Detecting antibody for CD6-resistant monoclonal antibody T1h and application

An antibody, whole antibody technology, applied in applications, biological tests, measurement devices, etc., can solve the problems of lack of neutralizing antibodies, no neutralizing antibodies, and inconvenient pharmacokinetic analysis.

Active Publication Date: 2016-04-20
BIOTECH PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This brings inconvenience to the pharmacology and pharmacokinetic analysis of T1h clinical trials; at the same time, there is no T1h neutralizing antibody,

Method used

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  • Detecting antibody for CD6-resistant monoclonal antibody T1h and application
  • Detecting antibody for CD6-resistant monoclonal antibody T1h and application
  • Detecting antibody for CD6-resistant monoclonal antibody T1h and application

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment 1

[0061] Specific Example 1, Biopanning of anti-T1h single-chain antibody

[0062] A series of gene cloning methods were used to transform the vector pcom3 vector (purchased from China Plasmid Vector Strain Cell Line Gene Collection Center) to be used for the construction and expression of phage single-chain antibody library. The transformed vector is named pScFvDisb-s, and its plasmid map is as follows: figure 1 As shown, and based on this vector, a fully synthetic phage antibody library was constructed.

[0063] Use T1h as the antigen to coat the immunotube, the amount of antigen coating is 5 μg / 500 μl / tube, and coat overnight at 4°C. The immunotube and the fully synthetic phage antibody library were then blocked with 4% skimmed milk powder / PBST for 1 hour at room temperature. The blocked phage antibody library was added to the immunotube for antigen-antibody binding, and the amount of phage input was about 10 9 ~10 12 , and reacted at room temperature for 1 h. Unbound ph...

specific Embodiment 2

[0066] Specific Example 2, Identification of Anti-T1h Phage Single-Chain Antibody Positive Clones

[0067] After three rounds of screening, well-separated monoclonal colonies were picked and inoculated in a 96-well deep-well plate supplemented with 2YTAG liquid medium, cultivated at 37°C and 220 rpm until the logarithmic growth phase, and about 10 10The helper phage M13KO7 was statically infected at 37°C for 30min. Centrifuge at 4000rpm at 4°C for 15min, discard the supernatant, resuspend the pellet with 2YTAK, and culture overnight at 28°C at 220rpm. The amplified phage supernatant was drawn for ELISA identification. Screened monoclonal antibodies 3E7, B10 and B2 with higher affinity, such as figure 2 As shown, the affinity of 3E7 from left to right is 0.0395 for IgG1Fc, 0.035 for IgG1, 0.0065 for CD6-ECD, 2.0065 for T1h, and 0.011 for BSA, and the affinity for T1h is higher. The 3E7, B10 and B2 monoclonal antibodies were identified as antibodies with different sequences ...

specific Embodiment 3

[0068] Specific example 3, gradient dilution phageELISA compares the affinity of anti-T1h single-chain antibody

[0069] The clones obtained in Example 2 were subjected to display and purification of monoclonal phage, and a phage gradient dilution ELISA experiment was performed to identify the affinity of phage-abs.

[0070] Coat T1h with pH 9.6 carbonate buffer solution, 200ng / well / 100μl, overnight at 4°C. Wash with PBST three times, block with 4% milk-PBST at 37°C for 1h. The purified phage was diluted five-fold with 4% milk-PBST, and 100 μl of the diluted sample was added to each well, and allowed to stand at room temperature for 1 hour. The ELISA plate was washed with PBST, the HRP-anti-M13 monoclonal antibody diluted in 4% milk-PBST was added to the ELISA plate, and left at room temperature for 1 hour. TMB color development kit was used for color development at room temperature for 10 min. with 2MH 2 SO 4 Stop color development, 50 μl / well. 450nm / 630nm readout. The...

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Abstract

The invention relates to the technical field of biological pharmacy, in particular to an obtaining method, a preparing method and a detecting method of a detecting antibody for a CD6-resistant monoclonal antibody T1h. According to the screening technology of a total-synthesis phage antibody library, single-chain antibodies of the antibody T1h are subjected to biopanning, single-chain antibodies of bacteriophage of the antibody T1h are subjected to positive clone identification, gradient-dilution phage ELISA is carried out, the affinity of the single-chain antibodies of the antibody T1h is compared, then all antibodies of the antibody T1h are prepared, and a new specificity monoclonal antibody of the antibody T1h is obtained. A heavy-chain variable region of the antibody comprises the amino acid sequence of SEQ.1, and a light-chain variable region of the antibody comprises the amino acid sequence of SEQ.2. The antibody is used for detecting the concentration of T1h in the blood and other body liquid in the clinical test, the blood concentration of the T1h can be sensitively and rapidly detected, a reliable research method is provided for the clinical test pharmacology and medicine generation analyzing of the T1h, and meanwhile the antibody can be used for neutralizing the T1h antibody.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to the acquisition, preparation and detection method of a detection antibody against CD6 monoclonal antibody T1h. Background technique [0002] CD6 is a type I integral membrane glycoprotein and a member of the scavenger receptor cysteine-rich superfamily SRCRSF (scavenger receptorcysteine-rich superfamily), with three cysteine-rich functional regions SRCR (scavenger receptorcysteine- rich), whose juxtamembrane domain (SRCR3) contains the ALCAM-binding site. CD6 is mainly expressed by T cells and interacts with human leukocyte activated adhesion factor ALCAM (activatedleukocyteladhesionmolecule, CD166); CD6 participates in T cell activation, regulates T cell function and mediates cell-cell adhesion. CD6 molecules can form complexes with TCR / CD3 and participate in the formation of immune synapses (immunological synapse, IS); mature immune synapses are divided into central...

Claims

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Application Information

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IPC IPC(8): C07K16/42C12N15/13C12N15/85C12N5/10C12N1/21C12N1/19G01N33/68C12R1/19C12R1/85
CPCC07K16/005C07K16/4241C07K2317/52C07K2317/55C07K2317/56G01N33/6854
Inventor 周海平李晓敏王婷张利萍裴爽吴俊玲田睿白义白先宏
Owner BIOTECH PHARMA CO LTD
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