Solid‐phase biotinylation of antibodies

Abstract

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.

Quantitative measurement of IgE antibodies to purified allergens using streptavidin linked to a high-capacity solid phase

“…This is in keeping with previous results on immunoglobulins but would need to be confirmed with other allergens. 13,17 The original identification of IgE was difficult because of the small quantities of this protein in serum. 1 In addition to being quantitatively lower than all other isotypes, total IgE is more variable and is profoundly influenced by allergic disease.…”

“…16,17 Typically, 5 mg Enzotin in 0.12 mL sterile water was added to 40 mg protein in 1 mL PBS. Assuming a molecular weight (MW) for the allergens of 30,000 d, this represents a molar ratio of 10 molecules Enzotin for each protein molecule.…”

Microfluidic immunoaffinity separations for bioanalysis

“…Avidin is a protein from egg white that has a high affinity for the vitamin biotin (K a = 10 15 /M), forming strong noncovalent bonds [19,20]. Streptavidin is a bacterial form of avidin with similar binding sites for biotin, but is also a form that has less nonspecific interaction with proteins.…”

“…Unlike the antibody binding of protein A or G, the streptavidin biotin bond is nearly irreversible. Antibodies must be linked to biotin in order to react with streptavidin, but their activity is unlikely to be affected due to the small size of biotin (244 Da) [20]. Numerous biotin derivatives for linkage to antibodies are available.…”

Simple and Portable Magnetic Immunoassay for Rapid Detection and Sensitive Quantification of Plant Viruses

“…Because the biotinylation reaction is based on the binding of random primary amine groups to the N-hydroxysuccinimide (NHS)-functionalized biotin molecule (48), it was necessary to ensure that epitope-binding activity was not significantly inhibited. Therefore, the binding efficiency and kinetic behavior of the biotinylated antibodies were evaluated by SPR spectroscopy (Fig.…”