WO2018036435A1 - Hybridoma cell strain scca1, monoclonal antibody secreted thereby, and application of same - Google Patents

Abstract

A hybridoma cell strain SCCA1, a monoclonal antibody secreted thereby, and application of same. The collection number of the hybridoma cell strain SCCA1 is CCTCC No: C2016131. The monoclonal antibody is secreted by the hybridoma cell strain SCCA1, and may be used in the preparation of a reagent for detecting a cervical cancer. The monoclonal antibody is highly specific. An in vitro diagnostic kit for detecting the cervical cancer built on the basis of the monoclonal antibody has a high detection speed, uses only a small amount of blood sample, and provides highly sensitive detection results. The in vitro diagnostic kit provides a rapid, specific, and sensitive detecting method for different clinical and practical requirements, and greatly improves cervical cancer monitoring and management.

Description

Hybridoma cell line SCCA1 and its secreted monoclonal antibody and application Technical field

The invention belongs to the technical field of clinical diagnosis, in particular to a hybridoma cell line SCCA1 and a preparation method thereof, a monoclonal antibody secreted by the hybridoma cell line SCCA1, and a monoclonal antibody in a kit for detecting cervical cancer Applications.

Background technique

Cervical cancer is the third most common malignant tumor in the world after breast cancer and colorectal cancer. It is the second most common malignant tumor in the developing world after breast cancer. It is the most common female reproductive tract. Malignant tumor.

Cervical cancer refers to a malignant tumor that occurs at the junction of the squamous epithelial cells of the cervix or the transitional zone and the columnar epithelial cells of the endocervix. After decades of extensive gynecological screening, the prevalence and mortality of cervical cancer have decreased by nearly 68%, but the incidence of cervical cancer is showing a trend of rejuvenation. The incidence of cervical cancer is high and the prognosis is poor. The most common pathological type of cervical cancer is squamous cell carcinoma, which accounts for about 75% of the total number of cervical cancers; followed by adenocarcinoma, which accounts for about 25% of cervical cancer patients.

Squamous cell carcinoma antigen (SCC) is a tumor marker that is well-specific and was the first to be used to diagnose squamous cell carcinoma. SCCA is a group of glycoproteins belonging to the serine/cysteine inhibitor family. It was originally isolated from squamous cell carcinoma by Kato et al. as a tumor marker for squamous cell carcinoma. For cervical cancer, pre-treatment serum. The level of SCCA can be used as an early prognostic indicator.

SCCA inhibits apoptosis and participates in the differentiation of squamous epithelial cells in normal squamous epithelial cells, and participates in tumor growth in tumor cells. It contributes to the diagnosis and monitoring of all squamous cell origin cancers, for example: Cervical cancer, lung cancer (non-small cell lung cancer), head and neck cancer, esophageal cancer, nasopharyngeal carcinoma, and squamous cell carcinoma of the vulva, etc., the serum SCC of these tumor patients increased, and its concentration increased with the increase of the disease period. Clinically used to monitor the efficacy, recurrence, and metastasis of these tumors and to evaluate prognosis.

SCCA has a high diagnostic value for cervical cancer: sensitivity to primary cervical squamous cell carcinoma is 44%-69%; recurrent cancer sensitivity is 67%-100%, specificity is 90%-96%; its serological level And tumor development, invasion The degree of SC and the presence or absence of metastasis, SCCA concentration decreased significantly after radical resection of cervical cancer; early recurrence was suggested, and SCCA concentration in 50% of patients was increased 2 to 5 months prior to clinical diagnosis. It can be used as an independent risk factor. application. In addition, SCCA quantitative detection of cervical cancer patients can also help to monitor disease status, reflect treatment efficacy and early detection of cancer recurrence, which has important reference value. Most cervical cancers SCCA and CA125 increase at the same time, but some cervical cancers only have a single tumor marker elevation. Therefore, the combination of the two can avoid the missed diagnosis of disease recurrence monitoring caused by the negative result of a single application.

SCCA assisted diagnosis of lung squamous cell carcinoma: the positive rate of lung squamous cell carcinoma is 46.5%, and its level is related to the degree of tumor progression. It can improve the sensitivity of diagnosis of lung cancer patients by combined detection of CA125, CYFRA21-1 and CEA. SCCA predicts esophageal squamous cell carcinoma and nasopharyngeal carcinoma: the positive rate increases with the progression of the disease. For advanced patients, the sensitivity is up to 73%. Combined detection of CYFRA21-1 and SCCA can improve the sensitivity of the test. The positive rate of cancer was 40%, and the positive rate in stage IV increased to 60%. Studies have shown that the positive rate of serum SCCA in patients with adenocarcinoma is significantly lower than that in patients with squamous cell carcinoma.

At present, the diagnosis of cervical cancer is mainly based on two detection methods. One is a transvaginal tissue examination. Including: 1, cervical smear cytology examination, is the main method of cervical cancer screening, should be taken in the cervical transformation area; 2, cervical iodine test, normal cervicovaginal squamous epithelium is rich in glycogen, after iodine solution It is brown or dark brown, and the unstained area indicates that the epithelium lacks glycogen and may have lesions. Biopsy in the iodine-unstained area can improve the diagnosis rate; 3, colposcopy, cervical smear cytology, Pap smear grade III and above, TBS classification as squamous intraepithelial neoplasia, should be under colposcopy Choosing a suspected cancerous area for cervical biopsy; 4. Cervical and cervical biopsy is a reliable basis for the diagnosis of cervical cancer and cervical precancerous lesions. The tissue should include interstitial and adjacent normal tissues, and the cervical scrapings are positive. However, the cervix is smooth or the cervical biopsy is negative, the small curette is used to cure the cervical canal, and the scraping is sent for pathological examination; 5. Cervical conization is suitable for cervical smear examination and positive for cervical biopsy; or cervical biopsy is Cervical intraepithelial neoplasia should be excluded from invasive cancer, cold knife resection, ring electric resection or condensing electrosurgical resection can be used. Such methods also require experienced clinical technicians to interpret the test results, and patients resist such invasiveness. an examination.

Another conventional method of detection is the detection of the biomarker SCCA. However, SCCA is specific Low and low sensitivity, and systemic squamous epithelial cells can make serum SCCA positive if there is lesion. Therefore, there is a need for a highly specific and sensitive marker to help increase the accuracy of detecting cervical cancer.

Summary of the invention

Based on the above, in order to overcome the defects of the above prior art, the present invention provides a hybridoma cell line SCCA1 and a preparation method thereof, a monoclonal antibody secreted by the hybridoma cell line, and a kit for detecting cervical cancer and a preparation method thereof .

In order to achieve the above object, the present invention adopts the following technical solutions:

A hybridoma cell line SCCA1, the cell number of which is deposited as CCTCC No: C2016131.

The present invention also provides a method for producing the above hybridoma cell line SCCA1, which is obtained by immunizing a mouse with an SCCA1 recombinant protein having the amino acid sequence of SEQ ID NO: 3 as an antigen.

The present invention also provides a monoclonal antibody which is secreted by the above hybridoma cell line SCCA1.

The invention also provides the use of the above monoclonal antibody in preparing a kit for detecting cervical cancer.

The invention also provides a kit for detecting cervical cancer, the kit comprising the above monoclonal antibody.

The invention also provides an enzyme-linked immunoassay kit for detecting cervical cancer, the kit comprising a microplate coated with the above monoclonal antibody.

The monoclonal antibody against cancer diagnosis of the present invention is secreted by the hybridoma cell line SCCA1, and the hybridoma cell line SCCA1 has been deposited with the China Center for Type Culture Collection (Wuhan University, China) on June 23, 2016. ), the deposit number is CCTCC No: C2016131.

Compared with the prior art, the present invention has the following beneficial effects:

1. The monoclonal antibody secreted by the hybridoma cell line SCCA1 of the present invention has good specificity, and the in vitro diagnostic kit for detecting cervical cancer based on the invention provides rapid and specific establishment for different clinical and practical needs. Sensitive detection method;

2. The kit for quantitatively detecting cervical cancer according to the present invention not only has a fast detection speed but a small amount of blood specimens, and uses a protein chip method to detect the cross-reaction of SCCA antibodies with other similar antibodies, and the sensitivity of the detection results is high, and the clinician is provided with high sensitivity. More accurate test results greatly improve the monitoring and management of cervical cancer.

DRAWINGS

Figure 1 is a graph showing the titer of mice after routine immunization four times in Example 1 of the present invention.

detailed description

The technical solutions of the present invention are further illustrated by the following specific examples, which are not intended to limit the scope of the present invention. Some non-essential modifications and adaptations made by others in accordance with the teachings of the present invention are still within the scope of the present invention.

Example 1 Preparation of hybridoma cell line SCCA1

Includes the following steps:

1. Preparation of antigen (SCCA1 recombinant protein)

Includes the following steps:

1) Construction of pET28a-SCCA1 recombinant vector

According to the DNA sequence of human SCCA1 (full length 1173 bp) provided by Genbank (NC_000018.10), the sequence was artificially synthesized, the N-terminal signal peptide was removed, and three pairs of primers were designed, and NdeI and XhoI were introduced from the 5' ends of the two primers, respectively. The cleavage site was amplified by conventional PCR method (vector pET-28a as template) to obtain four fragments of SCCA1 gene, which respectively expressed the four sequences of SCCA1 protein: the first segment: the 93rd to the 159th amino acid (SEQ ID No: 1); second paragraph: amino acids 44 to 102 (SEQ ID No: 2); third paragraph: amino acids 52 to 112 (SEQ ID No: 3); fourth paragraph: full length , amino acid from position 1 to position 390 (SEQ ID No: 4).

The vector pET-28a (commercially available) and the SCCA1 gene fragment purified by agarose gel were double-digested with NdeI and XhoI, and the purified digested product was ligated with T4 DNA ligase to obtain recombinant plasmid pET-28a. -SCCA1-1, pET-28a-SCCA1-2, pET-28a-SCCA1-3, pET-28a-SCCA1-4, transforming the ligation product into E. coli DH5α, containing ampicillin Clones were selected on LB plates, and plasmids were prepared in small amounts. Positive clones were screened by double enzyme digestion/PCR. The sequencing results showed that the recombinant SCCA1 fragments were identical to the designed sequences.

2), expression of SCCA1 recombinant protein

After the recombinant plasmid was verified by sequencing, it was transformed into E. coli (BL21) and cultured in LB medium containing ampicillin. Positive clones were selected on LB plates and identified by plasmid digestion. Plasmids were prepared in small amounts and digested with double enzymes. Positive clones were screened by PCR to obtain 4 recombinant plasmid engineering strains containing SCCA1.

Four recombinant plasmid-engineered strains containing SCCA1 were separately cultured in LB medium containing 100 μg/mL ampicillin, A ₆₀₀ was between 0.5-06, and then Isopropylβ-D-1-thiogalactopyranoside was added at a final concentration of 0.5 mM. IPTG) was induced at 37 °C for 4 h. After the induction, the bacterial solution was centrifuged at 4,000 rpm for 10 min. The cells were collected and washed with PBS. The pellet was resuspended in PBS, placed in an ice bath, centrifuged at 12000 rpm for 20 min after sonication, supernatant and sedimentation. SDS-PAGE electrophoresis showed that the expressed SCCA1 recombinant protein was cytosolic insoluble.

3) Purification and quantification of four groups of SCCA1 recombinant proteins

The cells expressed in a large amount were subjected to sonication, centrifuged, and then subjected to inclusion washing. After washing, the protein was purified using a GE Healthcare Smith Trap FF purification column (reagent preparation and purification according to the product specification). The finally obtained SCCA1 recombinant proteins were SCCA1-1, SCCA1-2, SCCA1-3, SCCA1-4, respectively, and analyzed by SDS-PAGE electrophoresis, and the concentration was measured by BCA protein quantification kit. Among them, the recombinant protein SCCA1-4 (SEQ ID No: 4) was used as a standard protein of the kit of the present invention.

2. Establishment of murine anti-SCCA hybridoma cell line

Includes the following steps:

a. 2 female BALB/c mice (1#, 2#, 3#) with 6-8 weeks old and about 18 g body weight were selected. After 1 week of adaptive feeding, negative blood was collected for comparison.

b. The immunization procedure uses 4 basic immunizations and 1 booster immunization. Using a medium-range immunization protocol (0.3 mL/only, 2 weeks/time), the first immunization (50 μg/mouse) was stirred with an equal volume of Freund's complete adjuvant. Emulsification, multiple injections in the back subcutaneous, immunogen using the first three groups of SCCA1 recombinant protein of step 1 (SCCA1-1, SCCA1-2, SCCA1-3, ie SEQ ID No: 1, SEQ ID No: 2, and SEQ ID No :3). Thereafter, the immunization was performed 3 times with the immunogen and an equal volume of Freund's incomplete adjuvant, and the titer was measured 7 days after the 4th routine immunization. The results are shown in Fig. 1 and Table 1. From the results of Fig. 1 and Table 1, it is known from Fig. 1 and Table 1. The titer of immunized mice is obviously above 1:320000, ready to strengthen immunity;

Table 1 Determination of titer after four immunizations in mice

Dilution of serum(1:X)	1#mouse	2#mouse	3#mouse
5000	2.306	2.442	2.016
10000	1.957	2.136	1.857
20000	1.592	1.596	1.498
40000	1.137	1.071	1.007
80000	0.822	0.779	0.753
160000	0.458	0.454	0.412
320000	0.355	0.289	0.212
Blank	0.076	0.096	0.082

c. Strengthen the immunization without adjuvant, strengthen the immunization dose to 50μg/only, and strengthen the eyeball for 3 days, take the eyeball to collect blood, separate the serum for preservation, and take the spleen at the same time;

d. Mix the spleen cells with the myeloma cells at a cell number of about 4:1, and fuse them under the fused action of polyethylene glycol (PEG, molecular weight 1450), and fuse the cells in HAT selective medium (25). The culture was carried out in -046-CI corning cellgro. After 10 days, positive hybridoma cells reactive with SCCA1 protein were screened by indirect ELISA, and the positive hybridoma cells obtained by screening were expanded and cultured. Two days later, the tagged protein was used (His -tag) exclusion of hybridoma cells to rescreen for hybridoma cells directed against the SCCA1 protein but not the tag;

e. The obtained positive hybridoma cells were subcloned at least twice more by limiting dilution method, and each subcloning was cultured with HT selective medium (25-047-CI corning cellgro), and subcloned 8-10 days later. The ELISA screen was selected until the monoclonal cell positive rate was 100%, and three hybridoma cell lines SCCA1-1, SCCA1-2, SCCA1-3 capable of stably secreting monoclonal antibodies were obtained.

3. Preparation of murine anti-SCCA monoclonal antibody and identification of monoclonal antibody subtype

Take 60ul of hybridoma cell line SCCA1 (3 hybridoma cell lines SCCA1-1, SCCA1-2, The culture supernatant of SCCA1-3) was identified as a subtype of monoclonal antibody secreted by the hybridoma cell line SCCA1 according to the mouse monoclonal antibody subtype identification reagent strip (purchased from Raybiotech, USA), and the results showed that the hybridoma cell SCCA1 The antibody subtype of the supernatant was IgG1.

Example 2 Preparation and Purification of Mouse Anti-SCCA Ascites Monoclonal Antibody

Includes the following steps:

a. 8-12 weeks old female healthy BALB/c mice were selected, intraperitoneal injection of norparin, 0.5 mL/head; after 7-10 days, each mouse was intraperitoneally injected with 1*10 ⁶ to 5*10 ⁶ The three monoclonal hybridoma cell lines of Example 1 were SCCA1-1, SCCA1-2, SCCA1-3, and two mice were injected per monoclonal hybridoma cell. Note that the cells should be blown from the culture dish or the cells should be diluted with PBS or Serum-free medium;

b. Centrifugal ascites at 10,000 r/min for 15 min to remove cellular components and other sediments, fats, oil layers, etc., and collect the intermediate layer;

c. Saturated ammonium sulfate precipitation: Pipette 5 mL of the intermediate layer into a small beaker, and add 5.0 mL of PBS containing 0.22 μm filter membrane while stirring; after mixing well, add 10 mL of saturated ammonium sulfate solution (pH 7.4) dropwise. Continue stirring slowly for 30 min; after standing for 2 h, centrifuge at 10,000 r/min for 15 minutes, discard the supernatant, resuspend the pellet with 0.22 μm filter in PBS, and then pass the resuspension through a 0.22 μm filter;

d. According to different subtypes of antibodies, different purification columns of GE Healthcare are selected. The columns of IgG and IgM have corresponding instructions. According to the instructions, different buffers are used for purification: IgG antibody purification is taken as an example. The column is equilibrated with buffer, elution buffer and regeneration buffer, usually equilibrating 5 column volumes, then the ascites resuspension of step c is loaded at 1 mL/min, and the binding buffer is equilibrated after loading. Then, wash the supernatant to the baseline position, collect the antibody peak; dialyze the antibody with PBS buffer, measure the antibody concentration with the BCA Protein Assay Kit 23225 Thermo Scientific, and dispense the antibody. Freeze at -70 °C.

Table 2 Comparison of three groups of monoclonal antibodies after purification

From the results in Table 2, the third group of SCCA1-3 recombinant protein (SEQ ID No: 3, antigen) was paired with a commercially available detection antibody (abcam, ab190756), and the SCCA1 protein was detected to have high expression in cervical cancer serum. While the other two groups of antibodies have a higher specific binding to the full-length recombinant protein SCCA1-4 (SEQ ID No: 4), they bind to the SCCA1 protein in the serum of cervical cancer. Therefore, the kit of the fourth embodiment of the present invention uses a monoclonal antibody secreted by the third group of hybridoma cell line SCCA1-3 as a capture antibody, which has been deposited with the China Center for Type Culture Collection on June 23, 2016. (in Wuhan University, China), the deposit number is CCTCC No: C2016131.

Example 3 Specific identification of the monoclonal antibody of Example 2

In the present embodiment, the SCCA1 recombinant antigen and the CA125, CA153, CEA, AFP, HE4, and GP73 proteins described in Example 1 were used as coating antigens, respectively, and the monoclonal antibody prepared in Example 2 was used as a recognition antibody. Indirect ELISA was used to detect SCCA.

1), the coating of the microplate

The coating solution was diluted to 1 μg/mL with a coating solution (Na ₂ CO ₃ 1.5 g, NaHCO ₃ 2.9 g, Na ₂ N ₃ 1.2 g, ddH ₂ O to 1 L, pH adjusted to 9.6), and mixed uniformly. To a 96-well microtiter plate, 100 μL per well, the plate was sealed at 4 ° C overnight.

2), the closure of the enzyme plate

A PBS containing 5% skim milk was used as a blocking solution. First, the overnight coated enzyme plate was patted dry, 200 μL/well blocking solution was added, blocked at 37 ° C for 2 h, and the plate was washed 6 times with a plate washer, and the plate was patted dry at 4 ° C, or -20 °C long-term preservation.

3), indirect ELISA detection method

The monoclonal antibody prepared in Example 2 was added to the sealed plate, 100 μL/well, and incubated at 37 ° C for 1 h. After washing the plate 6 times with a plate washer, the enzyme plate was patted dry, and then a certain concentration was added. Biotin-labeled goat anti-Mouse IgG antibody (purchased from Raybiotech, USA), 100 μL/well, incubated at 37 ° C for 1 h; after washing, streptomycin-labeled horseradish peroxidase (HRP) (purchased from Raybiotech, USA) 100 μL/well, incubated at 37 ° C for 1 h; after washing the plate, TMB coloring solution was added, and after color development was completed, 2 M concentrated sulfuric acid was added to terminate the color development, and OD _{450 was} measured with a microplate reader (Biotek, USA). The cross-reactivity was subjected to an alignment analysis, and the final result showed that the monoclonal antibody prepared in Example 2 recognized only SCCA1.

Example 4 Enzyme-linked immunosorbent assay kit for detecting cervical cancer

The enzyme-linked immunosorbent kit for detecting cervical cancer of this embodiment includes the following components:

1. ELISA microplate: coated antibody-monoclonal antibody, and blocked with 5% BSA, 0.2% sucrose in PBS, monoclonal antibody secreted by hybridoma cell line SCCA1, the accession number is CCTCC No :C2016131.

2. Specimen dilution 15 ml: Add 0.5% BSA, 0.05% Tween 20 in PBS buffer.

3. Detection antibody dilution 15 ml: Add 0.5% BSA, 0.05% Tween 20 in PBS buffer.

4. Detection antibody: anti-SCCA1 detection antibody lyophilized powder (abcam, ab190756) labeled with HRP.

5, standard: SCCA1 recombinant protein standard - dry powder recombinant protein SCCA1-4.

6. 10X washing solution 30 ml: 0.2 M Tris-HCl, 5 M NaCl, 0.5% Tween 20.

7. Substrate 20ml: TMB solution.

8. Stop solution 10 ml: 2M H ₂ SO ₄ .

9, kit product manual, packaging box, plastic film.

The operation procedure of the enzyme-linked immunosorbent kit for detecting cervical cancer of this embodiment is as follows:

1) Add SCCA protein standards and dilutions (1000 ng/ml, 333 ng/ml, 111 ng/ml, 37.1 ng/ml, 12.3 ng/ml, 41 ng/ml, 13 ng/ml, 0 ng/ml) to the microplate. 30 serum samples to be tested (samples from Zhongshan Second Hospital), two replicates per sample, 100 ul per well, and 40 minutes at 37 °C;

2) Configuring 1X washing liquid to wash the plate 5 times on the washing machine for 10 minutes;

3) Add the detection antibody dilution solution to the microplate and mix it in a microplate for 40 minutes;

4) Wash again and add substrate reaction for 10 minutes, add stop solution to develop color, and read on the microplate.

Calculate the standard curve based on the readings, and obtain a linear relationship between the reading and the SCCA protein standard. Substituting the OD value of the sample into a linear formula yields the sample content. The whole process is no more than one and a half hours.

Test Example 1 Mass analysis of the enzyme-linked immunoassay kit of Example 4.

1, the sensitivity of the kit

The experimental procedure was carried out according to the kit instructions. The zero calibrator (A) was used as a sample to measure 20 times, and the mean and standard deviation were calculated. The concentration obtained by substituting the absorbance value obtained by adding the average value of the A value plus the standard deviation of 2 times into the standard curve equation is the lowest detection amount.

According to the data, the scatter plot and the standard curve equation y=0.971x+1.473 are obtained, and the R-squared value is 0.996.

The average value of the dilution test A is 0.01025, the standard deviation is 0.003552242, the minimum test limit of the dilution test, and the sensitivity is OD value 0.017354483. That is, the minimum detection limit of SCCA detection by the enzyme-linked immunoassay kit of Example 4 is 0.58 ng/mL, and the linear range is: 0 ng/mL-70 ng/mL.

2, the specificity of the kit (cross reaction)

A. The CA125, CA153, CEA, AFP, HE4, and GP73 antigens were diluted to a certain concentration (see Table 4) and then tested using the kit of Example 4. The results are shown in Table 3 and Table 4, wherein Table 3, 1, 2 is a standard curve, and 3, 4, and 5 columns are data for detecting human serum samples by cross antigen. As a result, no significant cross-reaction was observed at a concentration of 1000 ng/mL or 1000 U/ml of each cross antigen.

Table 3 SCCA1 data results for cross-antigen test samples

Mark		Mark	3	4	5
2.214	2.129	0.047	0.049	0.052
1.531	1.352	0.048	0.047	0.051
0.789	0.739	0.047	0.046	0.049
0.391	0.287	0.049	0.049	0.047
0.166	0.147	0.062	0.054	0.047
0.101	0.097	0.049	0.048	0.053
0.094	0.062	0.05	0.053	0.057
0.05	0.05	0.061	0.054	0.055

Table 4 SCCA1 data results for cross-antigen test samples

Cross antigen	SCCA test value
CEA (1000ng/ml)	0.5
CEA (500ng/ml)	0.5
GP73 (1000ng/ml)	0.4
GP73 (500ng/ml)	0.5
AFP (1000ng/ml)	0.9
AFP (500ng/ml)	0.5
CA153 (414u/ml)	0.6
CA153 (207u/ml)	0.6
CA125 (1000u/ml)	0.5
CA125 (500u/ml)	0.5
HE4 (1000ng/ml)	0.5
HE4 (500ng/ml)	0.6

B. According to the relevant statistics, the reference value of the female SCCA1 healthy population is: 1.5ng/mL concentration, 1.5ng/mL as the negative judgment threshold, and more than 1.5ng/mL is true positive, by detecting the cervical scale Cancer patients (Zhongshan Second Hospital) serum 30 cases, the standard curve data shown in Table 5, the standard curve equation is obtained, the number of true positive samples divided by 30, that is, the total number of clinical diagnosis of cervical squamous cell carcinoma samples, is sensitive Sex, the results are shown in Table 6.

table 5

A scatter plot is obtained from the standard curve, and the standard curve equation y=1.127x+1.455, which converts the OD value into a concentration.

Table 6

From the results of Table 6, it can be seen that there are 23 cases greater than 1.5 ng/mL, 23/30 = 76.6%, that is, the sensitivity of SCCA1 as a detection of cervical cancer is 76.6%.

3, the stability of the kit

The concentration of the antigen standard was 1.09 to 70 ng/ml, and the OD value gave a standard curve equation of y=1.190x+1.276, and the R square value was 0.994.

Table 7

The kit was tested for stability after 7 days under heat at 37 ° C. Two controls were used, with a C1 concentration of 17.5 ng/ml and a C2 concentration of 4.37 ng/ml. The following tests were performed:

The minimum detection limit: the average OD value of 20 blank samples + 2SD as the signal value of the lowest detection limit, which is calculated as the lowest detection limit.

Repeatability: It is repeated for 10 samples of different concentrations, and the average value divided by the standard deviation is the repeatability measurement-precision.

Accuracy: The sample containing high concentration of SCCA is added to the low concentration sample, and the two sample volumes are not more than 1:9. The SCCA values of the three samples are measured, and the recovery rate is calculated by formula to evaluate the accuracy.

In the formula:

R—recovery rate;

V—the volume of the sample A solution;

V0—the volume of the sample B solution;

C———the concentration of the sample B after adding the liquid A;

C0—the concentration of sample B solution;

Linearity: After diluting a high-value sample close to the upper limit of measurement, no less than 5 gradients are used for measurement. The sample values and dilution ratios are fitted by least squares method, and R is not less than 0.99.

The results are shown in Table 8:

Table 8

Performance analysis after 37 days acceleration at 37 degrees

Minimum detection limit	0.6ng/ml
Repeatability C1	6%
Repeatability C2	3%
Accuracy	96.70%
Linear	0.9999

Test Example 2 Comparison of the correlation between the kit of Example 4 and the existing kit

The kit of the invention is compared with a diagnostic kit of an imported company commonly used in the market for the same batch of clinical samples (55 patients serum), and the test results are shown in Table 9:

Table 9 Comparison of test data of two kits

Using the sample detection value as the X-axis, the detection value of a diagnostic kit of an import company is plotted as a scatter plot on the Y-axis, and the trend line and the formula y=1.198x+0.072 are generated, and the R-squared value is 0.944, and the result shows that the reagent of the present invention The cassette has a good correlation with other kits and requires less time (the kit of the invention) It takes only one and a half hours, and an imported company diagnostic kit takes 2.5 hours).

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims (6)

1. A hybridoma cell line SCCA1, characterized in that the cell line is deposited under the number CCTCC No: C2016131.
2. The method for producing a hybridoma cell line SCCA1 according to claim 1, wherein the hybridoma cell line SCCA1 is obtained by immunizing a mouse with an SCCA1 recombinant protein having the amino acid sequence of SEQ ID NO: 3 as an antigen.
3. A monoclonal antibody produced by the hybridoma cell line SCCA1 of claim 1.
4. Use of the monoclonal antibody of claim 3 for the preparation of a kit for detecting cervical cancer.
5. A kit for detecting cervical cancer, characterized in that the kit comprises the monoclonal antibody of claim 3.
6. An enzyme-linked immunosorbent kit for detecting cervical cancer, characterized in that the kit comprises a microplate containing the monoclonal antibody of claim 3.

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