The overall goal of this scalable procedure is to isolate specific and high affinity fab phage clones from a phage displayed library using a high throughput approach that enables parallel selection against hundreds of antigenic target domains. This is accomplished by first immobilizing purified antigenic domains in micro well plates. The next steps are to expose, capture, release, and amplify fab phage clones that specifically bind to microplate immobilized antigen in successive rounds of selection.
During this process, the effectiveness of the selection is monitored using pooled methods at each step. The final step is to evaluate the specificity, affinity, and or activity of the isolated fab clones. Ultimately, specific high affinity fab fragments against large numbers of antigens are isolated in parallel, enabling the generation of antibodies useful in the analysis of entire classes of structurally or functionally related proteins.
The importance of this method lies in that it offers an alternative that can help to overcome the limitations and accelerate the pace of conventional antibody development technologies in either an academic or an industrial lab. The major advantage of this technique over conventional antibody development techniques such as hybridoma include that it doesn't require the use of animals. It can be used for virtually any protein, including toxic and non immunogenic proteins.
As we'll see, it's easily parallelized and ultimately yields renewable recombinant DNA encoding a high affinity antibody specific for particular antigen and functional and standard immunoassays in as little as six to eight weeks. Given the importance of antibodies in research in biomedicine, we saw an opportunity to adapt and scale antigen generation and antibody selection and identification to a high throughput approach while maintaining accessibility to laboratories ranging in both size and available resources. On the morning of the day of phage selection, pick a single colony of T one phage resistant e coli cells.
Add it to a milliliter of two yt media with tetracycline at 50 micrograms per milliliter. Let the colony incubate shaking at 200 RPM in a 2.5 centimeter orbital shaker. Once the culture's growth is visually evident, transfer cells to the required volume of media supplemented with tetracycline.
There needs to be enough for 100 microliters per selection well, or approximately 10 milliliters per 96. Well plate expand aliquots of the culture to both carbon penicillin and canin supplemented media. These cultures ensure against pre infection in the cells to be used for amplification.
When the culture's optical absorbance gets to 0.4 to 0.8 at 595 nanometers. After five to seven hours, the cells are ready to apply to the antigen plates. For this protocol to ensure the desired coverage of the library, prepare sufficient replicate antigen plates and non-specific control protein plates.
Using the displayed calculation details are provided in the text protocol. After overnight immobilization of target and nonspecific control proteins, remove protein solution from the plate. Add 200 microliters of blocking buffer and block for one hour with shaking and wash the plate four times prior to adding the phage library while the cells are growing.
On the day of phage selection, we pre-clear nonspecific or the tag specific binding phage displayed FAB clones from the library by transferring 100 microliters of phage library to the negative selection plates for added negative selection. Remove tag binding phage by adding an excess of soluble affinity tag. To do this, add a 10 micromolar final concentration of soluble GST to each.
Well then incubate the plates for one to two hours on a shaker at room temperature. To prepare the target protein plate aspirate protein solution from the plate, add 200 microliters of blocking buffer and block for one hour with shaking and wash four times prior to adding phage library. After the incubation, collect the phage snat from the plates.
Using a 96 channel liquid handler, transfer the phage supinate to antigen coated plates. Then incubate the plates for one to two hours on a shaker at room temperature. After the incubation, remove the unbound phage by washing the wells eight to 15 times using PT buffer.
This can be done manually or with an automatic plate washer. Then just prior to adding the cells, remove all the excess wash buffer when the cells and antigen plates are ready. Elute the phage by adding 100 microliters of growing cells to each well and let the plates incubate at 37 degrees Celsius for half an hour.
The first few rounds of selection are not expected to produce significant enrichment, but quantification of output phage titers can ensure the illusion of the minimum phage concentrations needed for successful selections. To do this, an aliquot of cells can be removed prior to the addition of helper phage in order to tighter the phage output by preparing and plating serial dilutions of the cells on antibiotic supplemented agar plates. After incubating for half an hour, add M 13 K oh seven helper phage to each well for a concentration of 10 billion colony forming units per milliliter.
Let the plates incubate another 45 minutes this time with shaking at 200 RPMs. Next, transfer the cells to a 96 well deep well block with a V bottom containing 1.2 milliliters of supplemented two YT media per well. Let the deep well plates incubate at 37 degrees Celsius overnight with shaking at 200 RPMs the following day.
Pellet the bacteria by spinning the plates at 4, 000 GS for 15 minutes at four degrees Celsius. Then from each replicate plate transfer equal volumes of supernatant containing the phage to a mini tube. 96 well microplate blue box to be used as input for subsequent rounds of selection to the pooled phage, add a one 10th volume of 10 XPBT and mix to neutralize.
Now, repeat the selection process from the library. Pre-clearance and antigen phage incubation three to four times using phage amplified from the previous round of selection. Do this until target binding enrichment is observed compared to binding of non-specific control proteins by Eliza Micro well plates coded with two micrograms per milliliter antigen or control protein as previously described in library pre-clearance and antigen phage incubation are then blocked and washed as before to blocked and washed antigen and control protein coated plates apply 100 microliters of diluted phage supt to the wells and let the plates incubate with shaking for 15 minutes at room temperature After the incubation, wash the plates eight times with PT buffer.
Then following the washes, add 100 microliters of anti M 13 HRP antibody at a one to 5, 000 lution in PBT to each well incubate for half an hour to allow antibody binding to antigen bound phage. Then wash the plates with PT buffer six times and twice more with straight PBS prepare, mix and apply 100 microliters of one to one TMB substrate to each of the wells. Let this react for five to 15 minutes, stopping the reactions when there is significant color change.
Stop the reactions by adding 100 microliters of one molar phosphoric acid. Now read the absorbance in the wells at 450 nanometers and compare the target binding signals to the binding signals of the control proteins Following enrichment. Individual clones can be isolated and characterized by sequencing phage, DNA conversion of phage bound to free fab, and the evaluation of direct binding to target using clonal FabuLisa as explained in the text portion of the protocol.
For laboratories in which higher throughput is required, this methodology can be scaled and automated. With this aim in mind, customized robotics have been developed that can expand the selection capacity by automating all aspects of the procedure, including blocking, phage, binding, and elucian and more. One approach to obtaining antigen for use in antibody selections is the use of synthetic gene technologies to obtain constructs for expression of isolated protein domains.
In most cases, this enables the high throughput expression and isolation of adequate quantities of sufficiently pure purified antigen for selection. During successive rounds of the selection process, eluded phage concentrations and enrichment of phage that specifically bound to antigen was monitored by either phage titration as shown previously or monitored with phage pools. In an ELA assay, a binding ratio greater than two generally indicates the presence of specific binding clones.
Conversely, a lower ratio can help determine the cause of a selection failure. Characterizing clone binding using a colormetric amino assay enables the comparison of binding fab phage or fab to immobilized antigen compared to binding negative control protein, thus yielding a measure of specificity when scaling and automating selections with robotic liquid handling units. The use of absorbing chromophores can help to detect when specific channels in the fluidics are either clogged or have lost their seal, resulting in partial volume delivery, and aid in troubleshooting during optimization of wash procedures, the use of known binding clones ensures that target binding is maintained while background binding is removed during the washes.
In addition, effective decontamination protocols ensure the absence of cross-contamination from successive rounds of selection or different selection procedures and are recommended additional troubleshooting parameters are reviewed in the text protocol. By scaling of this technique for industrial application, we hope to enable researchers to explore entire classes of structurally or functionally related proteins by accelerating the selection and identification of high affinity renewable antibodies with the ultimate aim of achieving maximum proteome coverage.
