Conjugation of Glucose Oxidase from Aspergillus niger and Rabbit Antibodies Using N-Hydroxysuccinimide Ester of N-(4-Carboxycyclohexylmethyl)-Maleimide

Abstract

Glucose oxidase from Aspergillus niger was conjugated with rabbit immunoglobulin G or its monovalent fragments (Fab'). The enzyme was treated with N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethy1)-maleimide to introduce maleimide groups, which were then allowed to react with thiol groups of reduced IgG or Fab'. More than 40% of immunoglobulin G, Fab' and enzyme used could be conjugated without self-coupling. The enzyme activity decreased about 26 and 15% upon conjugation with immunoglobulin G and Fab', respectively, and the ability of antibody to bind to antigen was well preserved in conjugates. Conjugate preparations purified by gel filtration contained little free form of immunoglobulin G, Fab' or enzyme. Both the cross-link and enzyme activity in Fab' conjugate were stable at pH 6 -7 at 4 "C for at least 6 months.Several maleimide compounds have been synthesized for cross-linking of proteins [l]. We selected one of them, o-phenylenedimaleimide, for the conjugation of antigens and antibodies with P-D-galactosidase from Escherichia coli. Thiol groups were generated in antigens or antibodies either by mercaptosuccinylation [2] or by reducing disulfide bonds [3,4] and treated with excess of o-phenylenedimaleimide. Maleimide groups thus introduced into antigens and antibodies were then allowed to react with thiol groups of b-D-galactosidase. Kitagawa and Aikawa have directly introduced maleimide groups into proteins using N-hydroxysuccinimide ester of m-maleimidobenzoic acid [5]. However, both thiol and maleimide groups are labile under conditions used in these studies.The present paper describes the preparation of rabbit IgG-glucose oxidase and Fab'-glucose oxidase conjugates using N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethy1)-maleimide under the condition where both thiol and maleimide groups are stable.Abbreviations. IgG, immunoglobulin G; F(ab')z, divalent fragments obtained by digesting IgG with pepsin; Fab', monovalent fragments obtained by reducing F(ab')z.Enzymes. P-D-Galactosidase (EC 3.2.1.23) ; glucose oxidase (EC 1.1.3.4); peroxidase (EC 1.11.1.7); pepsin (EC 3.4.23.1). Complete decomposition of excess acetic anhydride resulted in a clear brown solution. Then, 2 g charcoal was added and removed by filtration, while the mixture was hot. The filtrate was concentrated to 300ml in vmuo, cooled and allowed to stand overnight. The crystals formed were washed with 50 ml cold water and dried at 70°C overnight to yield 6.4 g (33.6"/,), m.p. 144.0-146.0 "C. Anal. Calcd. for C12H15N04: C, 60.75; H, 6.37; N, 5.91. Found: C, 60.24; H, 6.34; N , 5.65. MATERIALS AND METHODS Synthesis Synthesis oj N-Hydroxysuccinimide EsterA solution of 2.9 g (12 mmol) of N-(4-carboxycyclohexylmethy1)-maleimide and 1.9 g (16 mmol) of thionyl chloride in 30 ml of dry benzene was refluxed at 80 "C. After 1 h, benzene and thionyl chloride were removed under vacuum, leaving a residue, crude N-(4-chloroformylcyclohexylmethyl)-maleimide (3.0 g, 95.7 %). N-Hydroxysuccinimide (1.41 g, 10 mmol) in 30 ml of diglyme was...

Silica-Coated CdTe Quantum Dots Functionalized with Thiols for Bioconjugation to IgG Proteins

“…38 The reactive sulfo-NHS group creates amide bonds between the IgG and the cross-linker and leaves a long-lived maleimide reactive intermediate (Figure 2). The cyclohexane ring in sulfo-SMCC stabilizes the maleimide group and was found to be 50% stable in PB buffer after 64 h. 37 Here we have modified that protocol to allow our thiolated QDs to be easily and efficiently conjugated to IgG proteins and streptavidin-maleimide for interaction with polystyrene biotinylated microspheres.…”

Phosphorylation of αB-crystallin in Mitotic Cells and Identification of Enzymatic Activities Responsible for Phosphorylation

“…Rabbit antiserum against the amino-terminal dodecapeptide of ␣B-crystallin was raised in rabbits by injecting the peptide (MDIAIHHPWIRR-Cys) conjugated with hemocyanin (Sigma) using N-(4-carboxycyclohexylmethyl)maleimide (32), and the antibodies were purified by use of a column of bovine ␣B2-crystallin-coupled Sepharose as described previously (11). Antibodies that recognize each of the three phosphorylated serine residues in human ␣B-crystallin (Ser-19, -45, and -59) were prepared as described previously (24).…”

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“…Several factors may affect the conjugation process, such as concentrations of reactants (antibody, enzyme and cross-linker), pH, temperature, incubation time and purity of the enzyme. 26 In addition, amine groups of antibody and RBP randomly activated by glutaraldehyde and sulfo-SMCC in cross-linking reaction may lead to reduction of antibody and enzyme activity. 27,28 The periodate oxidation method is generally recommended for conjugation of glycoprotein as the carbohydrate moieties of antibody and enzyme are generally not required for their activities.…”