Evaluation of a simplified sucrose gradient method for the detection of rubella‐specific IgM in routine diagnostic practice

Abstract

Rubella-specific IgM was measured in a single fraction of serum from a sucrose density gradient. Haemagglutination inhibition (HAI) tests were performed on paired aliquots of the fraction untreated and after treatment with 2- mercaptoethanol, dilutions of the aliquots being incubated over night with rubella antigen before the addition of red cells. Of 822 sera tested, specific IgM was found in 249, but not in 492. When first tested, the remaining 81 sera gave unsatisfactory results because of contamination of the IgM fraction with IgG (6.0%), probable aggregation of IgG (3.5%), or the persistence of chick red cell agglutinins (0.4%). Tests were performed on 134 patients with rubella confirmed by a rise of HAI antibodies. Rubella-specific IgM was found at a titre of more than eight in the sera taken from 62 of 64 patients between 10 and 29 days after the onset of the rash but in only one of the sera taken between 80 and 119 days, and in none taken later. However, specific IgM was still to be found at lower titre in the sera of 13 patients collected between 80 and 162 days after the onset of the illness. In routine diagnostic tests over three years on the serum from 479 patients with suspected acquired rubella, specific IgM was found at a titre of more than eight in 51 patients and in only 10 instances (2.1%) did a lower level pose a problem in interpretation.

Enzyme-linked immunosorbent assay (ELISA) using antibody class capture for the detection of antitoxoplasma IgM.

“…The first four fractions collected were tested in the presence and absence of 2-ME. Of each fraction, 150 ,ul was added to two tubes, one tube received 50 Al of [0][1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] M NaCl and the other 50 ,l of 0.05 M 2-ME, the tubes were incubated at 37°C for one hour. The contents of both sets of tubes were examined by the DT, IgM-IFA and indirect haemagglutination test (IHA).…”

Use of the ultracentrifuge vertical rotor in the detection of rubella-specific IgM on a sucrose density gradient.

“…Twelve fractions of 0 4 ml were collected from each tube and tested as described below. Fractions 1-6 were also treated with 2-mercaptoethanol (2-ME) by the method of Caul et al (1974) in order to distinguish IgM from non-specific inhibitors or aggregated IgM (Caul et al, 1978).…”

“…The separation of immunoglobulins on a density gradient is a technique widely used in virology laboratories for the detection of rubella-specific IgM (Vesikari and Vaheri, 1968;Caul et al, 1974;Caul et al, 1978). However, the technique as originally described was time consuming.…”

Detection of rubella virus immunoglobulin G (IgG) and IgM antibodies in whole blood on Whatman paper: comparison with detection in sera

“…Another approach to the detection of recent rubella virus infection is the demonstration of rubella virus-specific immunoglobulin M (IgM). This can be accomplished by the separation of IgM from IgG by sucrose density gradient centrifugation (6,7), gel filtration (11), treatment of whole serum with 2-mercaptoethanol (2,6), and absorption of IgG from whole serum by staphylococcal protein A (1,20). All these techniques are cumbersome.…”