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...

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The clinical and laboratory features in five patients with non-excretory myeloma are described including plasma cell immunofluorescence and, in two cases, ultrastructure. Findings are compared with those in similar patients previously reported, and those in excretory disease. Clinical, haematological and biochemical features were similar to those found in excretory myeloma, showing differences only in relation to the absence of serum or urinary monoclonal immunoglobin. Cellular cytological and ultrastructural features allowed no differentiation from excretory cells. Within this non-excretory group distinction between secretory and non-secretory myeloma cells is possible on the basis of immunofluorescence. Differing patterns of intermittent excretion occurred in three of these patients.