Estimation of IgM rheumatoid factors by fluorimetry.

D J Lea,D J Ward

doi:10.1136/ard.37.3.247

Abstract

Sheep red cells coated with rabbit IgG antibody may be used to detect IgM rheumatoid factors either by agglutination or microscopically by the fluorescence of antihuman IgM antibody conjugate bound to rheumatoid factors on the cell surface. By substituting for red cells the plastic surface of a filter disc coated with rabbit IgG it was possible to elute the bound conjugate and measure the fluorescence in a fluorimeter. The results of both the sheep cell agglutination test and the fluorescence test agreed quite well for the majority of sera; both tests seemed to reflect IgM rheumatoid factor concentrations in these sera. The difficulties encountered in attempting to define and measure accurately rheumatoid factor concentrations in a serum are emphasised.

Highlights

Five sheep cell agglutination test (SCAT)-positive sera were assayed at one level (O- 8,.l) for IgG RFs, by using fluorescent anti-IgG conjugate, and gave fluorescence values between 0- 17 and 0- 355, which seemed unrelated to their SCAT titres
An unexpected combination of high fluorescence value and low SCAT titre was given by serum from a patient with myeloma and mixed connective tissue disease. The determination of both fluorescence values and SCAT titres were subject to experimental error, it was evident that a relationship existed between the titre of a serum and the amount of IgM in 1,d serum, which was bound to immobilised rabbit IgG under assay conditions
Whereas immunoassay techniques for determining the concentration of homogeneous antigens and simple chemicals may be judged in terms of specificity, accuracy, and precision, greater problems arise in the measurement of heterogeneous antigens, such as parathyroid hormone and serum immunoglobulins

Summary

Introduction

From a freshly prepared solution of anti-IgM conjugate and filtered normal rabbit serum in buffer (10 ,tl and 40 ,d respectively in 1 ml) a portion of 100 [l was added to each cup and disc, and after gentle agitation the covered racks were kept overnight at room temperature. Treatment in the same assay with two anti-IgM conjugates standardised by the manufacturer bu t with different batch numbers gave different mean fluorescence values.

Results

Conclusion