[0077]

[0078]

[0079] It can be seen from the above results that the performance test results of the kit involved in the present invention are better than that of the ELISA kit. It has good applicability and advanced.