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Methods and systems for generating, validating and using monoclonal antibodies

A monoclonal antibody, antibody technology, applied in the field of characterization and utilization of antibodies, production, production, can solve the problems of expensive and time-consuming antibodies

Active Publication Date: 2016-05-11
CDI LAB +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Antibody production can be expensive and time-consuming, so more cost-effective and less time-consuming methods of high-throughput antibody (especially highly specific antibody) production are desired

Method used

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  • Methods and systems for generating, validating and using monoclonal antibodies
  • Methods and systems for generating, validating and using monoclonal antibodies
  • Methods and systems for generating, validating and using monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0200] Example 1: Subcloning of approximately 17,000 full-length human ORFs into expression vectors .

[0201] Obtained approximately 16,000 unique ORF's ORF collection (Invitrogen, CA), while an additional 1,000 full-length human ORFs were subcloned. constructed for yeast Compatible expression vector (pEGH-A), which produces N-terminal 6xHis6::glutathione-S-transferase (GST) fusion protein in yeast after galactose induction ( image 3 ). Subsequently, all approximately 17,000 human ORFs were subcloned with a success rate of 99% as determined by restriction digestion. In particular, three replicates were prepared for this pool, from one of which the entry DNA (EntryDNA) was extracted, and the quality of the plasmid DNA was checked on an agarose gel. The resulting recombinants were then transformed into bacteria and single colonies were selected on plates containing Amp. For each LR recombinant, four single colonies were picked to generate glycerol stocks, two of which...

Embodiment 2

[0203] Example 2: Design and construction of a microarray of 5000 human antigens.

[0204] Preliminary experiments were performed to test the ability to rapidly purify properly folded recombinant proteins for microarray production. These proteins fall into 5 distinct functional classes: transcription factors and transcription co-regulators, RNA-binding proteins, protein kinases, chromatin-associated and chromatin-modifying proteins, and mitochondrial proteins. Proteins were placed into these categories based on primary sequence, literature and Gene Ontology annotations. Expressed ORFs represent up to 85% (in terms of transcription factors) of all human proteins in the relevant functional class. More than 90% of expressed proteins were purified at levels sufficient for array construction (Ho, S.W., et al., Linking DNA-binding proteins to their recognition sequences by using protein microarrays. Proc Natl Acad Sci USA, 2006. 103(26): p.9940-5).

[0205] Several functional as...

Embodiment 3

[0206] Embodiment 3: the preparation of human antigen protein chip

[0207] To purify the full set of 17,000 human protein antigens from yeast cells, the entire human ORF master set was cloned into yeast in pEGH-A, single colonies were picked and glycerol stocks were prepared. Yeast cells were induced for recombinant protein production and stored at -80°C. To monitor the quality of the induced cultures, 24 random strains were inoculated in duplicate for each batch of culture preparations and first underwent a protein purification step. Using immunoblotting and silver staining techniques, the success rate of culture induction was estimated ( Figure 4 A). Success was only indicated when at least 85% of the purified protein showed a major band within the expected molecular weight range in immunoblot and silver stain analysis. Otherwise, repeat the culture preparation for the failed batch. Using this standard, all 17,000 antigenic proteins were purified with an 85% success ...

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Abstract

Provided herein is an antibody library, wherein the antibody library may comprise a plurality of monoclonal antibodies, monospecific antibodies or immunoprecipitated antibodies. Also provided herein are methods of generating and using such antibody libraries.

Description

[0001] cross reference [0002] This application claims the benefit of US Provisional Application No. 61 / 355,329, filed June 16, 2010, which is hereby incorporated by reference in its entirety. [0003] Statement Regarding Federally Funded Research [0004] This invention was made with US Government support under grants RR020839 and GM076102 awarded by the National Institutes of Health. The government may have certain rights in this invention. Background technique [0005] There is a significant need in the biomedical community for reliable techniques capable of producing reproducible antibody reagents of the highest possible quality. Continued refinement of this technical route will directly benefit the health research community and the larger biomedical community. [0006] The detection or binding of epitopes, antigens or proteins is a large part of the research product industry as well as the diagnostic and therapeutic industries serving academia and the pharmaceutica...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B40/10C40B30/04
CPCG01N33/6845G01N33/6854C07K16/00C07K2317/33C07K1/047
Inventor 杰夫·D·伯克恒·朱塞斯·布莱克肖丹尼尔·J·艾兴格伊格纳西奥·皮诺
Owner CDI LAB
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