A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies

Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg-1 at 90% purity.
Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL-1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution.
We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.

One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice

It has been proposed that transgenic mosquitoes can be used as a “flying syringe” for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes.
We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect.

RFP (dsRed) Antibody

3993-100 Biovision each 516 EUR

RFP (dsRed) Antibody

3993-30T Biovision each 222 EUR

Red Fluorescent Protein (dsRed) Antibody

3994-100 Biovision each 451.2 EUR

DsRed- rab7 WT

PVT10355 Lifescience Market 2 ug 319.2 EUR

DsRed- Rab5 WT

PVT10499 Lifescience Market 2 ug 319.2 EUR

dsRed Fluorescent Protein

4997-100 Biovision each 489.6 EUR

dsRed Fluorescent Protein

4997-1000 Biovision each 3008.4 EUR

dsRed Fluorescent Protein

4997-5000 Biovision each 8626.8 EUR

dsRed Fluorescent protein

30R-2809 Fitzgerald 100 ug 589.2 EUR

pIRES2- DsRed- Express Plasmid

PVT1229 Lifescience Market 2 ug 289.2 EUR

Recombinant purified Red Fluorescent Proteins (RFP/dsRed) protein control for Western blot

RFP12-C Alpha Diagnostics 100 ul 343.2 EUR

pLVX- DsRed- Monomer- N1 Plasmid

PVT2312 Lifescience Market 2 ug 361.2 EUR

pBRPyCAG- Mettl3- dsRed- IRES- puro

PVT10449 Lifescience Market 2 ug 361.2 EUR

Recombinant (E. coli) Red Fluorescent Proteins (RFP/dsRed) protein for ELISA or Standards (>98%)

RFP15-R Alpha Diagnostics 25 ug 300 EUR

pMSCV- loxp- dsRed- loxp- eGFP- Puro- WPRE

PVT11052 Lifescience Market 2 ug 361.2 EUR

pCAG- DsRed1

PVT10763 Lifescience Market 2 ug 361.2 EUR

pLac- DsRed2

PVT11592 Lifescience Market 2 ug 364.8 EUR

pIZT- DsRed2- V5- 6*His

PVT11311 Lifescience Market 2 ug 444 EUR