Case Study

AlpalifeBio Generated VHH

Antigen targets that have been immunized for nanobody discovery by AlpalifeBio's Nanobody R&D team include but are not limited to PV16, HPV18, AAV2, AAV5, AAV8, AAV9, plant viruses, virus-like particles such as HPV58, Human claudin-18.2, Amyloid protein, Procalcitonin, Tac2, PD-1, PD-L1, CTLA4 (CD152), CD3 (epsilon& gamma), CD3 epsilon peptide fragment, Integrins (alpha 4 & beta 7), IL-2R beta & gamma, IL-5, IL-6, IL-25, IL-33, TSLP, PCSK9, KLH coupled peptides and tags (HA, Myc, Flag, V5, 6His), GST, CD5, CD7, CD19, CD20, CD22, CD24, CD28, CD33, CD38, CD40 , CD70, CD73, CD79, CD85d (LILRB2), CD120b, CD123, CD134, CD167, CD276 (B7-H3) VEGF, Tigit , B7-H4, B7-H7, CD276 (B7-H3), GRP78 (BiP, HSP5a ), RAGE, HSP70, HSP90, ROR1, IGF-1R, IGF-2R, IGFBP5, GPC3, SARS-CoV-2, EBV gH/gL, S100A8 & S100A9, KRAS, CXCL1, GUCY2C, hTPX2, Nectin-4, GPRC5D , CCR8, FGF, Trop2, EPO, DLL3, EGFR VIII, HSA, ADGRE2, apolipoprotein A-1, apolipoprotein B, ACE2, Carbonic anhydrase II, LRRC15, Hemoglobin, SERPINA3, Transferrin, human IgG1 Fc, mouse IgG2a Fc, GFP , mCherry, rabbit IgG and undisclosed antigens provided by customers. 

Case Study: Nanobody/VHH Discovery

Screening for nanobodies that can cross-react with human, cynomolgus and mouse antigens. Analyze the sequence and structure of the antigen, and determine the co-immunization scheme of human antigen and mouse antigen

01 -  Results

After 4 times of immunization, the titers of serum anti-human, cynomolgus monkey and mouse antigens all exceeded 104, and the titers of serum anti-human and cynomolgus monkey antigens both reached 105, indicating strong cross-species responses were produced.
 02 - VHH Phage Library
Total RNA was extracted from PBMCs isolated from 50ml of peripheral blood, and the electrophoresis of RNA sample was run to make sure of good quality mRNA.

The RNA was reverse transcribed into cDNA, and after two rounds of PCR amplification, Diverse VHH sequences were obtained for following ligation.
The VHH DNA sequences were ligated to the phagemid, and finally transformed into Escherichia coli competent cells to obtain a bacterial VHH library. After the helper phage infection and induction, the VHH phage library was obtained. The positive rate of cloning is 100% after single-colony PCR and there is no repetitive sequences among 51 positive clones after sequencing analysis. Phage library capacity > 108.
03 - VHH Selection

Screening Process: Human (H2) and mouse antigen (M2) with different labels, and monkey antigen (C) were used for three rounds of biopanning, respectively. BSA was used as a control to detect the enrichment. Afterwards, single clones were randomly picked for Elisa analysis to verify the cross-reactivity with the three antigens. 92 positive clones were sent for sequence analysis. Among 92 VHH sequences, 28 nanobodies with different amino acid sequences of CDR1, CDR2 or CDR3 were obtained.  

 ELISA verification results
04 - Expression and Verification

10 nanobodies selected for their cross-reaction with human, mouse, and cynomolgus antigens for expression and purification , and 5.5 mg of each purified nanobody protein was obtained for Elisa analysis. The nanobody with the highest affinity was selected to test the kinetic data (Octet).

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