A Quantitative ELISA for Measuring Red Cell-Bound Immunoglobulins

Abstract

An ELISA was developed for quantitating red-cell-bound IgG and IgA and its feasibility assessed on 50 blood donations and 32 clinical specimens with raised cell-bound IgG. Test and quality control samples and immunoglobulin standards (in red-cell lysate buffer) were assayed together. Calibration curves were derived from the standards, test values being read off and related to cell count. The working range was around 5-70 ng/ml, the upper limit being indefinitely extendable by dilution with lysate buffer. Ranges of results (and medians) in molecules per red cell were < 26-240 (40) for IgG and < 29-94 ( < 29) for IgA in the donations with 240-62,700 (1,200) for IgG and < 29-4,500 (73) for IgA in the clinical specimens. The method which can be adapted for any cell-bound immunoprotein should be particularly valuable in investigating autoimmune haemolysis.

Quantitation of red cell–bound IgG, IgA, and IgM in patients with autoimmune hemolytic anemia and blood donors by enzyme-linked immunosorbent assay

“…10 That study reported a median of < 29 molecules per RBC, which is comparable to the amount quantitated by our assay. 10 There was considerable variation in the number of IgG, IgA, and IgM molecules per RBC in patients with AIHA, demonstrating the ability of the assay to measure widely different degrees of sensitization in clinical samples. The results are comparable with previous studies where the reported ranges in patients with AIHA were from 230 to about 30,000 IgG molecules, from 20 to 168 IgM molecules, and from < 29 to 4500 IgA molecules per RBC.…”

“…Other methods gave similar results for IgG and IgM. 4,5,[8][9][10] There has been only one previous report of the number of RBC-associated IgA molecules on normal human RBCs. 10 That study reported a median of < 29 molecules per RBC, which is comparable to the amount quantitated by our assay.…”

“…3 Several methods to quantitate RBC-bound immunoglobulins have been reported, including an immunoradiometric assay, autoanalyzer assays, antiglobulin consumption assays, and enzyme-linked immunosorbent assay (ELISA). [4][5][6][7][8][9][10] Quantitation of immunoglobulins in a patient with AIHA would allow a more precise method of monitoring the disease or defining the synergistic effect of the different immunoproteins in causing RBC destruction. 11 We have developed an enzyme immunoassay for the quantitative determination of immunoglobulin isotypes in ether eluates made from RBCs.…”

“…4,5,[8][9][10] There has been only one previous report of the number of RBC-associated IgA molecules on normal human RBCs. 10 That study reported a median of < 29 molecules per RBC, which is comparable to the amount quantitated by our assay. 10 There was considerable variation in the number of IgG, IgA, and IgM molecules per RBC in patients with AIHA, demonstrating the ability of the assay to measure widely different degrees of sensitization in clinical samples.…”

Quantitation of red cell-bound immunoglobulins and complement in lymphoma patients

“…39 The method is similar to that used by others. 39,40 After measuring RBC concentration in samples tested, we calculated the extent of RBC sensitization molecules of IgG, IgA, IgM, C1q, and C3 per RBC.…”