Antiglobulin Consumption Test Research Articles

Guillain-Barré syndrome: demonstration of antibodies to peripheral nerve tissue.

Sera from 30 patients with acute Guillain-Barré syndrome were tested for the presence of antibodies to peripheral nerve tissue using the antiglobulin consumption test. An increased binding of IgG was observed in 15 out of 30 sera and in these sera F(ab')2 fragments prepared from IgG retained the capacity to combine with peripheral nerve tissue, suggesting an antigen-antibody interaction. The specificity for peripheral nerve tissue was demonstrated by absorption experiments. F(ab')2 fragments prepared from IgG of normal sera, however, failed to react with the tissue, suggesting a non-specific binding through the Fc part of the molecule. In sera from patients fully recovered from the Guillain-Barré syndrome and from patients with other neuropathies, no increased binding of IgG was observed.

Storage of Platelets in Earth Orbit. Effect of Gravity and the Formulation of Plastic Storage Containers on Platelet IgG and C3

We investigated the effects of gravitational force and three different plastic formulations of the storage bags on the quality of stored platelets by measuring the changes in platelet-associated immunoglobulin G (IgG) and two antigenic markers of the third component of human complement (C3), C3d and C3c. Pooled platelets were stored in parallel at microgravity (MG) and on the ground (lg) using three different plastic polymers: (1) polyvinylchloride (PVC) plasticized with di-2-ethylhexyl phthalate (DEHP); (2) PVC plasticized with trioctyl trimellitate (TOTM), and (3) unplasticized polyolefin (PO). The IgG and C3 were quantified by an automated antiglobulin consumption test in freeze/thaw disrupted platelets (total IgG or C3). The baseline values for platelet associated IgG and C3, measured after 2.5 days of storage at 1g just prior to the launch, fell within the normal range. Following an additional 6.5 day storage, platelets stored at MG had accumulated significantly less C3d than those stored at lg, suggesting that MG storage was beneficial. Specific plastic formulations also exerted a significant effect on the accumulation of these immunoproteins, the effect being particularly pronounced at MG. The smallest increases of IgG and C3 were seen in platelets stored in TOTM and the largest in those stored in DEHP. It is possible that further studies at MG would permit a clear characterization of the effects of other independent storage variables.

Quantitative studies on the Rh‐antigen D

Using an automated antiglobulin consumption test and Scatchard's analysis, the authors measured the number of D antigen epitopes on each of 46 samples of washed red cells (RBC) that represented six different Rh phenotypes. A clear D dosage effect was observed only when C was missing from the Rh genome, which confirmed the previously recognized suppressive effect of C. Thus, RBC of the Rh phenotype R2R2 had almost twice as many D epitopes (mean, 30,300/cell) as RBC of the Rh phenotypes R2r and R0r (means, 17,200 and 16,500 respectively). The data indicated that C in trans and cis suppressed similar amounts of D epitopes.

An autoanalyzer test for the quantitation of platelet-associated IgG.

A new quantitative antiglobulin consumption (QAC) test for the measurement of platelet-associated IgG is described. In this test washed platelets are incubated with anti-IgG at a final dilution of 1:2 million. The unneutralized fraction of anti-IgG remaining in solution is then measured with an Autoanalyzer and soluble IgG is used for calibration. The dose-response curves depicting the percent neutralization of anti-IgG by platelets and by soluble IgG were compared in detail and found to be nearly identical, indicating that platelet-associated IgG can be accurately quantitated by this method. The mean IgG values were 2,287 molecules/platelet for normal adults and 38,112 molecules/platelet for ITP patients. The Autoanalyzer QAC test is a sensitive and reproducible assay for the quantitation of platelet-associated IgG.

An Autoanalyzer Test for the Quantitation of Platelet-Associated IgG

A new quantitative antiglobulin consumption (QAC) test for the measurement of platelet-associated IgG is described. In this test washed platelets are incubated with anti-IgG at a final dilution of 1:2 million. The unneutralized fraction of anti-IgG remaining in solution is then measured with an Autoanalyzer and soluble IgG is used for calibration. The dose-response curves depicting the percent neutralization of anti-IgG by platelets and by soluble IgG were compared in detail and found to be nearly identical, indicating that platelet-associated IgG can be accurately quantitated by this method. The mean IgG values were 2,287 molecules/platelet for normal adults and 38,112 molecules/platelet for ITP patients. The Autoanalyzer QAC test is a sensitive and reproducible assay for the quantitation of platelet-associated IgG.

Enhancement of the indirect anti-platelet antibody test and its application to platelet immunology.

Comparisons in the same patients of platelet-bound anti-platelet antibody (APA) levels (direct test) with serum APA levels (indirect test) frequently do not give the same results. Indirect test results frequently are negative or marginally elevated even though platelet-bound antibody is greatly increased on direct testing. The most likely cause for these differences between the two tests is insufficient binding of serum antibody to test platelets in vitro. In an effort to enhance in vitro antibody binding, we examined four test modifications affecting platelet attachment. Rabbit anti-human platelet antisera (absorbed with their specific human donor leukocytes to remove HLA- and granulocyte-specific antibodies) were combined under various conditions with human platelets and tested in a modified antiglobulin consumption test utilizing rabbit IgG. The test conditions varied: incubation time of the serum APA-platelet mixture at 37 degrees C; age of the test platelet pool; ionic strength of the mixture (varied through incorporation of a low-ionic-strength solution (LISS); and vortex agitation for 30 seconds prior to the addition of sera. Optimal attachment in standard phosphate-buffered saline took place in 60 minutes using test platelets stored for either 3 or 30 days, and yielded mean values for rabbit immune sera of 9.9 fg of IgG per platelet. Preincubation controls yielded a mean of 0.7 to 1.5 fg per platelet. Incorporation of LISS or vortexing of test platelets did not affect the results of control, but both modifications substantially increased test values of immune sera to 15.8 and 17.3 fg of IgG per platelet, respectively. Combining LISS with vortexing did not further increase values.

Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G.

Immunoglobulin G (IgG) bound to platelets is usually detected by one of two general methods: binding of labeled anti-IgG or consumption of anti-IgG. The latter method gives, in general, values 5-10-fold greater than the former under the same conditions. To investigate these discrepancies, we have compared the detection of platelet-bound IgG by a labeled anti-IgG binding assay and by a quantitative antiglobulin consumption test using the same antibodies. The interaction of 125I-labeled monoclonal anti-IgG or polyclonal anti-IgG with washed and IgG-coated platelets was studied. The binding of these ligands to washed normal platelets was largely (50-80%) nonspecific; the binding was not saturable and was only partially inhibitable by excess unlabeled anti-IgG. The binding of anti-IgG to platelets coated with anti-PIA1, a platelet-specific IgG antibody, appeared to be saturable and inhibitable; the dissociation constant (KD) of this IgG-anti-IgG reaction was 4.9 X 10(-9) for monoclonal and 1.4 X 10(-7) for polyclonal anti-IgG. The ratio of sites present on the membrane (determined by 131I-labeled anti-PIA1) to the number of binding sites for anti-IgG determined by Scatchard analysis was 0.53 for monoclonal anti-IgG and 1.3 for polyclonal anti-IgG. The binding of monoclonal anti-IgG to platelet-bound immune complexes or IgG aggregates appeared to be complex. 131I-Labeled IgG was affixed to platelets and was detected by three tests: direct binding of radiolabeled monoclonal anti-IgG and quantitative antiglobulin consumption (QAC) tests, which were quantitated either by measuring directly the amount of radiolabeled anti-IgG consumed from fluid phase (direct QAC), or indirectly by reference to a calibration curve relating the consumption of anti-IgG by known amounts of fluid-phase, non-immune IgG (indirect QAC). The amount of platelet-bound IgG detected by the direct binding of 125I-labeled monoclonal anti-IgG and by the direct QAC approximated that known to be bound to the platelet. The results of the indirect QAC test were 10-fold greater. The discrepancy appears to be due to the fact that there is a difference between the IgG-anti-IgG interaction when IgG is bound to a platelet and when it is in solution or bound to plastic nonspecifically or specifically. This difference results in a falsely high value for platelet-bound IgG when fluid-phase or plastic-bound IgG is used to calibrate the antiglobulin consumption test.

Antibodies to peripheral nerve tissue in sera from patients with acute Guillain-Barré syndrome demonstrated by a mixed haemagglutination technique

The mixed haemagglutination technique (MA) was used to demonstrate antibodies to peripheral nerve tissue (PNS) in sera from patients with acute Guillain-Barré syndrome (GBS). Within the first 2 weeks of the disease IgG antibodies were detected in sera from 15 of 42 patients (36%). Antibodies were detected in 60% of the patients with severe GBS, and in 30% of those with a mild GBS. The highest titres were found in the sera from the most severely affected patients and could be detected after 48 days of the illness in one of the sera. Sera from healthy blood donors and from patients with various other diseases did not contain PNS. The MA technique is reproducible and is more sensitive than the indirect immunofluorescence test and the antiglobulin consumption test.

A quantitative determination for the detection of immunoglobulin (IgG) on the surface of platelets

A sensitive method for the quantitation of IgG on platelets had not been demonstrated until 1975, when Dixon, Rosse, and Ebbert described a quantitative antiglobulin consumption test useful in detecting platelet associated IgG (N Engl J Med 292:230, 1975). A modification of that technique has rendered the assay reproducible and removed the need for daily repetition of a standard IgG titration curve for quantitation. This modification utilizes 1-ethyl-3-3(dimethylaminopropyl)carbodiimide HCl (ECDI) (Sigma, E-7750), in place of chromic chloride, as a coupling agent for attaching IgG (Miles 64–145) to sheep cells (SRC), used as indicator cells. The ECDI consistently couples IgG to SRC and does not subject the SRC to sporadic spontaneous lysis, as does chromic chloride. This modification permits the detection of IgG on platelets (Direct Test), or in sera (Indirect Test) by incubation of a washed platelet pool with sera in vitro, and testing as in the Direct Test. Normal values of 0.01–1.56 and 0.14–1.6 femtograms (F) per platelet have been obtained for the Direct and Indirect Tests, respectively. In six cases of suspected ITP, values ranged 12.0–221.0 F and 2.9–37.6 F for the Direct and Indirect Tests, respectively. In conclusion, in disease states or other abnormal situations, quantities of IgG can be detected that are not usually present on the platelets of normal subjects.

Notre expérience de la consommation de l'antiglobuline (test de Dixon) dans la recherche des anticorps fixés sur les plaquettes

We measured the quantity of IgG bound to platelets (IgGP) by the antiglobulin consumption test (Dixon technic). In controls, the IgG level did not exceed 10 X 10(-15) g for one platelet. The amount of IgGP was often incraeased (more than 10 X 10(-l5) g) in some autoimmune diseases as lupus erythematosus, chronic lymphocytic leukemia and chronic active hepatitis. Among 26 patients presenting an idiopathic thrombocytopenic purpura (ITP) with a low number of platelets, 21 (77%) had a high titer of IgGP. In 6 ITP in remission, the IgGP titer was normal. After a review of the different technics detecting the IgG bound to platelets, we explain why we choose the antiglobulin consumption. This test is excellent for the research of anti-platelets auto-antibodies in ITP. Yet, the reaction remains negative in a minority of ITP. Several explanations are possible: low quantity of IgGP, presence of other anto-antibodies as IgM or IgA, cellular auto-immunity anti-platelets without antibodies, non immunologic ITP.

Subclasses of IgG antibodies in immune thrombocytopenic purpura (ITPO).

The subclasses of platelet-associated IgG were measured on the platelets of patients with immune thrombocytopenic purpura (ITP) by modification of the antiglobulin consumption test. All subclasses were found. In most patients, one subclass was predominant; this was usually IgG1. The platelet-associated IgG in normal donors is predominantly IgG1 and IgG2.

A new method for the simultaneous quantitation of platelet-bound immunoglobuin (IgG) and complement (C3) employing an enzyme-linked immunosorbent assay (ELISA) procedure.

An ELISA technique for the quantitation of platelet-bound IgG and C3 is described. This is an antiglobulin consumption assay using commercial horseradish peroxidase conjugated anti-IgG and anti-C3 antisera. The greater the amount of antiglobulin consumed by the reaction with platelets the less is available to bind to an immune adsorbent in the form of human serum-coated polystyrene balls. This test can be calibrated by adding known quantities of IgG of C3 to the specific enzyme-linked antibody and the unbound fraction of antiserum is quantitated by allowing it to react with a chromogenic substrate for the enzyme and measuring the intensity spectrophotometrically, thus establishing an inverse relationship with the amount of antigen. Platelets from normal donors and those with non-immunological thrombocytopenia gave values of 1.3--15.5 ng IgG/10(6) platelets and 0.22--0.96 ng C3/10(6) platelets which are in accordance with the normal ratio of these proteins in normal serum. Fifteen patients in whom immune destruction of platelets weas suspected had excess platelet-bound IgG ranging from 30 to 450 ng IgG/10(6) platelets. In none of these patients could excess platelet-bound C3 be demonstrated. Compared to the antiglobulin consumption test we have found this test to be superior both technically and in terms of sensitivity and reproducibility.

Tumor Fc receptors and tumor-associated immunoglobulins.

Tissue sections and cell suspensions from ten malignant tumors were tested for Fc receptors using sheep erythrocytes sensitized by rabbit IgG antibodies. Surface bound IgG on cells from the same tumors were estimated using an antiglobulin consumption test with 125I labelled human IgG as reference. The amount of IgG present per 10(6) cells varied from less than 100 ng to approximately 600 ng. When these amounts of IgG were plotted against the Fc receptor activity of corresponding tumors, seven of the tumors were distributed along a line showing an inverse linear relationship; i.e. tumors with large amounts of IgG on their cell surfaces had the lowest Fc reactivity and vice versa. Cells from three of the tumors had lower amounts of IgG on their surface than expected from this relationship. However, the lack of correlation could be explained by the focal distribution of the Fc positive tissue within non-reactive tissue. The cells from these areas presumably carry less IgG on their surface and thereby reduce the quantity of IgG calculated per 10(6) cells. Prolonged washing of tumor sections resulted in stronger Fc receptor activity, and correspondingly washed cells had lower amount of IgG on their surface. Presumably the Fc receptor can bind IgG in vivo.

Immunochemical study of incomplete platelet autoantibodies by the anti-globulin consumption test (AGCT) with specific anti-immunoglobulin sera

The indirect anti-globulin consumption test (AGCT) with specific immunoglobulin antisera (anti-IgG and anti-IgM) has been applied to the immunochemical characterization of incomplete platelet auto-antibodies in 33 patients with idiophatic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE) and autoimmune hemolytic anemia (AHA). In these cases indirect AGCT on platelets was positive with polyvalent gamma antiserum. The test with anti-IgG was positive in all cases except two, while always negative with anti-IgM, with no relation to the presence of complete platelet antibodies, the type of disease and the immunochemical type of erythrocyte autoantibodies in AHA patients. These results indicate that the incomplete platelets auto-antibodies were of the IgG class.

Myasthenia Gravis: Cross-Striational Fluorescence of Muscle Fibers Mediated by Fc Fragments

Abstract The antiglobulin consumption test was performed by using human skeletal muscle tissue incubated with Fc fragments of γG globulins prepared from normal human serum. An increased consumption was found indicating binding of the Fc fragments to the tissue. The indirect immunofluorescence test was employed for localization of the binding. A cross-striational staining was found. The intensity was weaker, but the pattern otherwise indistinguishable from that observed when using a positive myasthenia gravis serum.

Autoimmune neutropenia associated with chronic active hepatitis

Evidence was found for a serum immunoglobulin G (IgG) antibody against neutrophils in a patient with neutropenia and chronic active hepatitis. The patient's serum was fractionated on a DEAE chromatographic column. Each serum fraction was incubated with homologous neutrophils from the parents and siblings, and only the IgG fraction agglutinated the neutrophils. The patient's neutrophils were isolated utilizing a nylon fiber mesh. The antibody was eluted from the sensitized neutrophils and subjected to radial immunodiffusion analysis, which revealed IgG antibody. The neutrophilic antibody was further demonstrated in the serum by fluorescein-tagged anti-IgG and on the cells by means of an antiglobulin consumption test. The serum antibody was specific for human neutrophils and did not react against animal neutrophils, in contrast to systemic lupus erythematosus in which leukoagglutinins react against both human and animal granulocytes. The appearance of leukocyte autoantibodies is not surprising in a disease entity associated with a wide variety of abnormal serologic reactions.

Myasthenia gravis: results of serological investigations compared with clinical data.

Clinical data concerning twenty-nine myasthenia gravis patients were correlated with serological findings. The sera were examined for the presence of muscle antibodies by the antiglobulin consumption and indirect haemagglutination tests, and for thyroglobulin antibodies by indirect haemagglutination test. Positive reactions in the antiglobulin consumption test were found with 14 sera. The respective patients did not differ in any important clinical feature from the 15 seronegative. Positive reactions in the indirect haemagglutination test for muscle antibodies were obtained with 5 patient sera, including serum from one seronegative patient. Thyroglobulin antibodies occurred equally in sera from seronegative (3) and seropositive (3) patients. The theory of an immunological and a non-immunological form of myasthenia gravis was therefore not supported by clinical data.

Immunologic and morphologic studies of the acute effect of anti-lung serum

When normal CD strain rats are injected intravenously with rabbit antiserum to rat lung they develop lung and kidney lesions which resemble those of Goodpasture's syndrome. The present study shows that IgG prepared from this serum possesses the following properties: (1) It reacts with a globulin-free insoluble lung tissue extract in the antiglobulin consumption test and with a soluble lung extract in the complement-fixation test; (2) It localizes in alveolar septa and glomerular tufts; and (3) It is both pneumotoxic and nephrotoxic causing pulmonary hemorrhage and edema, and glomerulonephritis. Electron microscopic evidence shows that alveolar endothelial cells are injured during the acute pneumotoxic response and supports the view that these cells are primary immunologic targets in situ.

Demonstration by the Antiglobulin Consumption Test With Murine Antisera of Common Antigens in Tissues Infected With the Mammary Tumor Virus From Different Mouse Strains

Demonstration by the Antiglobulin Consumption Test With Murine Antisera of Common Antigens in Tissues Infected With the Mammary Tumor Virus From Different Mouse Strains

Direct Antiglobulin Consumption Test: Tests on Leucocytes and Platelets from Patients with Malignant Disease and Some Observations on Quantitation

Summary. Using the direct antiglobulin consumption test the leucocytes and platelets of 50 patients suffering from disseminated malignant disease have been tested for the presence of a coating gamma globulin. There was a positive incidence of approximately 50%. In 15 control normal subjects no positive results were obtained.No relationship was found between the presence of the globulin and either the type of neoplasm, the amount of radiation or the amount of transfusion.Elution experiments carried out failed to establish whether the globulin has antibody properties or specificity.A comparison of different methods of antiglobulin consumption suggests that this test is not reliable for the quantitative assessment of fixed antibody globulin.