Antibody screening process

Whether one uses hybridomas or phage libraries, screening and selection methods are crucial.In essence, the screen or selection determines what products will be obtained from a library, and a poorly organized screen or selection can severely hamper catalytic searches. CatELISA, which was developed from the conventional enzyme-linked immunosorbent assay (ELISA) for antibody affinity, uses substrate instead of hapten in the screening process. This then provides a more direct link to effectively screen for catalysis. 

By antibody screening of a Agtll cDNA expression library prepared from spinach poly (A+)-RNA cDNA clones encoding the entire precursor protein of CP24 were isolated. N-terminal amino acid sequence of the 20 kDa polypeptide of CP24 matched the corresponding sequence deduced from the nucleotide sequence. The nuclear encoded precursor protein is composed of 261 amino acids while the mature protein contains 210 amino acids (Table 1). When the isolated cDNA was translated vitro the product was imported into chloroplasts at high efficiency and processed correctly to its mature size (Fig. 1). 

A useful sensitive primary screening process for antibodies on the basis of their binding to immobilized antigen is the phage EUSA. Individual colonies generated by the selection process can be picked into 96-well format and phagemid rescue carried out. The resultant phage particles can then be used directly in an... 

Follman, D. K. and Fahrner, R. L. Factorial screening of antibody purification processes using three chromatography steps without protein A. /. Chromatogr. 1024 79-85, 2004. 

The use of a reporter line has multiple benefits for the high content screening process (a) assay development is facilitated as reporter expression can be followed in real time, (b) Assay controls are immediately visible before processing the plates as described, (c) Antibody-based read onts are costly and can be prohibitive when running larger screens. 

The importance of minimizing the number of covalent steps in the process to be catalysed is rather obvious. Single-step and double-step processes dominate the abzyme scene. However, there is substantial evidence that some acyl transfer reactions involve covalent antibody intermediates and so must proceed by up to four covalent steps. Nonetheless, such antibodies were not elicited by intentional design but rather discovered as a consequence of efficient screening for reactivity (Section 5). 

Screening in early work sought to identify high affinity of the antibody for the TSA, using a process known as ELISA. This search can now be performed more quantitatively by BIAcore analysis, based on surface plasmon resonance methodology (Lof s and Johnsson, 1990). A subsequent development is the catELISA assay (Tawfik et al., 1993), which searches for product formation and hence the identification of abzymes that can generate product. 

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

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