Quantification of Biomolecules
Quantitation applications are fundamental to numerous biological research protocols and in the production of pharmaceutical-grade biomolecules. It is frequently desirable to confirm the purification of antibodies or other biomolecules to characterize biologic processes or ensure the quality of starting materials1,2. Quantitation applications are thus key to a range of research and therapeutic protocols.
With ever-growing biotechnology capabilities, protein engineering is an increasingly important aspect of both research and biological therapies. Quantitation applications are paramount in providing confidence in protein purification methodologies before protein stduies3. Similarly, quantitation applications ensure that proteins destined for pharmaceutical use conform to rigorous quality parameters to ensure patient safety.
The majority of molecular biology experiments start with cloned DNA, which is then transferred to E.coli or a similar expression vehicle to obtain the protein of interest. To maximize the expression of the desired protein, the DNA construct must be designed to optimize the efficiency of expression by the vehicle selected. Furthermore, the purity of the DNA must be ensured through quantitation applications to increase the output of the target protein.
Adeno-associated virus (AAV) titer determination
AAV is widely used as the vector to introduce functional genes to humans with a disorder that stems from a genetic aberration. Such gene therapy is controlled by strict regulatory standards, including specifications related to vector purity and potency. The ability to accurately determine the dose of therapeutic AAV vector is thus critical to the gene therapy process. Accurate quantification of viral genome is fundamental to demonstrating purity and confirming dose4.
The determination of antigen-specific antibodies in plasma samples is an important diagnostic tool and provides important serosurveillance information during pandemic infections5. Analytic tests that provide quantitative results usually require complex and labor-intensive immunoassays. There is a need for a rapid, highly sensitive technique for antibody quantitation applications.
These quantitation applications and more are satisfied by a novel label-free technique using biosensors — biolayer interferometry (BLI) 6,7,8. BLI measures the interference pattern of white light reflected from the surface of a biosensor, which indicates the presence of biomolecular interactions. Used in conjunction with a standard curve of known concentrations, BLI provides a suitable tool for rapid data acquisition across numerous quantitation applications. BLI technology is suitable for quantitation applications involving complex matrices. It can be used for quantitation applications in samples that are crude supernatants or cell lysates, as well as purified proteins.
High sensitivity is achieved across numerous quantitation applications from small molecules to large biologic complexes. Furthermore, measurements are made in real-time, so changes in biomolecule concentration can be monitored with high precision. Only target biomolecules are measured, making BLI a powerful tool for a wide range of quantitation applications.
Gator Products for BLI Quantitation Applications
Gator offers a range of products to facilitate the use of BLI in quantitation applications. GatorPrime™ is a robust and maintenance-free BLI instrument for label-free analysis9. With automated biosensor loading and automated regeneration of biosensors, it offers a hands-free alternative to labor-intensive ELISA methodologies for quantitation applications.
GatorPrime incorporates eight independent spectrometers that enable quantitation applications to be conducted simultaneously on eight samples. With the capacity for assays sizes up to 168 samples per run, the GatorPrime system represents an efficient solution for quantitation applications. The system includes quick-start modes to support quantitation applications that are carried out by non-specialist operatives.
GatorPlus is a high-throughput device used in quantitation, kinetics, and epitope binning. Based on bio-layer interferometry (BLI) technology, this instrument provides rapid results.
In addition, Gator provides specialized solutions to further facilitate quantitation applications10. For example, Gator™ AAVX enables the direct capture and quantitation of different serotypes of AAV in crude lysates, column eluates, cell lysates and cell culture supernatants using the CaptureSelect™anti-AAVX ligand. The Gator™ Ni-NTA Kit enables rapid and continuous quantification of His-tagged proteins without the need for Ni2+ recharging.
The Gator BLI system uses software that complies with the FDA’s code of Federal Regulations (CFR) Title 2111. This is critical for quantitation applications involved in the monitoring and manufacturing of biological drugs. All data acquired is time-stamped and traceable and its enhanced audit trails, including recorded user sessions, comply with FDA guidance. The Gator system is thus suitable for use in environments where adherence to good manufacturing/laboratory practice is paramount and analyses must be CFR21-compliant.
- Westbrook JA, et al. Proteomics Clin Appl. 2015 Apr;9(3-4):295-300. Doi: 10.1002/prca.201400120.
- Heegaard NH, et al. Electrophoresis. 1999 Oct;20(15-16):3122-33.
- Lillehoj EP, Malik VS. Adv Biochem Eng Biotechnol. 1989;40:19-71.
- Jungmann A, et al. Hum Gene Ther Methods. 2017; 28(5):235-246.
- Dzimianski JV, et al. Sci Rep. 2020;10(1):21738.
- Qiao SP, et al. Chembiochem. 2021 Jun 2;22(11):1974-1984.
- Noy-Porat T, et al. STAR Protoc. 2021 Sep 15;2(4):100836.
- Dysinger M, et al. J Immunol Methods. 2012 May 31;379(1-2):30-41.
- Gator. https://www.gatorbio.com/products/gatorprime
- Gator. https://www.gatorbio.com/products/biosensors-and-reagents
- Gator. https://www.gatorbio.com/products/software