Duration: 5 min

Yajun Cao

Protein and Antibody Product Marketing

Dr. Yajun Cao graduated from Tsinghua University with a Ph.D. in biology. Yajun has published her work in journals, including Cancers (Basel) and Stem Cell Research. She joined GenScript's Antibody Department and has been working as a Senior Scientist since 2024. Yajun has over 10 years of experience in antibody discovery and antibody-based assay development. She is currently responsible for mouse monoclonal antibody platform and antibody sequencing platform. She has participated in the delivery of over 500 mouse mAb project.

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CONTENTS

• Polyclonal Anti-Id Technology
• Monoclonal Anti-Id Technology
• Antibody Library Technology

Idiotype antibodies (Id or Ab1) refer to antibodies that possess unique antigenic determinants, while anti-idiotype antibodies (Anti-Id or Ab2) are antibodies generated against the unique sites of antibodies. Since Anti-Id antibodies are also a type of antibody, the production methods are largely similar to those of conventional antiserum. Currently, the commonly used methods include polyclonal Anti-Id, monoclonal Anti-Id, and antibody library technology.

Polyclonal Anti-Id Technology

According to the immune network theory, after an antigen induces the production of antibodies in the body, the idiotype (Id) of these antibodies can further induce the production of anti- idiotype (Anti-Id). Therefore, a single immunization with monoclonal antibody (Ab1) can yield Anti-Id antibodies (Ab2). The advantage of this method is its low cost and simplicity. However, the drawback is that multiple preparations are required if a large quantity is needed. Statistics show that Anti-Id antibodies constitute only about 10% of the immunoglobulins in antiserum. If the goal is to select Anti-Id antibodies that can serve as artificial antigens or vaccines, particularly those targeting "common reactive idiotypes," the content is even lower. Additionally, the immunochemical properties of Anti-Id antibodies produced in different preparations lack uniformity, and the subsequent purification of the serum is relatively cumbersome.

Monoclonal Anti-Id Technology

Firstly, this method involves immunizing animals with an antigen to produce Ab1, and then using Ab1 as an immunogen to immunize another animal to produce Ab2. Since this process involves two rounds of immunization: antigen-animal I-Ab1-animal II-Ab2, some researchers also refer to it as the two-step immunization method.

As Ab1 used as the immunogen consists of both the Fab and Fc segments, each with a relative molecular mass of approximately 50 kDa, and since the idiotypic determinants are composed of amino acids in the hypervariable regions of the IgVL and VH domains of the Fab segment, their molecular weight is very small. The immunogenicity of these determinants is far inferior to that of the antigenic determinants on the larger Fc segment. Therefore, it is necessary to obtain highly purified Fab or F(ab')2 segments for immunizing animals.

Currently, different proteases are selected based on the specific characteristics of the antibodies to prepare Fab or F(ab')2 fragments. Commonly used enzymes include pepsin, papain, and ficin. Reports indicate that papain digestion of IgG may damage the binding sites of the Fab fragments. Pepsin operates at a low pH, which can easily cause denaturation of the antibody protein. Ficin (with a relative molecular mass of 25 kDa) is an emerging enzymatic cleavage technology that can catalyze various reactions and has a broad pH working range. During enzymatic cleavage, the pH can be adjusted to a range that maintains the activity of both the enzyme and the antibody ^[1].

Although the initial preparation process of monoclonal antibodies, including the preparation, separation, and purification of Fab or F(ab')2 fragments, is complex and costly, once successfully prepared, a sufficient amount of antibodies can be obtained easily and permanently. Therefore, this method offers higher practicality.

Antibody Library Technology

Antibody library technology refers to the use of gene cloning techniques to clone the complete repertoire of heavy and light chain variable region genes of a specific animal. These genes are then recombined into specific expression vectors, and functional antibody molecule fragments are expressed using E. coli or phages. Through affinity screening, specific antibody variable region genes are obtained. This technology allows for the screening of high-affinity antibodies from the constructed library while also identifying the genes encoding these antibodies. Due to its high throughput, operability, controllability, and diverse approaches, it has become one of the most dynamic fields in the development of antibody drugs ^{[2, 3]}.

Antibody library technology has evolved through several stages, including combinatorial antibody libraries, phage display antibody libraries, and ribosome display antibody libraries. Combinatorial antibody libraries involve randomly cloning the genes encoding antibody heavy and light chains into the same phage, which are then expressed as functional antibody fragments in engineered bacteria ^[4]. Shortly after their emergence, combinatorial antibody libraries were largely replaced by phage display antibody library technology, which was developed based on phage display technology ^[5].

Phage display antibody library technology involves inserting antibody genes downstream of the signal peptide coding region of phage membrane protein gene III or gene VIII, allowing them to be displayed as fusion proteins at the N-terminus of coat protein III or VIII. This technology, which uses phages and bacteria as hosts, is easy to operate and cost-effective, making it the most mature and widely used antibody library technology to date ^[6].

Ribosome display technology is a novel screening method that leverages functional protein interactions. The principle involves PCR amplification of a DNA library of target genes, incorporating promoters, ribosome binding sites, and stem-loops. The library is then incubated in a cell-free translation system capable of coupled transcription and translation, allowing the translation products of the target genes to be displayed on the surface of ribosomes, forming "mRNA-protein-ribosome" ternary complexes. Conventional immunological detection methods, such as immobilized target molecules, are used to screen the desired ribosome complexes from the ternary complexes. RT-PCR amplification is then performed, and through multiple cycles, high-affinity target molecules are ultimately selected [26-28]. Since this technology does not involve cell transformation, the constructed library capacity can be very large, reaching up to 1×1015. Additionally, mutations can be conveniently introduced during the amplification process after each round of screening, promoting antibody molecular evolution and affinity maturation ^[7].

GenScript——One stop Anti-ID antibody development service

GenScript's Anti-ID Antibody Development Service provides fast and affordable solutions for drug development. With our industry-leading manufacturing technology, you'll have anti-ID polyclonal antibody in just 7 weeks and anti-ID monoclonal antibody in 9 weeks—giving you more flexibility in your research timeline. What's more, GenScript’s rabbit anti-ID mAbservice is now the same price as our mouse anti-ID mAb! Rabbit immunization has a proven performance in antibody discovery compared to mouse. However, due to budget constraints, it is most often overlooked. Now you can take advantage of premier rabbit anti-ID mAbdevelopment for the price of mouse!

References

[1] Dainiak MB, Muronetz VI, Izumrudov VA, Galaev IY, Mattiasson B. Production of Fab fragments of monoclonal antibodies using polyelectrolyte complexes. Anal Biochem. 2000 Jan 1;277(1):58-66. doi: 10.1006

[2] Kim SJ, Park Y, Hong HJ. Antibody engineering for the development of therapeutic antibodies. Mol Cells. 2005 Aug 31;20(1):17-29.

[3] Hoogenboom HR. Selecting and screening recombinant antibody libraries. Nat Biotechnol. 2005 Sep;23(9):1105-16. doi: 10.1038/nbt1126.

[4] Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M, Burton DR, Benkovic SJ, Lerner RA. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science. 1989 Dec 8;246(4935):1275-81. doi: 10.1126/science.2531466.

[5] Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making antibody fragments using phage display libraries. Nature. 1991 Aug 15;352(6336):624-8. doi: 10.1038/352624a0.

[6] Thie H, Meyer T, Schirrmann T, Hust M, Dübel S. Phage display derived therapeutic antibodies. Curr Pharm Biotechnol. 2008 Dec;9(6):439-46. doi: 10.2174/138920108786786349.

[7] Edwards BM, He M. Evolution of antibodies in vitro by ribosome display. Methods Mol Biol. 2012;907:281-92. doi: 10.1007/978-1-61779-974-7_16.