Enzyme immunoassays for measuring complement-dependent prevention of immune precipitation (PIP) and solubilization of preformed antigen–antibody complexes (SOL)

Abstract

We have developed simple and sensitive enzyme-based methods for evaluating the ability of serum complement to prevent immune complex precipitation (PIP) or to solubilize preformed immune complexes (SOL). Alkaline phosphatase, serving both as antigen and label, is added to goat IgG anti-alkaline phosphatase antibodies, with serum present throughout the assay (PIP), or added after immune complex formation (SOL). After incubation at 37°C for 1 h followed by centrifugation, the enzyme activity of the supernatant, reflecting the amount of immune complexes in solution, is measured by colorimetry. Results are expressed with reference to a standard serum pool assigned 100 arbitrary units (AU). Intra- and inter-assay variabilities are within 10%. The normal ranges were 67-133 AU for PIP and 72-129 AU for SOL. These methods have been standardized for clinical use in relation to impaired complement function and immune complex disease, and adapted for measuring complement mediated binding of immune complexes to erythrocytes. They are sensitive, easy to perform and do not require expensive facilities. By measuring the interaction of complement with immune complexes, these methods may highlight aspects of the classical and the alternative pathway that are different from those detected using haemolysis as an endpoint.

Introduction

Complement is thought to play an important role in immune complex disease by either suppressing or exacerbating the disorder, depending on specific prevailing circumstances. On the one hand, activation of the classical pathway may prevent the formation and tissue deposition of large immune complexes at the time of the antigen–antibody reaction (Schifferli et al., 1980; Naama et al., 1984). Under special circumstances, however, when immune complex deposition is not prevented, activation of the alternative pathway may lead to solubilization of the deposited complexes (Miller and Nussenzweig, 1975). Both these functions of complement are regarded as being clinically relevant; the former, variably termed inhibition or prevention of immune precipitation (IIP or PIP) (Schifferli et al., 1980; Naama et al., 1984) is thought to form an important part of a mechanism whereby immune complexes are opsonized and eliminated without inflammation, whereas clearance by solubilisation (SOL) may result in inflammation through the generation of phlogistic complement fragments (Schifferli et al., 1986a). Assays which measure the capacity of serum to influence the size and solubility of nascent or preformed immune complexes are thus useful for studying the involvement of complement in the etiology and pathogenesis of immune complex disease (Lachmann and Walport, 1987; Lachmann, 1990; Sturfelt et al., 1992).

Current assays which measure the ability of serum to prevent immune precipitation (PIP) or solubilize complexes after their formation (SOL) are based on the use of ${}^{125}\text{I}$-labelled antigens (Miller and Nussenzweig, 1975; Takahashi et al., 1978; Schifferli et al., 1980; Naama et al., 1984, Naama et al., 1985), and have so far only been standardized for measuring SOL (Baatrup et al., 1983). These methods may lack sensitivity (Schifferli et al., 1986b) and they have the drawback of requiring access to radiometric facilities. We have developed and standardized methods for measuring PIP and SOL in which the enzyme alkaline phosphatase (AP) is employed as both antigen and label. The ability of sera to influence the size and solubility of nascent or preformed AP–anti-AP immune complexes is monitored by measuring enzyme activity in supernatants after centrifugation. This paper describes the development and standardization of these methods.