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A kind of anti-ca125 nanobody 5d2 and its application

A technology of CA125 and nano-antibodies, which is applied in the field of immunology, can solve the problems of specific antigen-antibody binding reactions and affect detection efficiency, and achieve excellent detection performance

Active Publication Date: 2022-04-22
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the large molecular weight of CA125 and the large number of glycosylation sites, it is difficult for conventional antibodies to fully recognize some epitopes hidden in gaps or cavities. If the antibody recognizes the epitope is too single or the sites are too close or Overlapping will cause the specific antigen-antibody binding reaction to be affected, which will seriously affect the detection efficiency

Method used

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  • A kind of anti-ca125 nanobody 5d2 and its application
  • A kind of anti-ca125 nanobody 5d2 and its application
  • A kind of anti-ca125 nanobody 5d2 and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Preparation of natural CA125 antigen

[0030] 1.1 Purification of CA125 in ascites of ovarian cancer patients by saturated ammonium sulfate precipitation

[0031] Stir 2L of ascites from a patient with ovarian cancer in an ice bath on a magnetic stirrer, slowly add 228g of ammonium sulfate to make the final concentration of ammonium sulfate 20%, let it stand overnight at 4°C, centrifuge at 10,000g for 10 minutes, and save the supernatant. Stir the supernatant in an ice bath on a magnetic stirrer, slowly add 524 g of ammonium sulfate to make the final concentration of ammonium sulfate 60%, let it stand overnight at 4° C., centrifuge at 10,000 g for 10 minutes, and retain the precipitate. Use about 400ml of PBS solution to blow and mix the precipitate, centrifuge at 10000g for 10 minutes, and save the supernatant. Concentrate the supernatant to 200ml with 4000g of 100kD ultrafiltration tube, and divide into 50ml / tube.

[0032] 1.2 Purification of CA125 in asc...

Embodiment 2

[0036] Example 2. Construction and screening of Nanobody phage display library

[0037] 2.1 Immunity of alpacas

[0038] A healthy adult alpaca was selected, and the CA125 antigen (GenBank: AAO52683.1, constructed on the pCDNA3.1 vector through HindⅢ and EcoRI restriction sites, expressed by human 293 cells using conventional molecular biology techniques) and Freund's adjuvant was mixed at a ratio of 1:1, and the alpaca was immunized with 6-7 μg / kg subcutaneously at multiple points on the back for a total of four immunizations with an interval of 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0039] 2.2 Isolation of camel-derived lymphocytes

[0040] Lymphocytes were separated from the collected alpaca peripheral blood using the Camel Peripheral Blood Lymphocyte Separation Solution Kit (Tianjin Haoyang Company, Cat. No. LTS1076). 7 Add 1ml RNA isolation reagent to each living cell, take 1ml for RNA ex...

Embodiment 3

[0069] Example 3. Preparation of Nanobody 5D2

[0070] 3.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL21(DE3)

[0071] Inoculate the original strain TG1 Glycerolbacterium containing Nanobody nucleic acid in 5ml of fresh LB-A medium at a ratio of 1:1000, and culture overnight at 37°C and 200rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the instructions. After verification, transform 1 μl of the above plasmid into 100 μl competent cells, mix gently, place on ice for 30 minutes, heat shock in a water bath at 42°C for 90 seconds, and cool in an ice bath for 3 minutes. Add 600 μl LB medium to the centrifuge tube, shake and incubate at 37°C for 60 minutes. Take 100 μl of the supernatant, spread it on the LB-A plate with a triangular spreader, and culture it upside down at 37°C overnight.

[0072] 3.2 Induced expression of nanobodies

[0073] The above monocl...

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Abstract

The invention discloses an anti-CA125 nanobody. The nanobody has three unique complementarity determining regions CDR1, CDR2, and CDR3. The invention also provides an expression vector containing the coding sequence of the nanobody variable region, and containing The host cell of the expression vector, and the application of the nanobody in detecting the CA125 content in serum. The nanobody provided by the present invention has specific recognition and binding ability to CA125, the affinity of the nanobody can reach 3.051E‑10, has a unique epitope recognition site, and can be obtained in CA125 serum detection, especially in the double antibody sandwich method Excellent detection effect.

Description

technical field [0001] The invention discloses a nanobody and belongs to the field of immunology. Background technique [0002] CA125 (cancer antigen 125) ovarian cancer-associated antigen was discovered in 1981, and it is a related antigen of ovarian epithelial carcinoid. It is secreted by epithelial cells in the embryonic stage, and it is not secreted or rarely secreted under normal circumstances. However, when malignant lesions occur in the ovary, even if there is no clinical manifestation or pathologically difficult to identify, the CA125 value will increase, so it is a better ovarian Cancer diagnosis and screening indicators, and is closely related to the metastasis and prognosis of ovarian cancer. CA125 was initially recognized and confirmed by Bast et al. through the ovarian cell line OVCA433 immunogen, which mediated the response of the mouse monoclonal antibody OC125. CA125 antigen is a glycoprotein with a relative molecular mass of 200ku, which has both membrane-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C12N15/70G01N33/535G01N33/543G01N33/574
CPCC07K16/3069C12N15/70G01N33/57449G01N33/54306G01N33/535C07K2317/565C07K2317/56C07K2317/569C07K2317/52C07K2317/92
Inventor 宋海鹏刘原源于建立王准曹慧古一李飞
Owner 深圳市国创纳米抗体技术有限公司
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