Patented Library Construction Method (WizAmpTM)

 

Every rabbit immune response is different, and a library only ever contains what the primers amplified. WizAmp™ (US Patent 9,890,414) replaces the mismatched primer pair of conventional PCR with a single non-gene-specific primer, and pairs it with a 39-primer first-strand set covering the widest rabbit VH FR1 diversity reported to date — including framework sequences that appear in no public database.

US 9,890,414
Patented amplification method
Matching 5′ and 3′ ends are appended during second-strand synthesis, so the entire repertoire amplifies with one primer at one melting temperature.
39
First-strand FR1 primers
Against 12 primers in a competitor’s published set and 13 across combined literature sources. Developed iteratively over hundreds of rabbit libraries.
0 → dozens
Hits from the same spleen
A 32-primer subset recovered no target-specific antibodies. The full 39-primer set recovered dozens, from identical immune tissue.

Same immunized rabbit, same panning — only the primer set changed

Library built withTarget-specific antibodies recovered after panning
Partial set — 32 primersNone
Full WizAmp™ set — 39 primersDozens of hits

Both libraries were constructed from the same immunized rabbit spleen. What decided whether the campaign produced anything at all was repertoire coverage at the cDNA step, not the panning conditions downstream — which is why Abwiz Bio treats primer coverage, rather than raw library size, as the limiting variable in immune library discovery.

Proprietary phagemid vectorNative, cell-based antigen panningRabbit monoclonal antibody developmentGPCRs, ion channels & difficult antigensVHH / nanobody discoveryAnti-idiotype antibodies25 peer-reviewed publications

WizAmpTM single-primer method amplifies antibody genes uniformly

We have developed a patented antibody library construction method termed WizAmpTM (US 9,890,414), which enables more uniform and robust amplification of antibody genes compared to traditional 2-primer PCR methods. In WizAmpTM, mRNA is purified from homogenized tissue and first-strand cDNA is first synthesized using a proprietary primer mixture recognizing rabbit antibody genes (see below). During second strand cDNA synthesis, matching nucleotide sequences at both 5’ and 3’ ends are appended, allowing for subsequent antibody gene amplification using a single non-gene-specific primer.

 

Traditional library construction methods employ 2-primer PCR. The use of two primers with mismatching sequences and even slight differences in melting temperature can cause non-specific and uneven amplification that will degrade the quality of the library. Unbiased, robust amplification by a single non-gene-specific primer creates high quality libraries that preserve the natural frequency and proportion of each antibody gene isolated from the immunized rabbit.

 

WizAmpTM recovers maximal antibody gene diversity from immune sources

Through iterative improvement after creating hundreds of rabbit libraries, we have created a primer set that covers the highest reported rabbit VH FR1 sequence diversity to date by including many FR1 sequences that are not currently in publicly available databases. These primers initiate first strand cDNA synthesis using mRNA purified from homogenized immune tissue, prior to the single-primer amplification step above. Compared to literature sources or a competitor’s published primer set, our primers cover many more rabbit antibody genes.

 

This primer set was developed iteratively – we have observed for a variety of antigens that when only a partial set of 32 primers are used for library construction, no target-specific antibodies can be isolated after phage panning. However, when the full set of 39 primers was used for library construction from the exact same rabbit spleen source, dozens of hits were obtained. Each immune response is unique – without full coverage of the immune repertoire, a target-specific antibody may be missed.

 

 

See how we pair these technologies with our proprietary phagemid vector to deliver rabbit Fab phage display for the first time.

 

See how we have used this cutting-edge library construction technology to build rabbit immune libraries to isolate rare, functional mAbs against GPCRs and ion channels and to make highly-specific companion diagnostic rabbit monoclonal antibodies.

From antigen to IgG expression vector

The full rabbit discovery workflow at Abwiz Bio. WizAmp™ is step 3 — the point at which the repertoire either survives into the library or is lost.

  1. Antigen preparationPeptide, recombinant protein, whole cells or lipid — prepared according to what the target actually is, not what is convenient to produce.
  2. Immunization and serum testRabbits are immunized and serum titers confirmed by ELISA, flow cytometry and other assays before any library is built.
  3. Library construction (WizAmp™)mRNA is purified from homogenized immune tissue and first-strand cDNA primed with the proprietary 39-primer set, followed by single-primer amplification.
  4. Phage display panning (Needle-in-a-Haystack™) and screeningAntigen capture, competitive selection and epitope masking are used to recover the rare functional clone rather than the abundant one.
  5. Sequence analysis and direct cloningPositive clones are analyzed with our own sequence analysis software, and lead candidates are cloned directly into IgG expression vectors.