Allele Mail Bag

What Does It Take to Bring New Nano Antibodies (nAbs) to the Hands of Researchers?

Judging from the hundreds of papers published using camelid VHH antibodies as reagents, there are probably thousands of researchers who have experience with this type of antibodies by now. We like to call the ~15kD camelid VHH antibody nano antibody or nAbTM. Once someone experiences how well a nAb works for co-IP using a fluorescent protein as tag, they often wonder what it takes to bring nAbs to broader use.

The success of a nAb project starts with the antigen presentation. It is critical to build the capability to produce large quantities of recombinant antigen for immunization. At Allele, our scientists also established some unique presentation formats for traditionally difficult targets (e.g. large membrane proteins).

After llama immunization, the next step is screening. With the goal of creating large scale nano antibodies against diverse targets, we have developed multiple high throughput screening methods to cover very large, diverse libraries generated from immunized animals. The technologies will continue to evolve as the scale of nAb generation continues to expand. We have the ability to functionally screen for site-blocking antibodies and antibodies that only recognized natively folded targets, or targets in their naturally occurring presentations.

A nAb isolation project does not end with the obtaining of a cDNA clone. Or, if it does, the nAb is probably not as great as what Allele Biotech has been offering. In our hands, all nAbs go through an engineering step beginning with the generation of a 3D structural model of the isolated clone. We use structure-guided design to alter the protein, allowing us to improve its properties. This includes increasing affinity, solubility, or altering the protein to improve performance for specific applications. We also like to use known structures of traditional monoclonal antibodies to assist camelid VHH antibody engineering against specific targets.

With a finalized clone in hand, the next step is to establish protocols for commercial production. The Allele team spends a tremendous amount of effort aimed solely at high-yield, low-cost recombinant VHH antibody production in a variety of formats, so that the costs for other scientists to take advantage of these great reagents can be kept as low as possible.

Last but not the least, nAb labeling, including conjugating stable soluble VHH antibody to solid supports for immunoprecipitation or to fluorophores for detection, requires additional expertise and tight operation control. However, our vision is to have a modular system for antibody labeling that will enable the end user to select from a variety of fluorophores and other detection tags, which can be instantaneously and irreversibly coupled via simple mixing.

Note added: we work with commercial (diagnostic and clinical) partners from developing nAbs all the way to the market. We have expert scientists available to customers and licensees for consultation and troubleshooting antibody- and imaging-related questions and problems.

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Allele Biotech Acquires BioCarta’s Distribution Business

Allele Biotechnology & Pharmaceuticals, Inc. is pleased to announce that as of January 1st, 2014, it has acquired the distribution business of BioCarta. This transaction will strengthen Allele Biotech’s presence in the antibody field, enhancing a broad customer and partnership base to further its plan in clinical diagnostics. Biocarta has been a leader in the field of gene expression for 10 years and has contributed immensely through its world leading effort of charting molecular biology pathways. The gene function maps published by Biocarta have been used and referred to by the NIH through the National Center for Biological Information and National Library of Medicine.

Among its well-regarded distribution business, for the past 11 years BioCarta had been the US and Canada’s exclusive distributor of Immune Function Assay Kits for Flow Cytometric Analysis by Glycotope Biotechnology, GmbH. These kits are clinically approved blood cell diagnostic assay products that are also heavily used for non-clinical blood studies.

Allele Biotech has recently launched a major business plan to create a large number of nano antibodies (nAbsTM). The nAbTM line will be great research tools for immunoprecipitation (co-IP), immunohistochemistry (IHC), Western blotting, co-crystallization, biologics purification. Additionally, nAbsTM willbe suitable for diagnostic assays because the single domain antibodies derived from camelid family animals are sturdy, specific, and low-cost. The inclusion of the BioCarta distribution channels in the antibody and pathway reagent fields will help speed up Allele Biotech’s expansion.

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Thursday, January 16th, 2014 Allele Mail Bag No Comments

We at Allele Biotech encourage you to help those in need

Typhoon Haiyan (Yolanda) has devastated vast areas in the Philippines and the local people are still suffering, particularly those who had minimum resources even before the disaster hit. Our company’s representatives’ recent trip to the country created a special bond between our employees and the people in the Philippines, and we will do what we can to help, including directly donate to agencies like the Philippine Red Cross. We encourage our clients, colleagues, and friends to do the same. Thank you.

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Wednesday, November 20th, 2013 Allele Mail Bag No Comments

Lab Skills You Stopped Being Proud Of

Molecular biologists who were in graduate school in the 90’s learned how to isolate plasmid DNA from E. coli cultures by a method called “boil-prep” during their first lab rotation. This process involved mixing the bacterial cell pellet in a little bit of detergent, salt and sucrose, dabbed with some fresh lysozyme, and then you are ready to cook, literally! Bacterial cell membranes are disrupted by boiling this soup in a beaker of water over a Bunsen Burner for one minute, and the debris (containing the broken cell membrane and attached chromosomal DNA) is collected by centrifugation in a microfuge at top speed for 10 minutes. Then comes the step that differentiates a true master of lab skills versus a rotation student—if you knew just the right amount of bacterial culture to begin with and handled the E coli pellet by the right techniques, a skillful lab person could collect nearly all the liquid without disturbing the pellet. Pouring out the plasmid-containing supernatant without dislodging the goo on the side/bottom of the tube was such a desirable skill that would not only give you your plasmid but also give you admiration from fellow lab members. That is, of course, if you were doing it before the mid-90’s, because after the introduction of miniprep spin columns by Qiagen, nobody, even the true masters of boil-preps (or its contemporary alkali prep that also involves pelleting by centrifugation and careful removal of tiny volume of liquids surrounding small pellets) would be showing off those skills any longer.

It is actually never easy or fun to collect liquid surrounding small amount of beads or pellets as you always have to struggle to remove as much liquid as possible while trying not to lose any of the beads

Some of the old-timers used to also be very proud of being able to pour a “sequencing gel” (a very thin ~40 cm x 30 cm polyacrylamide gel). I still remember the first time I reported to the second rotation lab at USC. After describing the lab research, the PI showed me around the lab and complained how “Sarah destroyed all my sequencing gel plates”. But consider this, in order to avoid any greasy spot on either plate, you needed to wash both of them fanatically if not religiously. Why? You would have just about a minute’s time to pour non-polymerized acrylamide without leaking from the sides or bubbles forming anywhere in the DNA running lanes, and then inserting a pair of paper-thin combs, all at a speed quicker than TEMED/AP-catalyzed acrylamide polymerization. Good thing that after capillary sequencing was invented, we all happily retired our sequencing-gel pouring skills with a collective sigh of relief.

Technology will always move forward, so will the skills lab researchers will be required to perfect. Using a spin column is very much a “skill-less” technique in contrast to collecting pellets and washing beads after centrifugation, but when there is a choice, people will chose the method that requires “less skills”, such as the spin-column format as the preferred platform for the new FP-nAb™ products.

BTW, like to have information on the spin column kit? Here it is:

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Is “100% Conversion” No Longer a Taboo Phrase in the iPSC Field?

“By removing a single protein called Mbd3, a team at the Weizmann Institute of Science in Rehovot, Israel, was able to increase the conversion rate to almost 100%”. A staggering statement written by Monya Baker for Nature News, after her journal’s online publication by Rais et al. reported their discovery. This discovery displays that by removing the expression of nucleosome remodeling and deacetylation repressor complex member Mbd3, all cells can be reprogrammed with the OSKM Yamanaka factors. What we have described last September in Nature’s Scientific Reports (Warren et al. 2012) that by fusing Oct4 with strong transcription activation domain, we achieved essentially the same results which we call “bulk conversion”. One can imagine that the strong transcription activation domain very likely effects such strong reprogramming through some type of chromosomal remodeling indirectly.

As we later learned, people in the field believe that reprogramming is a stochastic event and it is impossible to achieve something like “bulk conversion”. We are happy to see that sea change now that a separate group also demonstrated “bulk conversion”. Recently we have shown that instead of 2 weeks or more, as required when mRNA reprogramming was first reported or commonly practiced, we could start taking up iPSCs around a week without doing any traditional transfection—just change the medium everyday as for all stem cell cultures, with mRNA complex as a supplement to be added together with bFGF.

Knocking-down Mbd3 is potentially useful, especially in situations where we have difficulty reprogramming a particular line, and it could make reprogramming more successful in more demanding conditions such as leaving out B18R as we recently established. However one needs to be cautious about the unavoidable off-target effects of siRNAs when including siRNA against Mbd3 in their RNA mix.

Rais et al. Nature (2013)

Warren et al. Scientific Reports (2012)

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Thursday, September 19th, 2013 Allele Mail Bag, iPSCs and other stem cells No Comments