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You can pick out the top 0.001% clones from the entire splenocyte population with the help of our microfluidics plasma cell discovery (PCD) platform. The scale of phage display meets the power of in vivo affinity maturation--antibody discovery has never been so robust.
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Boster Bio's plasma-cell discovery (PCD) technology uses flow cytometry to screen individual cells treated with proprietary chemistry. This unique compound keeps B cells from secreting antibodies, retains antibodies on cell membranes. Subsequently the splenocytes are incubated with fluorochrome conjugated antigens and the plasma cells with the highest brightness are screened out for down stream screening and processing. We also have trade-secrete techniques to enhance the immunization efficiency.Get a free consultation
In theory we can screen the entire spleen of a rabbit. This makes a huge difference. A New Zealand White Rabbit's spleen is 50mm long on average, roughly 100 times larger than a mouse spleen, containing tens if not hundreds of billions of cells. Traditional rabbit hybridoma screening can investigate hundreds of successfully fused hybridomas. Our technology screens typically 10% of the whole spleen. The difference in scale is that of a village community survey vs. a world wide census. We can find the clones with the highest affinity for you. This is especially important if you have a difficult antigen to make antibodies against.
"Novel clinical biomarkers are increasingly more difficult to make. The easy ones have already been discovered and commercialized. Thus in antibody discovery, finding that top 0.1%, even 0.01% highest affinity B-cell population is key to breakthrough."
3 core concepts, move your mouse over to see details.
When building an antibody for diagnostic assays or therapeutic candidate, recombinant antibody is the go to choice. It has the following benefits:
Rabbit monoclonal antibodies have been publicized as a better alternative to mouse monoclonal antibodies. This advantage is attributable to the fact that rabbit has a bigger repertoire for hypervariable regions that codes the CDRs in the antigen-binding site. The more options to choose from, the higher the likelihood to come up with "the perfect solution"--a high affinity antibody with a bright clinical future.
The key to discovering highest affinity clones is to screen as many clones as possible. Boster Bio's microfluidics plasma cell discovery (PCD) platform can do just that. Because no fusion step is required, the cells do not need to stay alive. The cells are fixed our PCD platform can screen for as long as you need to. We can cover the entire spleen and have done so in some cases. Typically 7 days of screening is sufficient for generating very high affinity clones.
|Use||Diagnotics & Theraputics|
Recombinant rabbit monoclonal antibody production features high affinity, easy humanization and high scalability. The pricing is more than what one would expect for developing research only but it is very affordable for diagnostic and clinical antibody discovery projects. The turnaround time is around 6 months.
|Use||Diagnotics & Theraputics|
Even though it shares the same same CDR repertoire, hybridoma rabbit monoclonal technology falls short on the screening scale. The last generation rabbit monoclonal technique only screen thousands of average splenocytes that were lucky enough to successfully fuse while as we can screen the whole spleen if we want to. Our workflow does not require to keep the cells alive while screening and we go for the cream of the crop only -- plasma cells, with the brightest signals.
|Use||Diagnotics & Research|
Mice have limited CDR repertoires. In general mammals with longer reproductive cycles tend to have larger CDR repertoires due to the evolutionary pressure of survival long enough to reproduce. Mice excel in their innate immunity but their adaptive immunity is not as robust as that of rabbits. This puts mice at a disadvantage for generating antibodies with high affinities.
|Use||Research, stepping stone for rabbit monoclonal|
Long story short, polyclonal antibodies work for research but not for diagnostics. It is indeed a very low cost and fast turn around method for making antibodies against certain targets. Boster Bio does offer a $600 package for making a polyclonal antibody. Some claim that polyclonal antibodies suffer from specificity because they originate from multiple clones. This is a misconception because this "disadvantage" can be mitigated by using a single epitope for immunization and purification of the polyclonal antibodies. There are 3 main disadvantages of polyclonal antibodies in our experience. First is lot-to-lot variability. For any clinical assays or therapeutic assays it is almost impossible to get approval for an assay with polyclonal antibodies as core components. For research use however it is acceptable, and is often used as a stepping stone towards a monoclonal antibody. In our experience most antigens that prove to generate a good polyclonal antibody can also make a good monoclonal antibody. The second disadvantage is its inability to amplify only the best clones. This is important if the target protein is difficult to detect and requires high affinity. This is often the case in detecting secretive proteins and biomarkers in liquid biopsies, via ELISA or other similar immunoassays. During affinity chromatography, all antibodies with enough affinity to bind to the very concentrated antigens will be pulled out. Many of these antibodies might not have enough affinity to bind to the target protein in vivo where the target protein exists in low levels and masked by competing binding receptors in the matrix/in vivo environment.
|Affinity||++Can be better|
|Use||Diagnotics & Theraputics|
It lacks SOMATIC HYPERMUTATION. Even though phage display allows a reasonably large screening scale, it still falls short when compared to screening the full spleen, by at least a 1000 fold in scale. But the real problem is the CDR library phage display platforms have are "diamonds in the rough". Without going through SOMATIC HYPERMUTATION they will not be able to bind to the intended antigen with high affinity. Even though some claim they have secrete sauces in antibody engineering to improve antibody affinity after the fact, studies and our experience indicate that it is often no match to mother nature's affinity maturation process. It is a numbers game after all and the immune system can go through billions of iterations in vivo in a matter of weeks while as antibody engineering needs to experiment one amino-acid modification at a time, at weeks per iteration. Edited: some customers mentioned that one can train the phage library with certain antigen/pathogen groups to have better results. While this certainly helps, it is not a magic bullet to solve the intrinsic issues with phage display. I would also add that phage display does have an advantage, among many others, that it is great for generating antibodies against antigens that are not immunogenic or are autoimmunogenic, as it circumvents negative selection. See more information here Central Toloerance Wikipedia
Custom Rabbit Monoclonal Antibody Production Overview And Timeline.
Boster's custom rabbit monoclonal antibody development platform uses Single Plasma Cell Interrogation Technology, which screens the immune repertoire of rabbits. Our aim is to produce an antibody that works for your specific application. Our technical staff will guide you through the project, from designing the antigen, immunization, screening strategy, antibody validation, to the proper handling of the plasmid that expresses your antibody. We can also generate antigens for you. Our antigen synthesis service cover both peptides and recombinant proteins. We are capable to express the antigen in E. coli, baculovirus, and mammalian cell lines.
The final deliverable is the sequences of selected positive clones, and plasmids containing the cloned antibody genes. Recombinant antibodies ensure the preservation of genetic information.
|Peptide and Immunization: (2 rabbits)||Titer Data|
|Peptide synthesis 10mg, >90% purity||1 week||Titer Data|
|Peptide conjugation KLH for immunization and BSA-biotin conjugation for probing and screening||1 week||Titer Data|
|Immunization for 2 rabbits||2-3 months||Titer Data|
|Titer ELISA Assay Monitoring anti-sera titer and selecting the best animal for monoclonal|
|First Screening with FACS (> a million B cells)
|Supernatant production 0.25 ml scale, and Secondary screening with ELISA||1 week||ELISA data|
|Customer further screening with supernatant if necessary, choose three best clones.|
|Clone Deconvolution, cloning, expression/ELISA conformation; 30ml scale production, yield~ 0.5mg Ab per clone||3-4 week||Sequences|
What you will receive upon successful completion of above phases.