Cold Spring Harbor protocols最新文献

The antigen-binding fragment (Fab) is the ∼50-kDa monovalent arm of an antibody molecule. In the laboratory, the Fab can be produced via either enzymatic digestion or recombinant expression, and its use facilitates the accurate assessment of affinity and specificity of monoclonal antibodies. The high melting temperature of the Fab, together with its low tendency to aggregate and ready conversion to natural and nonnatural immunoglobulin (Ig) formats (without affecting antigen binding properties), have made it a preferred format for phage display, as well as a tool for accurate assessment of affinity, specificity, and developability of monoclonal antibodies. Here, we outline a strategy to clone, express, and purify human or chimeric nonhuman/human Fabs that have previously been selected by phage display. Fabs purified using this approach, which results in milligram amounts, enable a variety of downstream biophysical and biological assays that ultimately inform the success of phage display library generation and selection.

Rabbit monoclonal antibodies are attractive reagents for research, and have also found use in diagnostic and therapeutic applications. This is owed to their high affinity and specificity, along with their ability to recognize epitopes conserved between mouse and human antigens. Phage display is a powerful method for the de novo generation, affinity maturation, and humanization of rabbit monoclonal antibodies from naive, immune, and synthetic antibody repertoires. Using phagemid family pComb3, a preferred phage display format is chimeric rabbit/human Fab, which consists of rabbit variable domains (V_H, V_κ, and V_λ) fused to human constant domains. The human constant domains, C_H1 of IgG1 and C_L (C_κ or C_λ), not only provide established purification and detection handles but also facilitate higher expression in Escherichia coli compared to the corresponding rabbit constant domains. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of chimeric rabbit/human Fab libraries with randomly combined rabbit variable domains of high sequence diversity, starting from the preparation of total RNA from rabbit spleen and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 10⁸-10¹¹ independent chimeric rabbit/human Fab clones. As such, it can be used, for instance, for the generation of either specialized immune or large naive rabbit antibody libraries.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Monoclonal antibodies (mAbs) have exceptional utility as research reagents and pharmaceuticals. As a complement to both traditional and contemporary single-B-cell cloning technologies, the mining of antibody libraries via display technologies-which mimic and simplify B cells by physically linking phenotype (protein) to genotype (protein-encoding DNA or RNA)-has become an important method for mAb discovery. Among these display technologies, phage display has been particularly successful for the generation of mAbs that bind to a wide variety of antigens with exceptional specificities and affinities. Rather than multivalent whole antibodies, phage display typically uses monovalent antibody fragments, such as "fragment antigen binding" (Fab), as the format of choice. The ∼50-kDa Fab format consists of four immunoglobulin (Ig) domains on two polypeptide chains (light chain and shortened heavy chain), and exhibits its antigen binding site in a natural configuration found in bivalent IgG and other multivalent Ig molecules. The Fab fragment has a high melting temperature and a low tendency to aggregate, and can be readily converted to natural and nonnatural Ig formats without affecting antigen binding properties, which has made it a favored format for phage display for more than three decades. Here, I briefly summarize some of the approaches used for the generation and selection of phage display antibody libraries in Fab format, from human and nonhuman antibody repertoires.

The size of an antibody library, that is, the phage display-selectable diversity, is restricted mainly by its transformation into the host bacterial cells. Electroporation is the most efficient method for transforming Escherichia coli with plasmids, including phagemids. Here, we describe the preparation of electrocompetent E. coli for the generation of phagemid-encoded antibody libraries encompassing 10⁹-10¹¹ independent transformants. To become electrocompetent, the bacterial suspension has to have high resistance, i.e., low ionic strength, which is achieved by gradually and gently transferring bacteria grown to mid-log phase to 10% (v/v) glycerol in highly pure water. The electrocompetent E. coli must be F plasmid-harboring bacteria, referred to as F⁺ or male, in order to express F pili and be susceptible to infection by filamentous phage during library generation. In addition, it is necessary to apply antibiotic (e.g., tetracycline) pressure to retain the F plasmid, as it tends to segregate from bacteria. This protocol also includes assays for analyzing the prepared electrocompetent E. coli for competency, and evaluating potential contamination with helper phage, phagemid and phagemid-derived filamentous phage, and lytic phage.

Phage display selection of antibody libraries is a powerful method for generating and evolving monoclonal antibodies. The pComb3 phagemid family of phage display vectors facilitates the mining of antibody libraries in Fab format from human and nonhuman antibody repertoires. Here, we describe the screening for monoclonal Fab binders after selection of a polyclonal pool of Fab binders to an antigen of interest, with the goal of identifying and sequencing monoclonal antibodies that bind the antigen with high affinity and specificity. The screening cascade involves a phage ELISA, followed by a crude Fab ELISA and DNA fingerprinting and sequencing. The protocol outlines phage and crude Fab ELISAs using purified antigen immobilized on microplates, native antigen expressed on eukaryotic cells, or both.

The mining of naive, immune, and synthetic antibody repertoires by phage display has been widely applied to the de novo generation and in vitro evolution of monoclonal antibodies from multiple species. Once built, phage display antibody libraries can be selected by a variety of different strategies tailored toward the desired antigen binding properties. Here, we describe the selection of antibody libraries generated in a phage display vector of the pComb3 phagemid family. The approach includes panning procedures for immobilized antigens, biotinylated antigens in solution, and cell surface antigens. Although the typical format of these antibody libraries is human Fab or chimeric nonhuman/human Fab, the basic selection strategies provided in this protocol are compatible with a variety of formats.

Phage display is a powerful method for the de novo generation and affinity maturation of human monoclonal antibodies from naive, immune, and synthetic antibody repertoires. The pComb3 phagemid family of phage display vectors facilitates the selection of human monoclonal antibody libraries in the monovalent Fab format, which consists of human variable domains V_H and V_L (V_κ or V_λ), fused to the human constant domains C_H1 of IgG1 and C_L (C_κ or C_λ), respectively. Here, we describe the use of a pComb3 derivative, phagemid pC3C, for the generation of human Fab libraries with randomly combined human variable domains (V_H, V_κ, and V_λ) of high sequence diversity, starting from the preparation of mononuclear cells from blood and bone marrow. Depending on the complexity of the parental antibody repertoire, the protocol can be scaled for yielding a library size of 10⁸-10¹¹ independent human Fab clones. As such, it can be used, for instance, for the generation of a large naive human Fab library from healthy individuals or for the generation of a specialized immune human Fab library from individuals with an endogenous antibody response of interest.

Amino acids in maize can exist in both a free and protein-bound state. While most amino acids are part of a protein backbone, a small percentage of them remain free and play important biological roles, serving as signaling molecules, nitrogen transporters, osmolytes, and precursors for multiple primary and secondary metabolites. Their levels vary widely especially in maize leaves, depending on the developmental stage and in response to environmental conditions. Therefore, accurate and reliable quantification of free amino acids (FAAs) is vital in any effort aimed at studying their response to developmental and environmental cues. In this protocol, we describe a robust, high-throughput method that quantifies the 20 proteogenic amino acids (i.e., those that can be incorporated into proteins) that are found in the free form in maize tissue. This method consists of three major parts: first, aqueous extraction of FAAs from maize tissue; second, separation, detection, and quantification of all 20 proteogenic amino acids using ultraperformance liquid chromatography-tandem mass spectrometry; and third, data analysis and processing using the MassLynx data analysis software, TargetLynx.

In cereal crops, seed quality is determined by the composition and levels of protein-bound amino acids, which account for ∼90% of the seed total amino acid content. In maize particularly, seed quality is affected by the low levels of lysine and tryptophan, two amino acids that humans and animals cannot synthesize and must obtain from the diet. The low levels of these two amino acids in seeds is due to the dominance of seed storage proteins, namely zeins, which are deficient in these two amino acids. Many efforts have been deployed to improve the nutritional composition of maize kernels (i.e., seeds). Still, the lack of high-throughput and inexpensive methods for the quantification of amino acids that are found within proteins has limited those efforts, especially when large populations are targeted. In this protocol, we describe a robust, efficient, and high-throughput method for the quantification of all 20 proteogenic (protein-forming) amino acids from a crude protein extract. The method consists of four major parts: first, release of the 20 proteogenic amino acids from the protein backbone through hydrolysis; second, aqueous extraction of the released amino acids; third, separation, detection, and quantification of the released amino acids using a multiple reaction monitoring-based ultraperformance liquid chromatography-tandem mass spectrometry detection; and fourth, data analysis and processing using the MassLynx data analysis software, TargetLynx.