Preparation method of monoclonal antibody and kit thereof
A monoclonal antibody and kit technology, applied in the field of monoclonal antibody preparation, can solve the problems of time-consuming, poor quality, false positive sensitivity and specificity, and achieve the effect of simple operation, strong purpose and small workload
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Embodiment 1
[0051] The preparation of embodiment 1NT-BNP protein monoclonal antibody
[0052] (1) Animal immunity
[0053] The NT-BNP antigen was diluted to 1 mg / mL, mixed with an equal volume of Freund's complete adjuvant, and fully emulsified, and 5 6-week-old Balb / c mice were subcutaneously inoculated at multiple points, and the inoculation dose of antigen was 50 μg / mouse. After 3 weeks, the same dose of antigen treated with Freund's incomplete adjuvant was injected, and then the antigen was injected every 2 weeks, repeated 3 times, and the dose of each immunization was 25 μg per mouse. Three days before cell fusion, NT-BNP protein without adjuvant was injected intraperitoneally for a booster immunization, and then cell fusion was performed.
[0054] (2) Preparation of splenocytes
[0055] Three days later, blood was collected from the eyeballs of the mice, and the blood was placed at 37°C for 0.5h, at 4°C for 1h, centrifuged at 3000rpm for 5min, and the separated serum was used as p...
Embodiment 2
[0116] The preparation of embodiment 2NT-BNP protein monoclonal antibody
[0117] Repeat the test once according to the monoclonal antibody preparation method in Example 1,
[0118] Positive clones were screened by the indirect ELISA method of this experiment, and transferred to 24 wells for detection. The screened positive clones are as follows:
[0119] Positive clonal cell lines screened out in table 6
[0120] mother clone number
OD 450
mother clone number
OD 450
1
2.824
9
2.877
2
2.589
10
2.379
3
2.318
11
2.914
4
2.754
12
2.644
5
2.631
13
2.241
6
2.482
14
2.684
[0121] 7
2.863
15
0.07
8
2.375
Negative
3.097
[0122] 2. Establishment of NT-BNP protein competition ELISA method
[0123] Carry out competition ELISA test according to the method of Example 1, add 15C4-HRP to each ho...
Embodiment 3
[0144] The preparation of embodiment 3PCT monoclonal antibody
[0145] (1) Animal immunity
[0146] Dilute the PCT (procalcitonin) antigen to 1 mg / mL, mix it with Freund's complete adjuvant in equal volume, and fully emulsify it, and inoculate 5 6-week-old Balb / c mice subcutaneously at multiple points, and the inoculated antigen dose is 50 μg / Only. After 3 weeks, the same dose of antigen treated with Freund's incomplete adjuvant was injected, and then the antigen was injected every 2 weeks, repeated 3 times, and the dose of each immunization was 25 μg per mouse. Three days before cell fusion, PCT protein without adjuvant was injected intraperitoneally for a booster immunization, and then cell fusion was performed.
[0147] (2) Preparation of splenocytes
[0148] Three days later, blood was collected from the eyeballs of the mice, and the blood was placed at 37°C for 0.5h, at 4°C for 1h, centrifuged at 3000rpm for 5min, and the separated serum was used as positive serum. T...
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