Quantitation of red cell-bound immunoglobulin and complement using enzyme-linked antiglobulin consumption assay

Various techniques have been described for quantitating IgG or complement (C3) on red cells (RBCs). The techniques either are cumbersome, as the complement consumption test, or use radioactivity. This paper describes an antiglobulin consumption assay using an enzyme-linked immunosorbent method that can be used to quantitate IgG, IgM, and C3. With this technique RBCs from normal, healthy donors gave a mean value of 106 +/- 60 molecules of IgG per RBC, 4.5 +/- 3 molecules of IgM per RBC, and 37 +/- 28 molecules of C3 per RBC, respectively. The RBCs of hospital patients, particularly of those with infections or inflammatory conditions, contain increased amounts of nonspecifically bound immunoproteins. The availability of a common method to quantitate RBC-associated IgG, IgM, and C3 allows easy monitoring or study of the immune mechanism of autoimmune hemolytic anemia.     

Quantitation of red cell-bound IgG by an enzyme-linked antiglobulin test in human immunodeficiency virus-infected persons

Anemia, thrombocytopenia, and neutropenia have been observed in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex. To investigate whether red cells (RBCs) of patients with human immunodeficiency virus infection were coated with IgG and/or complement (C3), blood samples of 239 patients were tested. The prevalence of a positive direct antiglobulin test on RBCs was 16.7 percent. By use of an enzyme-linked antiglobulin test (ELAT) to measure more accurately the number of IgG molecules per RBC in a group of 67 patients, 30 of the 67 individuals were observed to have increased numbers (mean, 155) compared to normal controls and to patients with hypergammaglobulinemia due to multiple myeloma or chronic liver disease. Hemoglobin level was correlated with the number of IgG molecules per RBC (p = 0.008), but no correlation could be demonstrated between those numbers and serum immunoglobulin (p = 0.10) or circulating immune complexes (p = 0.38). Our results with ELAT suggest that some AIDS patients may have specific binding of IgG on the surface of their RBCs, rather than nonspecific uptake; further clinical correlations are necessary to confirm these findings.     

Ripley-like anti-Rh associated with red blood cell-bound IgG aggregates

A commercial Rho (D) immune globulin after heating at 63 C became Ripley-like in the Rh-positive red blood cells coated with this heated globulin carried biologic activities similar to those of red blood cells coated with Ripley anti-CD serum. These coated red blood cells fixed complement and were agglutinated by all 20 sera containing rheumatoid factor (RF). The RF-Rh-hemagglutinations were more readily inhibited by heated than by unheated human IgG. The heated globulin had no such effect on Rh-negative red blood cells. Fractionation studies by Na2SO4 precipitation and/or Sephadex G-200 gel filtration revealed that heat-induced IgG aggregates in heated globulin were responsible for the biological activities. In contrast, these activities in Ripley serum were carried by IgG monomers. Another anti-CD serum (Heyman), tested in paralledl, was found to be indistinguishable from Ripley. a pooled RF serum, after multiple adsorptions with red blood cells coated with globulin, lost its agglutination activity to red blood cells coated with Ripley or Heyman serum.     

The detection of cell-bound antibody on complement-coated human red cells

This study sought to elucidate the mechanism by which human red cells, in a variety of clinical settings, become coated in vivo with autologous complement components in the absence of anti-red cell autoantibodies demonstrable by standard methods. By means of a newly developed complement-fixing antibody consumption test, previously undetectable red cell-bound gammaG globulin could be detected and quantified. By this technique, the complement-coated red cells of 13 of 16 patients were shown to carry abnormally high numbers of gammaG molecules per cell, which were nevertheless below the level for detection by the direct antiglobulin test. Eluates were made from the red cells of seven of these patients and each eluate, when sufficiently concentrated, was capable of sensitizing normal human red cells (with gammaG antibodies) to give a positive indirect antiglobulin test with anti-gammaG serum. In the presence of fresh normal serum, six of the eluates so tested were capable of fixing complement to normal human red cells. The antibodies in the red cell eluates did not exhibit Rh specificity and did not react with nonprimate red cells. When studied by sucrose gradient ultracentrifugation, the gammaG antibodies to human red cells in these eluates sedimented in the 7S region. It is concluded that in many patients in whom direct antiglobulin tests reveal only cell-bound complement, the complement fixation is mediated in vivo by small quantities of "warm-reacting" erythrocyte autoantibodies of the gammaG class.     

Quantitation of red cell-associated IgG using an immunoradiometric assay

In this report, we describe a sensitive immunoradiometric assay (IRMA) for quantitating IgG on the surface of red cells. Washed red cells were prepared to a purity of greater than 99.9 percent. Varying dilutions of these cells were incubated with a fixed concentration of 125I-anti-IgG. After equilibrium was achieved, the unbound 125I-anti-IgG was measured by the addition of IgG covalently linked to agarose beads. The red cells were lysed by detergent, and the 125I-anti-IgG bound to the IgG- beads was measured. The amount of IgG on the red cells was determined by relating the concentration of test red cells causing 50 percent inhibition of binding of the 125I-anti-IgG to the IgG-beads to 50 percent inhibition of binding caused by the IgG standard. Using this assay, the red cell-associated IgG (RCA-IgG) of 20 healthy male and female controls with normal hemoglobin concentrations was 7.23 +/− 6.11 fg IgG per 10(3) cells (mean +/− 2 SD). The mean RCA-IgG on washed cells from 34 different tests performed on 19 anemic patients with clinically diagnosed autoimmune hemolytic anemia was 176.1 +/− 375.6 fg IgG per 10(3) cells. There was no correlation between the levels of RCA- IgG and the hemoglobin levels or reticulocyte counts in these patients.     

Quantitation of minor red cell populations using the single-channel autoanalyzer

An automated hemagglutination technique has been applied to the quantitation of the size of minor red cell populations. By choosing an antibody to an antigen present only on the minor population, it was possible to estimate the size of the minor population by measuring the antibody following incubation with the red cell mixture. The size was directly proportional to the amount of antibody consumed and was calculated from a regression plot established from standard mixtures. With this method, it was possible to detect minor red cell populations as dilute as 1:5000.     

Quantitation of the third component of complement on stored red cells

By means of an automated anti-C3c consumption technic, we quantitated the molecules of the third component of human complement (containing the C3c fragment) that accumulate on red cells (RBCs) during liquid whole-blood storage. Calibrated C-3 sensitized zymosan particles ( ZyC3 ) were used as standards for bound C3. Although the dose-response curves of anti-C3c neutralization by ZyC3 and stored RBCs were similar, mathematical analysis showed that the shapes of these two sigmoidal curves were significantly different. This indicated that the large C3 molecules bound to stored RBCs differed antigenically from those bound to ZyC3. Despite this difference, the former could be quantitated adequately by the automated anti-C3c consumption technic. Red cell- bound large C3 molecules were measured following various periods of storage at 4 degrees C of whole blood samples (n=102). An average of 48 molecules were detected on RBCs stored for 21 days. Statistical analysis of these data indicated that during storage at 4 degrees C there was a continuous accumulation of C3 on RBCs, followed by cleavage of C3c fragments. The degree of agglutination of stored RBCs with anti- C3c was proportional to the number of cell-bound large C3 molecules.     

Correlation between in vivo hemolysis and the amount of red cell-bound IgG measured by flow cytometry

A flow cytometry method was used to compare the amount of red cell (RBC)-bound IgG in 73 patients with and without immune hemolytic anemia (IHA). The positive results in 10 of the direct antiglobulin tests (DATs) were idiopathic, and those in 25 were due to methyldopa therapy; 38 of the 73 DAT-positive patients were babies born to women with IgG alloantibodies of potential clinical significance. Normal blood donors with (n = 30) and without (n = 121) positive DATs were also tested. RBCs that had been strongly sensitized (4+ indirect antiglobulin test) in vitro with different quantities of IgG anti-D, but that had similar antiglobulin test (AGT) titration scores, could easily be differentiated by flow cytometry. The mean percent fluorescence of RBCs, incubated with fluorescein-labeled anti-IgG, from neonatal patients with IHA was higher than that of RBCs from those without IHA, but there was no statistical difference in the other groups. There was considerable overlap in the respective ranges of percent fluorescence of RBCs from patients with and without IHA in all groups. It was not possible to define a clear quantitative threshold differentiating patients with IHA from those without. Although flow cytometry was more precise and reproducible than standard serology (e.g., AGT titration scores), correlations of the amount of RBC-bound IgG and in vivo hemolysis were similar.     

Quantitation of red cell porphyrins by fluorescence scanning after thin-layer chromatography

This paper reports a new method for the quantitative determination of erythrocyte and plasma porphyrins by fluoroscanning of the methyl esters separated by thin-layer chromatography (TLC). After esterification overnight in the dark the polycarboxylated porphyrin methyl esters were extracted into chloroform and aliquots applied to the TLC plates within the range of quantities shown in a preliminary study to be directly proportional to fluorescence intensity. The accuracy and reliability of the technique was tested by comparison of RBC protoporphyrin values obtained by TLC with an established quantitative porphyrin solvent extraction method and with a rapid method presently in common use. Good correlation was demonstrated between the solvent extraction and the TLC techniques. The high sensitivity and adaptability of the TLC technique and its ability to clearly separate all the porphyrins present in the samples are discussed, along with the fluorescence mechanisms involved and the effects of instrumentation.     

Quantitation of immunoglobulin classes and subclasses of autoantibodies bound to red cells in patients with and without hemolysis

The concentration of IgG, IgA, and IgM, as well as IgG subclasses, was measured by an enzyme-linked immunosorbent assay in autoantibodies eluted from red cells (RBCs); the number of molecules of each isotype per RBC was calculated. Three groups were analyzed: Group 1 included 23 patients with autoimmune hemolytic anemia (AIHA) associated with warm autoantibodies of IgG class; Group 2 included 11 patients without anemia but with a positive direct antiglobulin test (DAT); Group 3 included 10 healthy DAT-negative subjects. The mean number of IgG molecules per RBC in Group 1 (920) was about three times that in Group 2 (306) and about 17 times that in Group 3 (54). The range of RBC-bound IgG showed an overlap between the two groups of patients. The mean number of IgM and IgA molecules per RBC was low in the three groups. IgG1 predominated in all groups except in two patients with AIHA, in whom IgG3 made up at least 50 percent of total IgG. The mean number of IgG1, IgG2, and IgG4 molecules per RBC in Group 1 was about three times that in Group 2, whereas the mean number of IgG3 molecules per RBC was 10 times as high (p < 0.001). It follows that IgG3 was more common in patients of Group 1, but it was also detected in patients of Group 2.     

Quantitation of serum immunoglobulins

This article reviews critically the current "state of the art" in the quantitation of immunoglobulins in serum and other body fluids. The methodologies reviewed include (a) those occurring in solution, for example, automated immunoprecipitin and laser nephelometric techniques, rate analysis techniques and radioimmunoassays; (b) techniques involving radial diffusion in agarose gels, with and without secondary development steps; and (c) those techniques involving solid supports, such as fluoroimmunometric assays and solid phase radioimmunoassays. The theory of each technique is presented with an analysis of the strengths and weaknesses, particularly sources of error, and the techniques are compared in terms of accuracy, precision, sensitivity, test cost, equipment cost, feasibility for use in different laboratory settings, and ease of handling. Problems associated with the antisera used, with standards, and with quality controls are discussed and solutions suggested.     

Red blood cell-bound C3d in selected hospital patients

A radioactive anti-antiglobulin technique was used to measure C3d bound to the red blood cells of 227 hospitalized patients in 129 patients, with a wide variety of diseases, normal levels of RBC-bound C3d were found. Seventy-two patients had moderately elevated RBC-bound C3d; they generally did not have autoimmune hemolytic anemia but had diseases in which complement is thought to be activated. Patients with markedly elevated RBC-bound C3d (26 patients) usually had autoimmune hemolytic anemia with a positive antiglobulin test. Some patients with only moderately elevated levels of RBC-bound C3d had autoimmune hemolytic anemia and a negative antiglobulin test in individual patients the level of RBC-bound C3d correlated with both the severity of disease and the response to treatment. RBC-bound C3b was detected in two patients with a very high level of RBC-bound C3d. This study provides background data for assessing the significance of complement activation and fixation to RBC in health and disease.