Can the New Antibody Sequencing Technique Surprise the Market?

Discovering the structures and functions of antibodies in living organism is an important tool in understanding cellular processes, which allow drugs that target specific metabolic pathways to be invented more easily.

Antibody sequencing is a technique that determines the amino acid sequence of an antibody. Discovering the structures and functions of antibodies in living organism is an important tool in understanding cellular processes, which allow drugs that target specific metabolic pathways to be invented more easily.

This next generation antibody sequencing technique has now become a routine measurement that can be completed within 10 working days for IgGs. The method is applicable for all antibody formats, namely IgMs, fluorochrome conjugates, immobilized antibodies and mixtures. Creative Biolabs, an emerging qualified service provider in this field, provides researchers their unique technique in antibody sequencing.
With the development of biotechnology, scientists of Creative Biolabs have developed a novel strategy to determine the complete sequence of an antibody with unparalleled speed and accuracy. The technique which combines all the advantages of existing approaches is named "Database Assisted Shotgun Sequencing" (DASS).

In the first step, the antibody is fragmented to peptides by a special technique, which generates up to 5000 different peptides per chain. This set of peptides will be analyzed by high end mass spectrometers to generate extensive sequence information. MS/MS spectra are then de novo sequenced by the latest algorithms and matched against a database with related sequences. Special "in house" data mining tools allow Creative Biolabs to extract the sequence information from ten thousands of MS/MS spectra within hours.

De Novo Sequencing of the CDR3 Region

While the CDR3 of the light chain is mostly encoded by the germline sequences, the CDR3 of the heavy chain is usually not available in databases. It is encoded by the so called D-segments, but these are modified by nucleases and terminal transferases. Typically, only 1-4 AA of a D-segment remain in the matured antibody. The rest of the D-segment is "artificial" and has to go through phage display techniques. Their method generates many overlapping peptides during the fragmentation process, enabling them to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows them to read the CDR3 like a book. The technique is so powerful, that they were able to sequence a 20 kDa protein, which had no homologue in the database.

Sequencing of the V and J and C segments by "Database Assisted Shotgun Sequencing"

The V and J and C gene segments of antibodies are available in public databases. During the maturation of an antibody, the B-cell introduces hypermutations into the sequence to optimize the affinity. Creative Biolabs's mapping algorithm is error tolerant and can match the "mutated" peptides to the corresponding germline reliably.

Because of the great number of peptides, Creative Biolabs is able to get sequence information for ever bond in the antibody. Typically, 20-70 different MS/MS spectra are generated for each amino acid (AA) position. Hence, even the hardest sequences of proline and arginine rich peptides can be resolved.
Learn more about de novo sequence.

About Creative Biolabs


Creative Biolabs is specialized in providing custom biotechnology and pharmaceutical services that cover the full scope of biotechnology needs of early drug discovery and development, including antibody production , membrane protein preparation, etc.

DNA Immunization Technique in Diseases Research

DNA immunization is a technique for protecting an organism against disease by injecting it with genetically engineered DNA to produce an immunological response. It has a number of advantages over conventional immunization, including the ability to induce a wider range of immune response types.

DNA immunization, also known as gene immunization, polynucleotide vaccine and DNA vaccine, is a newly established immunological theory and technique first discovered in the 1990s. Compared with current protein vaccine, DNA immunization is safer and more inexpensive. Besides, it induces a more effective immune response and is easy to be prepared. With the potential application in some specific field, DNA immunization is not only been widely used in anti-virus, bacteria, fungi, parasites as well as other anti-infection immunity, but also plays a n important role in tumor immunology and autoimmune disease. Like all other groups, Creative Biolabs follows up with the updated technique in DNA immunization, and made their own innovation by putting DNA immunization in the production of antibody.

Creative BioLabs has developed Direct Antibody Technology™ (DAT) to produce custom polyclonal and monoclonal antibodies. As a highly optimized service, this DNA immunization technology relies on a proprietary genetic immunization procedure using plasmid DNA encoding the target protein of interest. The immunized hosts then produce the encoded protein and raise antibodies in vivo.

To ensure the accomplishment of the genetic immunization, the cDNA-encoded protein must be secreted by the transfected cells in immunized animals or expressed on the surface of the transfected cells. Creative BioLabs introduces the gene in the form of a cDNA directly into an animal, which translates this cDNA into protein thus stimulating an immune response against the foreign protein. As a result, synthesis and purification of protein immunogens is not necessary for this genetic immunization approach.

Creative Biolabs’ Direct Antibody Technology™ maximizes the likelihood of producing and maintaining the native structure of the antigen. The foremost advantage of this antibody production approach is its high success rate in generation of high-affinity antibodies recognizing membrane proteins (such as 7-membrane-spanning GPCR proteins, ion channels and other multiple membrane spanning proteins) in their native conformation, unknown proteins whose genes have been obtained, toxic proteins, insoluble proteins, proteins containing disulfide bonds, post-translational modified proteins, or large protein domains. For therapeutic antibodies, however, the antigens in their native conformation should be targeted with a high-affinity.

Creative Biolabs is a leading service provider in the field of DNA immunization. 

Creative Biolabs released membrane protein expression service

Creative Biolabs released cell-free membrane protein expression service for protein related research.

Membrane proteins occupy one fourth of total encoding genes in genome. They localize on the cell membrane, play various roles such as signal transduction, transportation and ligand-binding. It is estimated more than half of the drug candidates target membrane proteins, mainly G-protein coupled receptors (GPCRs) and ion channels. However, due to the nature of membrane protein, traditional protein expression methods encounter obstacles such as low amount of expression, inadequate post-translational modification, misfolding, and loss of bio-activity.

Aiming to solve the problems in the conventional methods, Creative Biolabs designs a novel cell-free membrane protein expression system. In this unique system, the neonatal membrane protein inserts into liposome, which is well designed to imitate the in vivo membrane features, keeping the native conformation and bio-activity to the greatest extent.

The cell-free expression system also possesses other advantages: high purity of target protein, rapid and stable expression, high yield and good repeatability. The system is also easy to be adapted to diverse requirement from our clients, such as the size and modification of liposome, the therapeutics encapsuled in the liposome and type of solvent reagents. Our system is tailored for customers in membrane protein projects of antibody screening, ligand discovery, drug delivery, vaccine development, and structural determination.

About Creative Biolabs

Creative Biolabs is specialized in providing custom biotechnology and pharmaceutical services that cover the full scope of biotechnology needs of early drug discovery and drug development. As a trusted provider of the most cost-effective outsourcing solutions, Creative Biolabs has been working for a large number of satisfied clients from biotechnology and pharmaceutical companies as well as government and academic research laboratories all over the world. It was founded by scientists who are dedicated to the conquering of cancer. We believe to build up a custom-service-centered business model is important for optimizing the drug development process, leveraging accessible resources, and forming a team of various background to conduct drug discovery in future. Our commitment to this long-term goal motivates us to deliver the highest quality results to our clients with speed.

How to Increase Protein Production Capability?

With the common effort of the staff scientists and researchers, Creative Biolabs released its Single Protein Production (SPP) ^TM technology into the protein production market. This service is developed mainly for the R&D of high-throughput protein production.

In the SPP system, live E. coli cells are converted into a bioreactor producing only a single protein of interest in a high yield. A yield of 20–30% of total cellular protein can be obtained with our technology, which overwhelms all protein production methods known so far.

Its technology involves the introduction of an mRNA endoribonuclease or interferase in E. coli cells to disrupt the endogenous protein production. However, the cells retain full metabolic activity for RNA and protein synthesis. Therefore, when the mRNA for a protein of interest is engineered to be devoid of the mRNA endoribonuclease recognizing sequence without altering the amino acid sequence of the protein, the cells start to produce any single protein of your choice.

With the evolution of this technology, Creative Biolabs is now capable of producing any proteins with the SPP system. With the technology, protein yields can be kept without being affected even if the culture is condensed up to 40-folds, reducing the cost of protein production by up to 97.5%. The technology provides isotope-labeled proteins at a very high signal-to-noise ratio. More than 90% of the isotope can be incorporated into the target protein in the SPP system. Furthermore, a refinement of this technology has eliminated lengthy purification steps in the production of membrane proteins suitable for structural studies. 

Learn more about Single Protein Production (SPP) ^TM technology of Creative Biolabs.

About Creative Biolabs

Creative Biolabs, a US biotech company established in 2005, is focused on the research and development of antibody engineering and protein production. Learn more about Creative Biolabs. 

The Future of Sequencing Technology in Clinical Application

As science develops, traditional Sanger sequencing has failed to meet the requirements of low cost, high throughput and fast in speed. It is under this circumstance that the next-generation sequencing technology (second-generation sequencing) appears. As a comparatively new industry, the next-generation sequencing technology can be applied for clinical genetic testing, health industry, industrial and agricultural use of gene-oriented study as well as scientific research and development.

Recent years, with the discovery and promotion of second-generation sequencing technology, gene sequencing speeds up greatly while achieving a substantial decline in costs, making large-scale application of genome sequencing possible. Now, the cost of personal whole genome sequencing is about 5,000$, and is expected to decreased to less than $ 1,000 in the next few years.

The rapid development of sequencing technology will promote the massive accumulation of DNA sequencing data, accompanied by the accumulation of the corresponding individual signs, diseases and other data at the same time. When enough data is accumulated, how to understand these data will be critical. On the micro level, generations of molecular biologists' studying the effects of apparent biological traits genes exert on utilizing technologies (such as gene knockout) has made breakthroughs in many crutial aspects. On the macro level, statistics and other data analysis techniques are introduced to study the relationship between gene sequences and biological phenotype. The accumulation of basic scientific research gradually brings breakthroughs in clinical applications.

There are now two types of clinical applications mainly, one aims at disease screening of ordinary people. It detects people’s risks of getting cancer in the future by measuring the known genes associated with a disease loci. The other aims at the diagnosis cancer and other deadly diseases. It finds in a series of drugs or plans the most effective one for certain patients by testing the loci of certain genes.

Data from BBC research shows that total global gene sequencing market increased from $ 7.941million in 2007 to $ 4.5 billion in 2013, and is predicted to reach $ 11.7 billion in 2018 with the CAGR up to 21.2%.

Currently, the market of next-generation sequencing platform is mainly taken by several major manufacturers, including the Illumina, Ion Torrent / Life Technologies (was the acquisition of Thermo Fisher in 2014), 454 Life Sciences / Roche and other small ones like Creative Biolabs and CD Genomics.

Current Situations of Genome Sequencing for New Born Babies

Since last century, newborn babies have gotten a heel-prick test in which their blood is screened for dozens of congenital diseases. Routine newborn baby screening has basically eliminated the risk of death or irreversible brain damage that some of these disorders can pose if they are not identified right away. Even though, it has brought about great controversies in this field.

Last December, Mercy Children’s Hospital of Kansa released its results of genome sequencing for sick new born babies on Science Translational Medicine. In this research, they practiced whole genome sequencing or exome sequencing for children with severe neurological developmental disorders from 100 families, among which there are families that have been seeking for diagnostic methods for their children for years. According to the data, 45% of the families have experienced genome sequencing. Moreover, 73% of the babies with congenital disease have accepted this brand new testing approach.

Doctor Stephen F. Kingsmore, leader of this project hopes that the whole genome sequencing project could be carried on to around 14% of the newborns observed in ICU among the total 4 million per year.

Researchers from the USA are also investigating families’ attitudes towards genome sequencing. Last December, they made a research on parents of 514 healthy babies born within the past 48 hours in Brigham and Women's Hospital. This research was aimed to get a general knowledge of how much do these parents know about their babies’ genome information, risks of getting genetic diseases and the meaning of genome information. While being asked about whether they would be willing to attend the project of whole genome sequencing for newborn babies, results showed that 83% said yes and were happy to be in this project.

Moreover, Donald Chaplin of Acton, the farther a 17 months old baby, was also interested in it. He himself is a pharmacist, and he worried that the gene data might have the risk of been misused. Even though, he still wants to learn more about the gene information of his son. Jamaican engineer Nicholas Catella, however, said that he wouldn’t care much about the result unless it shows that his babies have great risks of getting severe diseases. Mr. Catella has two children, one is three old and the other 16months. He knows that gene sequencing is able to reveal the risks of getting chronic diseases like Alzheimer's, but we do have no good invention methods at present.

We hope, one day when this new testing skill develops greatly, that the gene sequencing data since the birth of babies can accompany throughout all their life, and that gene sequencing can lead the growing process of newborn babies. 

Bivalent and Bispecific Single Chain Antibody in Solving the Problems of scFv

Antibody is not a stranger to everyone, even a new born baby. It is the black hole on the biological world, as there is always something new that attracts biologists and researchers.

Since the first success achieved in single chain antibody fragment（scFv）research by Bird and Huston in the 1988, tremendous achievements have been made in the research and development of single chain antibody technology.

However, in terms of application, there are still problems remain unsettled. The scFv fragments derived from phage display antibody libraries usually have short half-life and less affinity. However, multivalency of antibody molecules turns out to be a desirable property in many in vitro and in vivo applications. After years of experience and great efforts, scientists and researchers from Creative Biolabs have carried out a series of approaches for bivalent and bispecific scFv and Fab construction, which may greatly contribute to the current situation. (Bivalent and Bispecific scFv/Fab)

According to its newest data, there are three approaches of generating genetically engineered and dimerized scFv antibody fragments, miniantibody, diabody and Tandem scFv.

Figure 1. Schematic diagram of bispecific antibody

Miniantibody

Bivalent or bispecific (scFv) 2, the so-called miniantibody, is produced by the combination of two scFv molecules with two modified dimerization domains. Leucine zippers are utilized to mediate dimerization of scFv in a miniantibody form. It constructed dimerization cassettes which allow the conversion of scFv antibodies from all its phage display libraries to bivalent or bispecific antibodies. During this procedure, either Fos or Jun leucine zippers are fused to scFv proteins. Two cysteine residues were engineered in the Fos and Jun zipper domains to produce disulfide-stabilized homodimers, which usually leads to efficient production of stable, secreted homodimers that are able to retain their specificity as assessed in a number of assays.

Diabody

Diabody is a non-covalent dimer of single-chain scFv, fragments that consists of the heavy chain variable (VH) and light chain variable (VL) regions connected by a small peptide linker. 14–15 amino acid residues’ common linkers are long enough to span the distance between the N- termini and C-termini of the variable domains in a scFv. However, utilizing linkers of 3–12 amino acid residues in length can lead to the formation of diabody.

When two ScFvs linked with the short linkers are expressed in the same cells, dual-functional antigen-binding sites will be formed through crossover pairing of the variable light-chains and heavy-chains. The bi-specific diabody, which is constructed with heterogeneous scFvs, is also an important and commonly used form of recombinant bi-specific antibody. A distinct feature of diabody is that it has a rigid structure and can be expressed at high yields in bacteria. See figure 2.
It is worth mentioning here that bispecific T cell engager (BiTE) is a unique form of tandem scFv. In a BiTE molecule, one of the scFvs binds to T cells via CD3 receptor and the other to a tumor cell via a tumor specific molecule. As this procedure brings together the T cell and cancer cell, it has great possibilities in cancer therapy. (BiTE). See figure 2.

Tandem scFv

Tandem scFv (taFv) is produced by connecting two scFv molecules with a short linker. This form of scFv has a very flexible structure and is comparatively easy to be generated. Both bacterial expression and refolding and eukaryotic expression are able to produce tandem scFvs. With years of experiences, Creative Biolabs has successfully constructed over 100 tandem scFvs.

Figure 2. Schematic diagram of Diabody and Tandem scFv

About Creative Biolabs

Creative Biolabs is a professional biotech service provider. Since been established in the year of 2005, it has been focused on the research and development of single chain antibody technology. Learn more about Creative Biolabs.