D. McCarthy

Intrinsically stable IgG aggregates

Measurement of the absorbance due to light scattering at 40 nm proved to be a simple and reliable way of assessing the extent of aggregation in heat-treated IgG solutions. Using this technique the rate of aggregation was demonstrated to be markedly temperature dependent with a sharp inflexion in the curve close to 63 degrees C. During heating at 63 degrees C the concentration of unaggregated IgG fell in a manner consistent with a first-order process and the mean size of the IgG aggregates increased with time. IgG aggregates could be selectively removed from heat-treated IgG solutions and concentrated by precipitation with 3.5% polyethylene glycol without altering their size distribution. Furthermore, and in contrast with a previous report, the IgG aggregates examined in this study were remarkably stable in the absence of any other protein such as serum albumin. In consequence, several important practical recommendations concerning the production of heat-aggregated IgG for use in immune complex assays are made.

The effect of IgG aggregate size and concentration on reactivity in immune complex assays

The reactivity of heat-aggregated IgG of known size, in the Raji cell assay, the C1q binding assay and the C1q solid phase radioimmunoassay as a function of concentration, has been investigated. Marked differences were found in the way that the three assays behave when the IgG concentration and aggregate size are varied. These findings indicate the pitfalls in attempting to express the results of immune complex assays performed on biological fluids in terms of equivalent concentrations of aggregated IgG.

A rapid preparation technique for leucocytes

A procedure is described for making leucocyte preparations from blood samples fixed ex vivo. Briefly, blood is treated with buffered formaldehyde and the erythrocytes and platelets removed subsequently by lysis and differential centrifugation. The fixed leucocyte preparations can then be processed or fixed further for various types of microscopy as required. In stained preparations examined by light microscopy, the different leucocytes, i.e. lymphocytes, polymorphonuclear leucocytes (PMN) and monocytes, can readily be identified by their characteristic nuclear morphology, and polarized PMN can easily be recognized by their non‐spherical shape. Experiments with chemotaxins in vitro indicated that the procedure could be used to determine whether polarized PMN circulate in the blood of normal individuals and patients with inflammatory disease. A study of the blood from eleven normal subjects showed that 84.0 ± 10.8% (mean ± 2 SEM) of circulating PMN were spherical, while the majority of the remainder (14.3 ± 10.5%) were only ‘slightly’ polarized.

The production of small IgG aggregates by glutaraldehyde cross-linking

Reaction conditions have been determined for the production of soluble IgG polymers in the size range 10 S to 30 S by covalent cross-linking with glutaraldehyde. This size range is comparable with that of the immune complexes which are frequently found in the circulation of patients with certain autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. The yield of IgG aggregates in this size range is far greater than has been reported for cross-linking by other bifunctional reagents or for aggregation by heating. Glutaraldehyde cross-linked IgG polymers are stable and biologically reactive. They can also be labelled with fluorescein and freeze-dried with minimal loss of integrity or reactivity.

A simple procedure for assessing the stability of heat-aggregated IgG preparations

Abstract A simple procedure is described for distinguishing between IgG preparations which on heating form aggregates that are subsequently either stable or unstable in the absence of additional protein. Of 4 commercially available IgG preparations tested in this manner, only one gave IgG aggregates that were unstable.

A tannic acid based preparation procedure which enables leucocytes to be examined subsequently by either SEM or TEM

A modification of the glutaraldehyde‐osmium tetroxide‐tannic acid‐uranyl acetate (GOTU) fixation procedure is described which allows human leucocytes to be examined subsequently by either transmission electron microscopy (TEM) or scanning electron microscopy (SEM).

Soluble IgG aggregates produced by heating remain stable on freeze-drying

IgG aggregates produced by heating gamma globulin solutions were freeze-dried, kept at 4 degrees C and reconstituted up to 4 months later. By comparison with frozen (-20 degrees C) preparations, only minimal changes in biological reactivity and in physical integrity occurred during this period. These results demonstrate that freeze-dried preparations of heat-aggregated IgG are potentially useful as a reference reagent for the comparative evaluation and standardisation of immune complex assays.

Centrifugation of normal and rheumatoid arthritis blood on Ficoll-Hypaque and Ficoll-Nycodenz solutions.

Attempts to use the rapid single-step Ficoll-Hypaque centrifugation procedure for the purification of mononuclear and polymorphonuclear leucocytes from the blood of normal individuals and rheumatoid arthritis patients have sometimes been unsuccessful, largely because the erythrocytes would not sediment through the centrifugation medium. Re-evaluation of the factors (e.g. Ficoll concentration, temperature, and ratio of the diatrizoate salts) which affect these separations showed that under our conditions it was advantageous to use a medium with a lower viscosity (Ficoll concentration) and/or a higher osmotic strength (increased sodium diatrizoate: meglumine diatrizoate) than had been recommended previously (Ferrante and Thong, 1978; 1980; Ferrante et al., 1982). Higher osmotic strength media must be used for separating the components of blood from rheumatoid arthritis patients than from normal individuals because rheumatoid arthritis erythrocytes have a lower buoyant density than normal erythrocytes.

Binding of monomeric and aggregated immunoglobulin to enzymes: A source of artefact in antibody assays

Pepsinogen has previously been shown to bind non-specifically to immune complexes and aggregated immunoglobulins. We demonstrate here using a solid-phase immunoassay that immunoglobulins aggregated by heat or glutaraldehyde bind non-specifically to several different enzymes. Some of these, including pepsinogen (marketed as pepsin), hyaluronidase and trypsin, are used in the breakdown of tissues or biochemical preparations during the preparation of antigens. Contamination of impure antigens by enzyme is likely to lead to products which bind non-specifically to immune complexes. This can cause misidentification of complexes as antibodies. We recommend that all tests for specific antibody involving the use of antigens prepared by these or other enzymes should include a control with aggregated immunoglobulin substituted for the test serum.

Quantitation of solid-phase immunoassays using a fibre-optic reflectance spectrophotometer

A reflectance measuring instrument (fibre-optic spectrometer linked to a personal computer) which will quantitate the results of immunoassays that result in the deposition of a coloured chromophore on a membrane or other similar solid phase (SP) is described and the reflectance spectra of some chromophores which are commonly produced or used in SP immunoassays are presented. The instrument has been used in conjunction with silver-enhanced SP-immunogold capture assays to determine the concentrations in serum of IgG and of IgM rheumatoid factor (RF) and the results have been compared with those obtained by rate nephelometry and latex agglutination assays, respectively. It is concluded that fibre-optic reflectance photometry is an accurate and useful means of quantitating SP immunoassays in which coloured chromophores are produced and that the results obtained from such immunoassays are comparable to those given by established procedures.