Enzyme-linked Antiglobulin Test Research Articles

Immunohematological characterization of direct antiglobulin test-negative autoimmune hemolytic anemia through simple, sensitive methods: Experience from a tertiary care hospital in India

Background and Objectives: A negative DAT does not rule out AIHA. The incidence of this clinical entity known as DAT negative AIHA has been reported to be 2-4%. Sensitive techniques like enzyme linked antiglobulin test (ELAT), flow cytometry (FC), complement-fixation antibody consumption test etc. have been described for diagnosis of DAT negative AIHA. Majority of blood bank laboratories lack these advanced methods. Here we share our experience of investigating DAT negative AIHA using simple, sensitive methods which are otherwise less practised. Methods: The prospective study included 377 anemic patients clinically suspected of AIHA. Blood samples received in blood bank were subjected to polyspecific DAT using both conventional tube test (CTT) and column agglutination technique (CAT). Polyspecific DAT negative results were further evaluated using sensitive but simple methods. Hematological and biochemical parameters of all patients were obtained from hospital information system. In vivo hemolysis was categorized as per the criteria established by previous workers. SPSS statistical software (version 13, USA) was used for all statistical evaluation. Results: Of the final 353 clinical AIHA patients evidence of autoimmunization by CTT was observed in 335. Where DAT negative AIHA was observed in 18 (5.1%) patients, 14 showed evidences of autoimmunization by extended sensitive methods. Four patients responded well to AIHA therapy despite DAT negativity by available methods. The mean Hb, Retic, S. Bil and LDH were found to be 6.9 g/dL, 3.25%, 2.8 mg/dL and 512 IU/mL respectively. Conclusion: We conclude that DAT negative patients with clinical suspicion of AIHA and positive laboratory evidences should be evaluated for the presence of autoantibody by alternate sensitive methods which are otherwise less practiced. Blood banks may establish these useful simple techniques and stick to the defined protocols to diagnose DAT negative AIHA.

Inhibitors of Bruton's Tyrosine Kinase Reduce Anti-Red Blood Cell Response in a Murine Model of Autoimmune Hemolytic Anemia

Autoimmune hemolytic anemia (AIHA) is an antibody-mediated condition where the mainstays of treatment are corticosteroids and immunosuppressive therapies. This leads to substantial toxicity in patients with refractory or chronic disease and thus there is a need for improved therapies. BTK is a critical kinase in the B-cell receptor signaling cascade and we hypothesized that inhibition would decrease autoantibody responses. Supporting this, CLL patients taking ibrutinib, a first generation BTK inhibitor, experience very low rates of AIHA (Leukemia 2016). Based on this rationale we evaluated two BTK inhibitors, ibrutinib (ibr) and acalabrutinib (acal), in mouse models of AIHA to validate BTK as a target in this disease.Young C3H/B6 F1 mice were treated with acal, ibr, or vehicle (veh) via drinking water for 2 weeks then injected intraperitoneally with washed rat red blood cells (RBCs) weekly for 4 weeks using the method of Playfair and Marshall-Clarke (Nature New Biology 1973) to induce a response against autologous RBCs starting 14 days after initial injection and peaking days 28-35. Drug treatment was continued throughout the study and using this delivery method BTK occupancy was 99.1% for acal, 96.1% for ibr, and 0% for veh. Quantitation of immunoglobulin on mouse RBCs was performed by a direct enzyme-linked anti-globulin test (DELAT) and by flow cytometry for surface immunoglobulin as a confirmatory method. Results from DELAT are shown in figure 1. A repeated measures model was applied to log-transformed data. On day 7, prior to autoimmune response, there were no statistically significant differences between treatment groups. However, on day 21 significantly less autoantibody was detected on RBCs in the acal group vs. both veh (p=0.001) and ibr (p=0.008); the ibr group showed a trend toward less autoantibody compared to veh but this did not reach statistical significance (p=0.542). This relationship between treatments held true at days 42 and 56 (acal vs. vehicle, p=<0.001 and p=0.001; acal vs. ibr, p=0.008 and p=0.004; ibr vs. veh, p=0.542 and p=0.542). These results were confirmed by flow cytometry. Hematocrit tests were performed by capillary tube centrifugation with no mice developing anemia and no differences between treatment groups excluding rapid RBC turnover as a cause of reduced immunoglobulin on RBCs.As ibrutinib has been shown to alter macrophage function, we used a passive model to determine if BTK inhibitors changed clearance of antibody coated RBCs from circulation. After two weeks of treatment with acal (n=13), ibr (n=12), or veh (n=12) mice were injected with a single dose of anti-RBC antibody (anti-ter-119) and hematocrit was measured daily for 7 days. Median hematocrit values at baseline for the three groups were 53.7 (SD=1.3), 52.8 (SD=1.4), and 52.8 (SD=0.9) for acal, ibr, and veh respectively and fell to a low of 18.4 (SD=2.7), 18.0 (SD=3.2), and 15.8 (SD=1.9) on day 3 before recovering. A repeated measures model was applied to the data and no significant differences between drug treatment groups were found (p=0.177), indicating no effect of BTK inhibition on RBC clearance.To examine anti-rat RBC antibody response washed rat RBCs were incubated with mouse plasma and analyzed by flow cytometry. Treatment groups were compared at day 42 after initial rat RBC injection. An analysis of variance (ANOVA) model was applied to log-transformed data. There was significantly less anti-rat RBC antibody in the acal group vs veh (p=0.008). In addition, there was decreased, albeit not statistically significant, antibody production with ibr compared to veh and acal compared to ibr (p=0.193 and p=0.102, respectively). This is the same trend as for autoantibody production and argues that BTK inhibition may reduce antibody responses.In summary, treatment with acalabrutinib significantly reduced anti-RBC autoantibody response in our model compared to vehicle and ibrutinib, and autoantibody was consistently decreased in ibrutinib treated mice compared to vehicle although this did not reach statistical significance. The differences between inhibitors may be due to potency of BTK inhibition or effects on other targets of ibrutinib which is a less specific drug. This mechanism is under active investigation. These results support BTK as a potential therapeutic target in AIHA and the further development of acalabrutinib for this indication. [Display omitted] DisclosuresWoyach:Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding.

Enzyme-linked antiglobulin test for the evaluation of the amount of IgG autoantibodies on red blood cells.

An enzyme-linked antiglobulin test (ELAT) was adapted to the assessment of IgG autoantibodies on red cells. Due to the worked out standard curve and the performance of the test on microplates, ELAT became a simple and inexpensive technique. It can be used for the detection of IgG within the range from 70 to 37,500 molecules per red cell. This is a simple quantitative method that can be used in immunohaematological studies of red cells.

The statistical analysis of immunohaematological data.

Almost 30 years ago, I submitted one of my first research papers on immunohaematology for publication in a prestigious journal. After a few weeks, I received back the referee's comments, with many criticisms, including the fact that I had used tests. The referee had been disappointed because statistical tests are not usually performed in immunohaematological studies. For that time, the referee was actually right. Even in more recent years, however, immunohaematologists continued to apply statistics very sparingly. For example, consider a table such as table I, which is frequently encountered in immunohaematological articles. It contains rows representing antibody specificities, columns representing two or more techniques for antibody detection, which are being compared, while data are titration end-points. When there are many rows (say, more than six), a fleeting reader would greatly appreciate a summary statistic. However, I remember just a few papers in which such a statistic was presented1–3, and in only one of them1 was it the most appropriate (the geometric mean)4. Table I Summary data (reciprocals of titre end-points) from a comparison of three techniques (Aggl: standard tube test; ELAT-W: enzyme-linked antiglobulin test; ELAT-G: like ELAT-W but with gradient centrifugation instead of washing) for the indirect antiglobulin ... The analysis of immunohaematological studies is, however, straightforward, provided adequate consideration is given to the type of measurement and the distribution of the data. In most cases, non-parametric tests are appropriate. In this paper, I shall briefly review the modern software that is freely available to researchers and then show the use of tools in the most common situations. First of all, however, I shall discuss the contribution of statistics to the presentation and analysis of immunohaematological data.

Quantitation of red cell-bound IgG by an enzyme-linked antiglobulin test in the patients with warm-type autoimmune haemolytic anaemia

We have previously reported the evaluation of a gel-direct antiglobulin test and enzyme-linked antiglobulin test (ELAT) in the laboratory diagnosis of autoimmune haemolytic anaemia (AIHA). We now report our experience with quantitative ELAT performed on a large group of patients under long-term observation. The number of IgG molecules/red blood cell was determined in 658 blood samples from 268 randomly selected patients with warm-type AIHA. Eighty-six patients were tested every 2-4 weeks for several months. Laboratory signs of haemolysis were present in 65.7% of blood samples with a small amount of red cell-bound autoantibody (< 200 IgG molecules/red blood cell) and in 70.4% of blood samples with moderately coated red blood cells (200-1000 molecules/red blood cell). Haemolysis was demonstrated in 87.9% samples with > 1000 IgG molecules/red blood cell, which were predominantly IgG3 and C3 complement, the qualitative factors that may increase haemolysis. In 79% of periodically tested patients, the number of IgG autoantibody molecules/red blood cell decreased and this correlated with the improvement of haemolysis parameters. The number of IgG molecules varied in 21% of AIHAs and was associated with poor prognosis.

RBC-associated IgG in patients with visceral leishmaniasis (kala-azar): a prospective analysis.

Despite the fact that anemia is one of the most striking clinical features of visceral leishmaniasis (kala-azar), the factors involved in its pathogenesis are not fully understood. Although the cause of the anemia seen in these patients is often multifactorial, sequestration and destruction of the RBCs in the enlarged spleen, immune mechanisms, and alterations in RBC membrane permeability have been implicated. To investigate whether RBCs of patients with kala-azar were coated with IgG, blood samples of 67 patients were tested, prospectively, before (Day 1), during (Day 30), and after (Day 90) antimonial therapy, to examine the presence of RBC-associated IgG, circulating immune complexes (CICs), and rheumatoid factor (RF). The prevalence of a positive DAT on Day 1 was significantly greater than the prevalence of a positive DAT performed on Day 30 and on Day 90 (32.8 vs. 4 vs. 0%, p < 0.001). With an enzyme-linked antiglobulin test (ELAT) to measure the number of IgG molecules per RBC more accurately, it was found that the amount of IgG molecules per RBC was increased (mean, 298 molecules of IgG per RBC) in the group of patients with kala-azar tested before antimonial therapy, but was considered normal (<50 molecules of IgG per RBC) in all patients tested 90 days after treatment. The prevalence of a positive eluate test was low (15.0%) in DAT (anti-IgG)-positive patients and the positivity of DATs and ELATs correlated with the presence of either RF or CICs, respectively. These data suggest that a nonspecific adsorption of CICs on the RBC surface is probably the most important factor involved in the increased amount of RBC-associated IgG in patients with untreated visceral leishmaniasis; however, further prospective studies are required to establish the exact role of the RBC-associated antibodies, CICs, and RF as contributing factors of the anemia seen in these patients.

Autoimmune hemolytic anemia caused by warm-reacting IgM-class antibodies

Warm IgM autoantibodies occur in association with IgG-class and/or IgA-class immunoglobulins in approximately 30 percent of patients with warm-type autoimmune hemolysis. They may be classified as agglutinins or hemolysins, which may be incomplete or complete, depending on in vitro serology; they almost always bind complement. Autoimmune hemolytic anemia solely due to warm IgM autoantibodies is exceedingly rare. We report two cases of the incomplete agglutinin type. The autoantibodies were confirmed as IgM by their ability to rebind to normal red blood cells (RBCs) after elution; the absence of small increases in RBC-bound IgG and IgA was shown by a sensitive enzyme-linked antiglobulin test. Patient 1 was a 64-year-old female with non-Hodgkin's lymphoma, with a hemoglobin of 50 g/L and haptoglobin of < 0.1 g/L. Direct antiglobulin tests were positive for IgM, C3d, and C3c; only IgM was present in an eluate. The serum contained a weak autoantibody at 37 degrees C and tests for hemolysins were negative. The patient suffered chronic hemolysis and required intensive treatment, including splenectomy. Patient 2 was a 65-year-old female; the hemoglobin was 78 g/L and the haptoglobin was < 0.1 g/L. Direct antiglobulin tests were positive for IgM and C3d; an eluate contained only IgM. No free autoantibody was present in the serum and tests for hemolysins were negative. Two serious infections occurred and the hemolysis remained chronic, requiring continuous treatment during the 4 months she was followed.

Autoreactive T cell specificity in autoimmune hemolytic anemia of the NZB mouse

Splenic T cells from Coombs'-positive New Zealand Black (NZB) mice proliferated consistently in vitro in response to the integral red blood cell (RBC) membrane protein Band 3, the antigen previously shown to be the target for the pathogenic RBC autoantibodies. The responding cells predominantly express CD4 and the proliferative response is blocked by antibodies to the NZB major histocompatibility complex class II but not by antibodies to an irrelevant H-2 haplotype. NZB splenic T cells also proliferated in response to the internal membrane skeleton protein spectrin. By contrast, T cells from BALB/c and DBA2 mice, which bear the same H-2 haplotype as NZB mice, but which do not develop autoimmune hemolytic anemia (AIHA), fail to respond to Band 3. It is considered that these results support the hypothesis that Band 3-reactive T cells provide help for the production of pathogenic anti-Band 3 autoantibodies in NZB mice. T cells from Coombs'-negative NZB mice as young as 3 weeks old proliferated in response to Band 3; moreover, the RBC from Coombs'-negative mice bore elevated levels of autoantibody as judged by a sensitive direct enzyme-linked anti-globulin test. Thus, the pathology of AIHA develops at a much earlier age than was thought previously.

Red cell-reactive non-specific immunoglobulins and autoantibodies in the sera of normal and anaemic dogs

The levels of red blood cell (RBC) membrane-reactive IgG were measured in sera from normal and anaemic dogs using an enzyme-linked immunosorbent assay with erythrocyte ghosts as the target antigen (g-ELISA). The anaemic dogs were classified as cases of primary autoimmune haemolytic anaemia (AIHA), other anaemias with elevated levels of RBC-bound immunoglobulin detected by a direct enzyme-linked antiglobulin test (DELAT), or DELAT-negative anaemias. The g-ELISA detected IgG capable of binding RBC membranes in all the serum samples tested, and the levels were significantly higher ( P < 0.05) in dogs with AIHA than in cases of DELAT-negative anaemia or in healthy animals. When sera were preabsorbed with RBC ghosts, g-ELISA readings were reduced in two of three AIHA cases that were tested, but not in dogs from any other group. There was no correlation between g-ELISA results and serum IgG levels in the cases of AIHA, but there was a significant relationship ( r s = 0.74, P < 0.001) between these parameters in the dogs with other DELAT-positive anaemias. When spectrin(s) from the internal RBC membrane skeleton was used as the target antigen in the s-ELISA, sera from all groups of anaemic dogs yielded a wide range of values. Although the levels of spectrin-reactive IgG were significantly higher ( P < 0.05) in AIHA cases than normal dogs, suggesting that haemolysis provoked production of anti-membrane skeleton antibodies, the highest reading was recorded in a healthy animal. It is concluded that all canine sera contain IgG non-specifically reactive with RBC membranes and that serum antibodies against the erythrocyte surface can also be detected in some dogs with primary AIHA. Distinct spectrin-reactive serum IgG antibodies can also be demonstrated in normal and AIHA positive dogs.

Effects of papain on the agglutination of canine red cells with serum autoantibodies

The papain test, which detects incomplete antired blood cell ( rbc) autoantibodies in serum, was positive in 12 out of 16 anaemic dogs. Positive results were significantly correlated ( x 2=11·1, P<0·001) with increased levels of rbc-bound immunoglobulin, but in three of these cases it was considered that a diagnosis of autoimmune haemolytic anaemia could not be justified. Furthermore, enzyme-linked antiglobulin test measurements of rbc-reactive serum IgG were increased in only three of the dogs with a positive papain test. Papainised canine rbc did not consistently take up more serum IgG than untreated cells in an indirect enzyme-linked antiglobulin test. However, the zeta potential of the RBC was reduced after enzyme treatment, and electrophoretic analysis revealed that glycophorins, which bear a strong negative charge, were cleaved from the cell membrane. It is concluded that a positive papain test alone is not reliable in the diagnosis of canine autoimmune haemolytic anaemia and that the enzyme increases the agglutinability of rbc by reducing the mutually repulsive electrostatic forces between the cells, rather than by increasing the amount of autoantibody bound.

Red cell-bound immunoglobulins and complement measured by an enzyme-linked antiglobulin test in dogs with autoimmune haemolysis or other anaemias

A direct enzyme-linked antiglobulin test ( DELAT) was used to measure IgG, IgM, IgA and C 3 bound to the red blood cells ( RBC) of anaemic dogs. One or more of these parameters was raised in 78 per cent of the cases tested, and elevated levels of bound IgG, IgM, IgA and C 3 was the most common pattern recorded. When the DELAT-positive animals were divided into two groups on the basis of clinical findings, the two categories differed serologically. Cases diagnosed clinically as primary autoimmune haemolytic anaemia ( AIHA) were typified by the demonstration of specific antibody binding to RBC, had significantly higher levels of RBC-bound IgG (P<0·001) and IgM (P<0·02) and included a significantly (P<0·01) higher proportion of the dogs with at least three DELAT parameters raised. The second group comprised the DELAT-positive dogs with other diseases. In most of these cases, RBC-specific antibody was not detectable and there was a significant (r s=0·62, P<0·001) relationship between RBC-bound and serum IgG levels, suggesting that the immunoglobulin was reacting with the RBC non-specifically in a concentration-dependent manner. It is concluded that elevated levels of RBC-bound immunoglobulin and, or, complement are detectable with the DELAT in most anaemic dogs, but that this finding is not necessarily associated with autoimmune haemolysis.

The Quantification of Fetomaternal Hemorrhage by an Enzyme‐Linked Antibody Test with Glutaraldehyde Fixation

A modified enzyme-linked antiglobulin test (ELAT) for quantifying D-positive red blood cells in the circulation of D-negative pregnant women is described. The antibody-antigen bond was stabilized and the problem of hemolysis eliminated by the use of 0.05% glutaraldehyde. The r value for the standardization curves for measuring cord RBC in mixtures was 0.98; coefficient of variation 5.8%. Reproducible quantitation of 0.125% D-positive cord RBC mixed with D-negative RBC was demonstrated. In comparative studies of artificial mixtures, Kleihauer-Betke results for more than 1% cord RBC were 50-100% greater than the true values. In several examples of large fetomaternal hemorrhages, the ELAT results matched the clinical data more closely than the Kleihauer-Betke based estimates. 2 ml of fetal RBC in a 1,600-ml red cell mass can be quantified using the modified ELAT.

An enzyme-linked antiglobulin test to detect red cell globulins after glutaraldehyde fixation.

The purpose of this study was to develop an enzyme-linked antiglobulin test (ELAT) for IgG on RBC without the hemolysis caused by the high pH of the alkaline phosphatase reaction. This was achieved by fixing the RBC with 0.05% glutaraldehyde after attachment of the antibodies. Assays using anti-D reference standards demonstrated the sensitivity to be 1-2 ng of antibody as compared to 7.5 ng for the manual indirect antiglobulin test. The coefficients of variation of these assays ranged from 10.4 to 20.1%. The mean background absorbance at 405 nm of 105 normal RBC samples was 0.08 +/- 0.03 SD. There was an increase in sensitivity of the test after the fixed RBC were stored. Dilute glutaraldehyde stabilizes the RBC membrane and the antigen-antibody linkage resulting in a more sensitive ELAT.

Autoimmune haemolysis in the dog: Relationship between anaemia and the levels of red blood cell bound immunoglobulins and complement measured by an enzyme-linked antiglobulin test

A direct enzyme-linked antiglobulin test (DELAT) was used to measure the levels of red blood cell (RBC) bound IgG, IgM, IgA and C 3 in dogs with autoimmune haemolytic anaemia (AIHA). At presentation, one or more DELAT parameters was raised in each AIHA case, and the RBC were typically coated with immunoglobulin of more than one class, together with C 3. There was no relationship between the levels of RBC-bound IgG, IgM or IgA and the severity of the anaemia, although a significant negative correlation ( r s = −0.66, P < 0.02) was found between bound C 3 and blood haemoglobin concentration. These results indicate that the level of sensitisation of erythrocytes with IgG alone is not a reliable predictor of the severity of haemolysis in different cases, and that the pathogenesis of AIHA can be complex, involving multiple immunoglobulin classes and complement in the destruction of RBC. A significant relationship ( r s = 0.63, P < 0.02) was found between serum IgG concentration and haemoglobin levels, and it is suggested that this may be due to free IgG inhibiting the interaction of IgG-sensitised RBC with macrophages. Serial measurements from individual AIHA cases during treatment revealed that the levels of RBC-bound immunoglobulins fell simultaneously with improvements in anaemia. In one dog, a relapse was associated with increases in bound IgG and IgM. Transient relative reticulocytopenia at presentation was common, but was not related to the severity of the anaemia. However, in other cases there was a persistent failure to increase RBC production, which was associated with slower recovery.

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.

Use of a direct enzyme-linked antiglobulin test for laboratory diagnosis of immune-mediated hemolytic anemia in dogs

Summary Detection of autoantibody, complement, or both bound to rbc is an essential requirement for unequivocal diagnosis of immune-mediated hemolytic anemia in dogs. An enzyme-linked antiglobulin test was adapted for laboratory diagnosis of this disease. The refinement and routine use of this assay have allowed further observation of the pathogenesis of the disease process. In particular, degree of hemolysis can be related to the degree of rbc sensitization associated with primary immune-mediated hemolytic anemia, and this correlation is highest for IgG autoantibody. Results indicate that autoantibody isotype might have an important role in the hemolytic process.

The Quantification of Fetomaternal Hemorrhage by an Enzyme-Linked Antibody Test with Glutaraldehyde Fixation

A modified enzyme-linked antiglobulin test (ELAT) for quantifying D-positive red blood cells in the circulation of D-negative pregnant women is described. The antibody-antigen bond was stabilized and the problem of hemolysis eliminated by the use of 0.05% glutaraldehyde. The r value for the standardization curves for measuring cord RBC in mixtures was 0.98; coefficient of variation 5.8%. Reproducible quantitation of 0.125% D-positive cord RBC mixed with D-negative RBC was demonstrated. In comparative studies of artificial mixtures, Kleihauer-Betke results for more than 1% cord RBC were 50-100% greater than the true values. In several examples of large fetomaternal hemorrhages, the ELAT results matched the clinical data more closely than the Kleihauer-Betke based estimates. 2 ml of fetal RBC in a 1,600-ml red cell mass can be quantified using the modified ELAT.

A review: the enzyme-linked antiglobulin test (ELAT) and its applications for reference laboratories

A review: the enzyme-linked antiglobulin test (ELAT) and its applications for reference laboratories

Evidence that Wra and Wrb are antithetical.

Studies on 24 Wr(a+b+) and 23 Wr(a-b+) blood samples, using anti-Wrb in the enzyme-linked antiglobulin test (ELAT), have shown that Wr(a+b+) red cells bind, on average, a little over half the amount of anti-Wrb bound by Wr(a-b+) red cells. Similarly, ELAT studies using six different anti-Wra and 10 Wr(a+b+) samples, as well as red cells from the original Wr(a+b-) proposita, have shown that Wr(a+b+) red cells bind about half the amount of anti-Wra bound by Wr(a+b-) red cells. Various pitfalls that can arise when the ELAT is used to measure antigen ratios on red cells have been avoided but are described. This conclusive evidence that Wra and Wrb have an antithetical relationship is discussed in light of the knowledge that a ficin-resistant portion of MN sialoglycoprotein (SGP), when carried in liposomes, can inhibit anti-Wrb. It is possible that Wra, Wrb, or both may encode a post-translational change in MN SGP, or production of transferases that glycosylate membrane lipids that affect in situ orientation of MN SGP, or production of protein band 3 that then forms a complex with MN SGP at the red cell membrane surface.

An enzyme-linked antiglobulin test for the detection of erythrocyte-bound antibodies in canine autoimmune haemolytic anaemia

A method has been developed which allows identification and quantitation of red cell-bound immunoglobulins and complement in canine blood. The technique utilizes ELISA methodology and the assay identifies cases of autoimmune haemolytic anaemia which are negative by the Direct Coombs test. Further, the amount of antibody present on the red cells shows a close correlation to the haemoglobin level; suggesting that the degree of sensitization of the RBCs influences their rate of destruction. Favourable response to treatment correlates with a decrease in the levels of bound antibody and complement.