The purity of all our antibodies is routinely evaluated using SDS-PAGE. We also clean all our hybridomas to guarantee they are free of mycoplasma and other bacteria. Additionally, our hybridomas and recombinant antibodies are extensively sequenced to validate each clone further.
One aspect that sets NeoBiosciences apart from other antibody manufacturers is that we sequence all our hybridomas and recombinant antibodies. After producing the hybridoma, we validate it using HuProt, Immunohistochemistry (IHC) and Western Blot, sequence (see below), validate the sequence, and validate using HuProt and other applications. This workflow ensures that our products are highly specific and will yield reliable and reproducible results.
Our sequencing protocol requires amplifying the paired VDJ receptor from RNA extracted from the hybridoma. Combining Illumina dual and primer-specific indices, each hybridoma is barcoded with a unique identifier in 96- or 384-well plates. The resultant libraries are pooled and sequenced as a large multiplex pool as single-end reads on Illumina 500-cycle or 600-cycle kits. Then, an automated pipeline demultiplexes the sample barcodes and maps the resulting reads, yielding the sequences of the paired heavy and light antibody sequences inclusive of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, and the isotype of the antibody. These sequences are deposited into a secure, user-friendly database for each client.