Complementarity determining regions (CDRs) are the most important structure parts of an antibody that recognize antigens. The specificity of an antibody is mostly governed by its largest CDR, i.e. CDR H3. Compared with antibodies from other species, bovine antibodies are unusual in having exceptionally long CDR H3 loops; about 10% of bovine antibodies have ultralong CD3 H3 regions (35-60 AAs). Evidence has shown that these ultralong antibodies may be more effective in defense against disease than typical antibodies.
Picture source: Current Opinion in Structural Biology
Alpha Lifetech Inc. is a recognized leader in the field of antibody discovery. We have developed proprietary procedures for antibody discovery. Our optimized library construction and screening processes with phage display technology guarantee successful isolation of high affinity antibodies in a short time. Alpha Lifetech Inc. has successfully produced multiple positive clones for a variety of antigens, ranging from large protein molecules (>400kD) to small haptens (small chemical molecules of ~200 dalton), many of the binders have sub-nanomolar affinities measured by ELISA.
Bovine Ultralong CDR3H Antibody Generation Service Details
|
Steps |
Specification |
Timeline |
|
Step1: Bovine Immunization |
--A healthy, full-aged bovine was immunized 4-7 times
--Serum titers were measured by ELISA and collected peripheral blood |
6-8 weeks |
|
Step2: cDNA synthesis (Clients can provide cells) |
--Total RNA extraction
--RT-PCR
--PCR amplification with Actin-specific primers to identify the quality of cDNA |
1 day |
|
Step3: Ultralong CDR3H antibody Library construction |
--Primer design & synthesis
--PCR amplification of variable region genes of heavy using cDNA as a template
--Plasmid construction & transformation: after enzyme digestion, VHH and phagemid vector were ligated and transformed into TG1 host bacteria by an electric shock to construct antibody libraries
--Identification: 20-50 clones were randomly selected, PCR identification, sequencing and analysis of antibody sequences |
3-4 weeks |
|
Step4: Ultralong CDR3H antibody Library Screening (3 Rounds) |
--The antigenic limit pressure method is used for 3 rounds of panning
--Identification: 96 clones were randomly selected for ELISA titer determination
--Up to 96 positive clones were sequenced, and the 10-15 clones were selected for prokaryotic expression transformation
--10-15 positive clones were expressed to construct VHH antibody supernatant |
3-4 weeks |
|
Ultralong CDR3H Antibody Production (optional) |
--Gene synthesis, subcloning, plasmid preparation
--Transient expression and purification
--QC analysis |
2 weeks |
|
Affinity Ranking (optional) |
--Affinity measured by Biacore 8K |
1 weeks |
Deliverables
--Experimental report: including detailed construction procedures and representative sequence information
--Ultralong CDR3H antibody library 2ml, >1⨯1013 /ml phage particles, Effective storage capacity>109
--10-15 positive clones were expressed to prepare VHH antibody supernatant
--10-15 expressing strains of positive clone
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