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Single domain antibody for resisting CEACAM-5 and application of single domain antibody

A CEACAM-5, single-domain antibody technology, applied in the field of immunoglobulin, can solve the problems of affecting detection efficiency, easy aggregation, low affinity, etc., and achieve the effect of improving detection efficiency and ensuring specificity

Active Publication Date: 2018-10-16
长春力太生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low molecular weight of single-domain antibodies, some structural defects such as low affinity, easy aggregation, and short serum half-life hinder the further application of single-domain antibodies.
In the specific application of CEACAM-5 immunoassay, if the antigenic determinant of CEACAM-5 recognized by the anti-CEACAM-5 antibody is too single or the sites are too close or overlapped, it will cause the specific antigen-antibody binding reaction to be infected, thus Seriously affect the detection efficiency

Method used

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  • Single domain antibody for resisting CEACAM-5 and application of single domain antibody
  • Single domain antibody for resisting CEACAM-5 and application of single domain antibody
  • Single domain antibody for resisting CEACAM-5 and application of single domain antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction and Screening of Anti-CEACAM-5 Single Domain Antibody Phage Display Library

[0026] 1.1 Immunization of alpaca: Select a healthy adult alpaca, mix the recombinant protein CEACAM-5 antigen with Freund's adjuvant at a ratio of 1:1, and inject 6-7 μg / Kg subcutaneously at multiple points on the back The alpacas were immunized four times in total, and the immunization interval was 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0027]1.2 Separation of camel-derived lymphocytes: analyze lymphocytes from the collected camel-derived anticoagulated whole blood according to routine procedures in this technical field, every 2.5×10 7 Add 1mL RNA isolation reagent to each living cell, take 1mL for RNA extraction, and store the rest at -80°C.

[0028] 1.3 Extraction of total RNA: extract total RNA according to routine procedures in this technical field, and adjust the concentration to 1...

Embodiment 2

[0056] Example 2 Preparation of Anti-CEACAM-5 Antigen Single Domain Antibody 2E5

[0057] 2.1 Amplification of single domain antibody original strain TG1 and transformation of single domain antibody recombinant plasmid into Escherichia coli BL21(DE3)

[0058] Inoculate the original strain TG1 Glycerobacterium containing single domain antibody nucleic acid in 5 mL of fresh LB-A medium at a ratio of 1:1000, and culture overnight at 37°C and 200 rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the instructions. After verification, 1 μl of the above plasmid was transformed into 100 μl of competent cells, mixed gently, placed on ice for 30 minutes, heat-shocked in a water bath at 42°C for 90 seconds, and cooled in an ice bath for 3 minutes. Add 600 μl LB medium to the centrifuge tube, shake and culture at 37°C for 60min. Take 100 μl of the supernatant, spread it on the LB-A plate with a triangular spreader, and culture it upside down at 3...

Embodiment 3

[0063] Example 3 Affinity Activity of Anti-CEACAM-5 Single Domain Antibody and CEACAM-5 Antigen

[0064] 3.1 Chip antigen coupling

[0065] Prepare CEACAM-5 antigen with different pH sodium acetate buffer (pH 5.5, pH 5.0, pH 4.5, pH 4.0) to make 20μg / mL working solution, and prepare 50mM NaOH regeneration solution at the same time, use Biacore T100 protein interaction analysis The template method in the system instrument is used to analyze the electrostatic binding between the antigens at different pH conditions and the surface of the chip (GE company). The amount of signal increase reaches 5 times RL as a standard, and the most suitable neutral pH system is selected and Adjust the antigen concentration as necessary as the conditions for conjugation. The chip was coupled according to the template method included in the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the designed theoretical couplin...

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Abstract

The invention discloses a single domain antibody for resisting CEACAM-5. The single domain antibody has three unique complementary determining regions CDR1, CDR2 and CDR3. The invention further provides an expression vector containing a variable region coding sequence of the single domain antibody, a host cell containing the expression vector, fusion protein of a variable region of the single domain antibody and human alkaline phosphatase, application of the single domain antibody in preparing a CEACAM-5 detection kit, a method for CEACAM-5 immunological detection by applying the single domainantibody as well as a corresponding detection kit. The single domain antibody for resisting the CEACAM-5, provided by the invention, has specific recognizing and binding capability for the CEACAM-5;the affinity of the single domain antibody can reach 4.51E-09; the single domain antibody has unique antigenic determinant recognition site and can obtain an excellent detection result in CEACAM-5 immunological detection, in particular to a double antibody sandwich method.

Description

technical field [0001] The invention discloses a polypeptide, more specifically, the invention discloses an immunoglobulin. Background technique [0002] Carcinoembryonic antigen (CEACAM-5, abbreviated as CEA, also known as CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEACAM-5 is a member of the immunoglobulin superfamily and contains seven domains linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. The seven domains include a single N-terminal Ig variable domain and six domains (A1-B1-A2-B2-A3-B3) homologous to the Ig constant domains. CEACAM-5, originally classified as a protein expressed only in fetal tissues, has now been identified in several normal adult tissues. Overexpression of CEACAM-5 has been observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Therefore, CEACAM-5 has been identified as a tumor-associated antigen. CEACAM-5 is readily cleaved from ...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K19/00C12N15/13C12N9/16G01N33/574
CPCC07K16/3007C07K2317/22C07K2317/33C07K2317/56C07K2317/565C07K2317/569C07K2317/92C07K2319/61C12N9/16C12Y301/03001G01N33/57473
Inventor 李文秋谢英林徐雪松王珂刘永红
Owner 长春力太生物技术有限公司
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