Microplate and In Vitro Diagnostic Kit for HPV 16/E7 Oncoprotein Detection and Preparation Method Thereof

Abstract

The invention relates to a microplate, an in vitro diagnostic kit for HPV type 16 E7 oncoprotein detection and a preparation method thereof The in vitro diagnostic kit is comprised of a microplate pre-coated with an HPV type 16 E7 antibody and an HRP-labeled HPV type 16 E7 antibody with concentration of 0.05-0.4 μg/ml, wherein the amount of the antibody in each microwell of the microplate is 0.05-0.5 μg. The in vitro diagnostic kit is used for directly detecting the expression of high-risk HPV-associated oncoprotein and the expression level, and thus has a clear judgment on the infection degree of high-risk HPV, and facilitates subsequent treatment.

Claims

1. A microplate for an HPV type 16 E7 oncoprotein detection, wherein the microplate is pre-coated with an HPV type 16 E7 antibody.

2. The microplate according to claim 1, wherein an amount of the HPV type 16 E7 antibody in each microwell of the microplate is 0.05-0.5 μg.

3. A method for preparing the microplate according to claim 1, comprising: taking an HPV type 16 E7 antibody, adding a coating buffer to microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h); washing the microwells with a washing solution five times at an amount of 300 μl/microwell; adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2h for blocking; suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300.

4. A method for preparing the microplate according to claim 2, comprising: taking an HPV type 16 E7 antibody, adding a coating buffer to microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h); washing the microwells with a washing solution five times at an amount of 300 μl/microwell; adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2 h for blocking; suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300.

5. An in vitro diagnostic kit for an HPV type 16 E7 oncoprotein detection, comprising: a microplate pre-coated with an HPV type 16 E7 antibody, wherein an amount of the HPV type 16 E7 antibody in each microwell of the microplate is 0.05-0.5 μg; and an HRP-labeled HPV type 16 E7 antibody with a concentration of 0.05-0.4 μg/ml.

6. The in vitro diagnostic kit according to claim 5, further comprising a human cervical exfoliated cell lysis buffer.

7. The in vitro diagnostic kit according to claim 6, wherein the human cervical exfoliated lysis buffer is comprised of 0.05%-l% of surfactant, 10 mM-1 M of buffer and 1 mM-10 mM of protease inhibitor.

8. The in vitro diagnostic kit according to claim 7, wherein the surfactant is one or more of Tween 20, Triton-100 or sodium dodecylsulfonate, the buffer is phosphate-buffered saline, Tris-HCl or a carbonate solution, and the protease inhibitor is one or more of cystatin, PMSF or Antipain.

9. The in vitro diagnostic kit according to claim 5, wherein a method for preparing the microplate pre-coated with the HPV type 16 E7 antibody is comprised of following steps: taking the HPV type 16 E7 antibody, adding a coating buffer to the microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h); washing the microwells with a washing solution five times at an amount of 300 μl/microwell; adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2 h for blocking; suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300.

10. The in vitro diagnostic kit according to claim 6, wherein a method for preparing the microplate pre-coated with the HPV type 16 E7 antibody is comprised of following steps: taking the HPV type 16 E7 antibody, adding a coating buffer to the microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h); washing the microwells with a washing solution five times at an amount of 300 μl/microwell; adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2 h for blocking; suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300.

11. The in vitro diagnostic kit according to claim 7, wherein a method for preparing the microplate pre-coated with the HPV type 16 E7 antibody is comprised of following steps: taking the HPV type 16 E7 antibody, adding a coating buffer to the microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h); washing the microwells with a washing solution five times at an amount of 300 μl/microwell; adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2 h for blocking; suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300.

12. The in vitro diagnostic kit according to claim 8, wherein a method for preparing the microplate pre-coated with the HPV type 16 E7 antibody is comprised of following steps: taking the HPV type 16 E7 antibody, adding a coating buffer to the microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h); washing the microwells with a washing solution five times at an amount of 300 μl/microwell; adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2 h for blocking; suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300.

13. A preparation method of an in vitro diagnostic kit for an HPV type 16 E7 oncoprotein detection, comprising: 1) preparing a microplate pre-coated with an HPV type 16 E7 antibody which comprises taking the HPV type 16 E7 antibody, adding a coating buffer to microwells at an amount of 100 μl/microwell and standing at 4° C. overnight (16-18 h); washing the microwells with a washing solution five times at an amount of 300 μl/microwell; adding a blocking solution to the microwells at an amount of 300 μl/microwell, and standing at 37° C. for 1-2 h for blocking; suction-drying in a drying room (18-26° C.) and sealing in vacuum (2-8° C.) to obtain a microplate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 0.5-5 mg of HPV type 16 E7 antibody to 1000 ml of buffer, and the buffer is comprised of 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution is comprised of 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300; 2) preparing calibrators and quality control samples which comprises preparing HPV type 16 E7 oncoprotein detection calibrators 0, 1, 2, 3, 4 and 5: taking lyophilized powder as the calibrators, and respectively adding 1 ml ddH.sub.2O for dissolving, wherein linear concentrations of calibrator solutions are 0, 10, 20, 40, 80, and 160 ng/ml, respectively; preparing HPV type 16 E7 oncoprotein detection quality control samples 1 and 2: taking lyophilized powder as the quality control samples, and respectively adding 1 ml ddH.sub.2O for dissolving, wherein concentrations are 10 and 80 ng/ml, respectively; 3) preparing an HRP-labeled HPV type 16 E7 antibody which comprises labeling with a sodium periodate method, adding an HRP stabilizer, mixing thoroughly and storing at 2-8° C., wherein a concentration of the HRP-labeled HPV type 16 E7 antibody is 0.05-0.4 μg/ml; 4) preparing a human cervical exfoliated lysis buffer which comprises preparing 0.05%-1% (vol/vol) of surfactant with 10 mM-1 M of buffer and adding a protease inhibitor with final concentration of 1 mM-10 mM before use, wherein the surfactant is one or more of Tween 20, Triton-100 or sodium dodecylsulfonate; the buffer is phosphate-buffered saline, Tris-HCl or a carbonate solution, and the protease inhibitor is one or more of cystatin, PMSF or Antipain; 5) preparing a concentrated washing solution (20×) which comprises adding 28.8 g of Na.sub.2HPO.sub.4.12H.sub.2O, 4.8 g of KH.sub.2PO.sub.4, 160 g of NaCl, 4 g of KCl, 10 ml of Tween-20, and 6 ml of Proclin 300 to ddH.sub.2O, diluting to 1000 ml, mixing thoroughly and storing at room temperature; and 6) directly obtaining a substrate solution and a sealing film by purchasing.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0056] FIG. 1 is the standard curve equation obtained by taking the calibrator concentration in the Embodiment 4 as the abscissa and the luminescence value as the ordinate and by adopting the four-parameter logic fitting method.

DETAILED DESCRIPTION OF THE INVENTION

[0057] The invention will be described in further detail with reference to the embodiments, but the embodiments are not intended to limit the scope of the invention.

Embodiment 1

[0058] A method for preparing the microplate coated with the HPV type 16 E7 antibody includes the following steps: [0059] taking the HPV type 16 E7 antibody, adding a coating buffer to the wells at an amount of 100 μl/well and standing at 4° C. overnight (18 h); washing with washing solution five times at an amount of 300 μl/well; adding blocking solution to the wells at an amount of 300 μl/well, and standing at 37° C. for 2 h for blocking; suction-drying in a drying room (26° C.) and sealing in vacuum (4° C.) to obtain a microwell plate coated with the HPV type 16 E7 antibody, and drying and storing, wherein the coating buffer is prepared by adding 5 mg of antibody to 1000 ml of buffer, and the buffer includes 1.59 g/L of Na.sub.2CO.sub.3 and 2.93 g/L of NaHCO.sub.3; the washing solution includes 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 0.5 ml/L of Tween-20, and 0.3 ml/L of Proclin 300; the blocking solution includes 1.44 g/L of Na.sub.2HPO.sub.4, 0.24 g/L of KH.sub.2PO.sub.4, 8 g/L of NaCl, 0.2 g/L of KCl, 10 g/L of BAS, 25 g/L of sucrose and 0.3 ml/L of Proclin 300;

Embodiment 2

[0060] Preparation of the human cervical exfoliated lysis buffer and lysate [0061] preparing 1% of Tween 20 with 50 mM of PBS; and adding the protease inhibitor PMSF with final concentration of 5 mM before use. 500 μl of lysate was added into a cervix exfoliated cell collection tube, the collection tube was vortex-shaken for 2 min, placed in a −20° C. freezer for 10 min, then placed in a 37° C. oven for 2 min, shaken again, and centrifuged (20 min, 13,000 rpm, 4° C.), and the supernatant was collected and detected.

Embodiment 3

[0062] A preparation method of the in vitro diagnostic kit for the HPV type 16 E7 oncoprotein detection [0063] {circle around (1)} preparation of the microplate pre-coated with the HPV type 16 E7 antibody, wherein the amount of the antibody in each microwell was 0.5 μg, and the microplate was a bar-shaped microwell plate (12 bars×8 wells) and arranged in a frame; and the preparation method was the same as the Embodiment 1; [0064] {circle around (2)} preparation of the HPV type 16 E7 oncoprotein detection calibrators 0, 1, 2, 3, 4 and 5, wherein the freeze-dried powder served as the calibrators, 1 ml ddH.sub.2O was respectively added for dissolving, and the linear concentration of the calibrator solution was 0, 10, 20, 40, 80, and 160 ng/ml, respectively; [0065] {circle around (3)} preparation of the HPV type 16 E7 oncoprotein quality control samples 1 and 2, wherein the lyophilized powder served as the quality control samples, 1 ml ddH.sub.2O was respectively added for dissolving, and the concentration was 10 and 80 ng/ml, respectively; [0066] {circle around (4)} preparation of the HRP-labeled HPV type 1.6 E7 antibody: labeling with the sodium periodate method, adding an HRP stabilizer, mixing thoroughly and storing at 2-8° C., wherein the concentration is 0.4 μg/ml, one bottle and 12 m1 per bottle;

[0067] {circle around (5)} preparation of the human cervical exfoliated lysis buffer wherein the preparation method was the same as the Embodiment 2; [0068] {circle around (6)} preparation of the substrate solution: chemiluminescent substrate A and B (Huzhou innoreagents Technology Co., Ltd.); [0069] {circle around (7)} preparation of the concentrated washing solution (20×): adding 28.8 g of Na.sub.2HPO.sub.4.12H.sub.2O, 4.8 g of KH.sub.2PO.sub.4, 160 g of NaCl, 4 g of KCl, 10 ml of Tween-20, and 6 ml of Proclin 300 to ddH.sub.2O, diluting to 1000 ml, mixing thoroughly and storing at room temperature; [0070] {circle around (8)} selection of the sealing film, wherein the sealing film was a viscous film for sealing the micro ell plate during incubation.

Embodiment 4

[0071] Detection of one cervical sample

[0072] The of the microplate and the lysis of the cervical sample were the same as the Embodiments 1-2. The detection was performed by (1) taking the required pre-coated microplate from the 4° C. refrigerator and standing at room temperature for 15 min. (2) Adding 100 μl calibrator/quality control sample/sample to each well, mixing thoroughly and incubating at 37° C. for 60 min. (3) Remove liquid from each well, adding 300 μl of washing solution o each well, standing and incubating for 1 min, the Remove liquid from each well, drying by clapping on the absorbent paper, and repeating for 5 times. (4) Adding 100 μl of HRP-labeled antibody solution to each well and standing and incubating at 37° C. for 60 min. (5) Remove liquid from each well, adding 300 μl of washing solution to each well, standing and incubating for 1 min, remove liquid from each well, drying by clapping on the absorbent paper, and repeating for 5 times. (6) Adding 50 μl of chemiluminescent substrate A-and 50 μl of chemiluminescent substrate B into each well, placing in the chemiluminescence reader and reading the luminescence value; obtaining the standard curve equation by taking the calibrator concentration as the abscissa and the luminescence value as the ordinate and by adopting the four-parameter logic fitting method, which is shown in FIG. 1. The HPV type 16 E7 oncoprotein concentration in the to-be-detected sample was calculated to be 109.42 ng/ml according to the standard curve equation. According to the result interpretation, the sample is positive, and the patient needs positive treatment.

TABLE-US-00002 concentration calculated antigen according to the concentration luminescence standard curve ID (ng/ml) value (ng/ml) Cal0 0 1060 Cal1 10 6912 Cal2 20 10721 Cal3 40 22244 Cal4 80 44363 Cal5 160 68556 sample to 55124 109.42 be detected

[0073] The cervical exfoliated cells of a part of sample patient were detected by using the kit of the invention (for example the in vitro diagnostic kit of the Embodiment 3), and the detection data results are shown below:

TABLE-US-00003 TABLE 1 detection data results of the cervical exfoliated cells of sample patient HPV16 E7 antigen detection concentration sample number (ng/ml) No. 1 sample patient 1.17 No. 2 sample patient 3.01 No. 3 sample patient 4.43 No. 4 sample patient 8.04 No. 5 sample patient 8.26 No. 6 sample patient 0.66 No. 7 sample patient 19.26 No. 8 sample patient 3.62 No. 9 sample patient 0.03 No. 10 sample patient 1.24 No. 11 sample patient 4.89 No. 12 sample patient 0.04 No. 13 sample patient 10.22 No. 14 sample patient 1.51 No. 15 sample patient 1.69 No. 16 sample patient 0.43 No. 17 sample patient 13.38 No. 18 sample patient 0.05 No. 19 sample patient 2.54 No. 20 sample patient 0.04 No. 21 sample patient 16.99 No. 22 sample patient 0.03 No. 23 sample patient 0.07 No. 24 sample patient 1.16 No. 25 sample patient 0.26 No. 26 sample patient 0.08 No. 27 sample patient 0.10 No. 28 sample patient 9.99 No. 29 sample patient 0.09 No. 30 sample patient 0.59 No. 31 sample patient 1.92 No. 32 sample patient 0.52 No. 33 sample patient 13.50 No. 34 sample patient 14.18 No. 35 sample patient 4.20 No. 36 sample patient 3.50 No. 37 sample patient 0.41 No. 38 sample patient 1.17 No. 39 sample patient 0.09 No. 40 sample patient 0.04 No. 41 sample patient 0.06 No. 42 sample patient 0.63 No. 43 sample patient 1.11 No. 44 sample patient 3.79 No. 45 sample patient 23.80 No. 46 sample patient 0.91 No. 47 sample patient 21.73 No. 48 sample patient 9.30 No. 49 sample patient 6.42 No. 50 sample patient 17.58 No. 51 sample patient 0.15 No. 52 sample patient 2.97 No. 53 sample patient 12.61 No. 54 sample patient 0.15 No. 55 sample patient 2.74 No. 56 sample patient 5.18 No. 57 sample patient 0.24 No. 58 sample patient 0.30 No. 59 sample patient 8.16 No. 60 sample patient 0.03 No. 61 sample patient 2.56 No. 62 sample patient 1.05 No. 63 sample patient 0.37 No. 64 sample patient 0.57 No. 65 sample patient 0.32 No. 66 sample patient 0.40 No. 67 sample patient 1.60 No. 68 sample patient 0.25 No. 69 sample patient 3.89 No. 70 sample patient 0.17 No. 71 sample patient 5.23 No. 72 sample patient 1.93 No. 73 sample patient 3.70 No. 74 sample patient 0.99 No. 75 sample patient 0.91 No. 76 sample patient 1.15 No. 77 sample patient 1.29 No. 78 sample patient 10.33 No. 79 sample patient 8.46 No. 80 sample patient 6.06 No. 81 sample patient 9.76 No. 82 sample patient 2.66 No. 83 sample patient 2.25

TABLE-US-00004 TABLE 2 Detection data results of the cervical exfoliated cells of patients HPV16 E7 antigen detection sample pathological concentration number data (ng/ml) No. 1 patient squamous 125.03 carcinoma of the cervix No. 2 patient CIN II 82.46 No. 3 patient CIN III 26.85 No. 4 patient squamous 152.50 carcinoma of the cervix No. 5 patient CIN III 45.98 No. 6 patient CIN II 52.20 No. 7 patient CIN I 19.19 No. 8 patient CIN II 15.25 No. 9 patient CIN I 1.89 No. 10 patient CIN II 92.15 No. 11 patient CIN II 19.45 No. 12 patient CIN III 12.64 No. 13 patient CIN I 16.85 No. 14 patient squamous 112.05 carcinoma of the cervix No. 15 patient CIN I 2.43 No. 16 patient CIN II 62.20 No. 17 patient CIN III 102.54 No. 18 patient CIN III 75.25 No. 19 patient CIN II 8.56

[0074] Data Analysis

[0075] 1. According to the detection values of the sample patient, the average value, the standard deviation, and the Cutoff value were calculated. The detection results of cervical exfoliated cells of 83 cases of sample patient were averaged, and the standard variance SD was calculated. When the Cutoff value of the kit was determined, the sum of the average value of the sample patient and twice of the standard variance was selected as the judgment standard of the negative and positive expression of the HPV16 E7 protein, and the results are as follows:

[0076] The calculation formula of Cutoff is cutoff=average value+2*SD standard variance

TABLE-US-00005 TABLE 3 statistics of detection data table 1 average value 4.06 SD standard 5.48 variance Cutoff 15.02 ng/ml

[0077] 2. The negative and positive expression of the HPV16 E7 protein is determined by the Cutoff value, and the expression is considered to be positive when exceeding the Cutoff value, namely the HPV16 E7 protein is expressed, as compared with the pathological data.

[0078] 3. Diagnosis results of the cervical exfoliated cells of the patients in Table 2, judged according to the Cutoff value (“+” shows that the test results are positive; “−” shows that the test results are negative)

TABLE-US-00006 judgement according to the HPV16 E7 Cutoff antigen value detection negativity sample pathological concentration and number data (ng/ml) positivity No. 1 patient cervical 125.03 + cancer No. 2 patient CIN III 82.46 + No. 3 patient CIN II 26.85 + No. 4 patient cervical 152.50 + cancer No. 5 patient CIN III 45.98 + No. 6 patient CIN II 52.20 + No. 7 patient CIN I 19.19 + No. 8 patient CIN II 15.25 + No. 9 patient CIN I 1.89 − No. 10 patient CIN II 92.15 + No. 11 patient CIN II 19.45 + No. 12 patient CIN III 12.64 − No. 13 patient CIN I 16.85 + No. 14 patient cervical 112.05 + cancer No. 15 patient CIN I 2.43 − No. 16 patient CIN II 62.20 + No. 17 patient CIN III 102.54 + No. 18 patient CIN III 75.25 + No. 19 patient CIN II 8.56 −

[0079] Accuracy ratio formulated according to the diagnosis result positivity and negativity of the cervical exfoliated cells of the patients

TABLE-US-00007 pathological number detection of data of HPV16 E7 protein result people positivity negativity CIN I 4 2 (50%) 2 CIN II 7 6 (85.7%) 1 CIN III 5 4 (80%) 1 cervical 3 3 (100%) 0 cancer 19 15 (78.9%) 4

[0080] As shown in Table 2, in the experiment, the total number of the detected patient cervical exfoliated cells is 19 cases, the detection results of 14 cases using the kit for the HPV16 E7 protein are positive, and the diagnostic sensitivity is up to 78.9%, in which the diagnostic sensitivity is up to 80% for the case samples of CIN II or above.