Total Antibody Activity Research Articles

Antibody Affinity, Total Antibody Activity, and Protein Content to Anti-HBs Antigen Among Sudanese and Chinese Using Terminal Antibody Method.

Generally, HBV infection serum markers have been globally measured, and the analysis of entire an-tibody details include the affinity, total protein content and antibody activity are rarely measured between two different ethnic groups. We detected and determined the entire characteristics of anti-HBs (antibody to HBs anti-gen) among Sudanese and Chinese HBV resolved patient's using a terminal antibody (TA) method. Serum samples of Sudanese and Chinese resolved HBV infection positive anti-HBs were collected. All se-rum samples were diluted in serial dilutions (20, 40, 80, and 160 dilutions). Anti-HB markers were measured with enzyme-linked immunosorbent assay (ELISA), antibody affinity, total protein content, and total antibody activity to anti-HBs were calculated according to the results obtained for each dilution. The antibody affinity to HBV statistically showed higher significance among Sudanese than Chinese (p < 0.05). The total antibody activity to HBV among Sudanese was higher statistically than Chinese patients (p < 0.05). Statistically, there was a high correlation between age and antibody affinity to HBV among Sudanese compared to the Chinese group (p < 0.05). The measurement of the antibody affinity, total antibody activity, and protein content of anti-HBs among Sudanese and Chinese, two different ethnic groups, may predict HBV infection status among African race and Asian race, and in addition, may play an important role in a high or a low incidence of the disease between different ethnicities.

Obinutuzumab (GA101) More Potently Engages Phagocytic-Lineage Cells Resulting In Enhanced Monocyte and Macrophage Activity When Compared To Rituximab and Ofatumumab

Therapeutic antibodies possess several clinically relevant mechanisms of action including cell death induction, perturbation of tumor cell signaling, activation of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP) and induction of adaptive immunity. Obinutuzumab (GA101) is a novel humanized, glycoengineered Type II anti-CD20 monoclonal antibody engineered for displaying enhanced FcγRIIIa (CD16) binding affinity and characterized by stronger induction of ADCC and direct tumor cell death when compared to wild-type, Type I anti-CD20 antibodies rituximab and ofatumumab. In light of the important role of phagocytic lineage cells in the mechanism of action of therapeutic antibodies, we compared GA101, rituximab and ofatumumab for their ability to trigger FcγR-dependent monocyte and macrophage effector functions. We show that, due to glycoengineering, GA101 displays superior CD16-dependent binding to monocytes, M1 and M2c macrophages in presence of nonspecific, competing, human endogenous IgGs, a situation that more closely mimics physiological conditions. Subsequently, GA101 more strongly engages monocytes and macrophages and leads to significantly higher elimination of CD20-expressing tumor cells as shown by assays detecting total antibody activity (ADCP, ADCC and direct effects). In support of the stronger GA101 activity, higher nitric-oxide (NO) levels are also detected in supernatants of tumor/macrophage co-cultures treated with antibody. Taken together, our data show that in addition to stronger NK-cell mediated ADCC and direct cell death induction due to Type II CD20 binding, GA101 more potently engages phagocytic-lineage cells resulting in enhanced monocyte and macrophage activity under conditions that more closely resemble physiological settings. Disclosures:Herter:Roche: Employment. Klein:Roche Glycart AG: Employment. Umana:Roche: Employment, Equity Ownership. Bacac:Roche: Employment.

The novel use of a routine quantitative system to analyze the activity, content and affinity of an antibody to hepatitis B core antigen

BackgroundCurrently, quantitative measurement of serum markers of HBV infection has been widely used, but commonly used analysis for specific antibodies only involves the measurement of the total antibody activity, and the binding affinity or protein content is rarely analyzed. ObjectiveTo investigate the detailed features of an antibody to hepatitis B core antigen (anti-HBc) during different periods of hepatitis B virus (HBV) infection with a new method of analysis. Study designSerum samples were collected from patients that were positive for the anti-HBc antibody. On the basis of the other serological markers in the samples, all patients were divided into a hepatitis B surface antigen (HBsAg)-positive group and an antibody to hepatitis B surface antigen (anti-HBs)-positive group. All samples were diluted 2-, 20- and 200-fold. Anti-HBc quantification was measured with a chemiluminescent microparticle immunoassay; total anti-HBc activity, protein content and affinity were calculated according to a measured value of each dilution. Serum HBV DNA load and alanine aminotransferase (ALT) levels were also measured. ResultsThe total anti-HBc activity in the HBsAg-positive group was statistically higher than that in the anti-HBs-positive group (p<0.05). The anti-HBc protein content during active HBV infection was statistically higher than during the convalescence stage (p<0.05), while anti-HBc affinity during HBV infection was lower than during recovery. There were correlations among total activity, affinity, protein content of anti-HBc, and ALT, HBV-DNA (p<0.05). ConclusionsIt is potentially possible to predict the status of HBV infection by measuring total activity, protein content and affinity of anti-HBc.

A simple method to measure antibody affinity against the hepatitis B surface antigen using a routine quantitative system

The aim of the study was to establish a simple method to measure antibody affinity to hepatitis B surface antigen (anti-HBs) by using a routine quantitative system to explore the significance of anti-HBs affinity. Serum samples of hepatitis B patients who had been discharged from the hospital were collected at the time of re-examination. The 60 patients with resolved HBV infection were divided into two groups based on the serological markers measured in the samples: an anti-HBs group (anti-HBs-only positive) and an anti-HBs positive with anti-HBc group. The serum samples were diluted prior to the measurement of total anti-HBs by using a commercial assay with the AxSYM device. The total anti-HBs (mIU/mL) of all dilutions were measured, and the positive data (>10mIU/mL) were transformed into logarithmic values (lgU). The relative affinity of anti-HBs was calculated and derived from the mass action formula. The absolute value of (lgU1−lgU2) positively correlated with the antibody affinity. The results indicated that affinity of anti-HBs in the samples obtained from the anti-HBs-alone positive group was statistically higher than that of the other group (P<0.05), though no significant difference was noted in regard to the total antibody activity (P>0.05). This simple approach to measurement of anti-HBs antibody affinity may serve as a new tool in clinical laboratories and contribute to the management of patients with hepatitis B.

Establishment of a new method for the detection of the affinity of antibody to hepatitis B e antigen by a routine quantitative system

BackgroundThe commonly used quantitative analyses for specific antibodies only involve measurement of the total antibody activity, and the binding affinity is rarely analyzed. We established a method for the detection of the affinity of the antibody to hepatitis B e antigen (anti-HBe). MethodsA method to detect anti-HBe affinity was established by using a routine quantitative system (Architect Analyzer). Serum samples were collected from hepatitis B patients with anti-HBe. On the basis of the other serologic markers in the samples, patients were divided into HBsAg positive and anti-HBs positive groups. The total anti-HBe activity and anti-HBe affinity were each measured. ResultsThe affinity of anti-HBe in the anti-HBs positive group was higher than that in the HBsAg positive group (P<0.05); conversely, the total anti-HBe activity in the anti-HBs positive group was lower than that in the HBsAg positive group (P<0.05). ConclusionsThe anti-HBe affinity arising from an immune response to HBV infection may vary significantly between different situations. This new approach will be suitable for determining antibody affinity in clinical laboratories.

Incorrectly placed C-spine collar

The IgG subclasses displayed by autoantibodies were examined in patients with systemic rheumatic diseases. Solid-phase assays performed with purified antigens were combined with a set of four mouse monoclonal antibodies specific for each human subclass to provide quantitative data for all the major autoantibody specificities. IgG1 accounted for an average of 55% of the total antibody activity to native and denatured DNA, Sm antigen, and histone and constituted significantly more anti-SS-B and anti-nRNP (84% and 92%, respectively). The remaining antibody activity consisted largely of IgG3, and this subclass was particularly prominent with anti-histone and anti-Sm in patients with systemic lupus erythematosus. In contrast, IgG2 constituted 3 to 12% of the anti-native and anti-denatured DNA and less than 5% of the anti-SS-B/La activity in only three patients with Sjogren9s syndrome. IgG2 was essentially undetectable in antibodies to Sm and RNP antigens. IgG4 was also uncommon, although this isotype was significantly more prevalent in anti-histone from patients treated with procainamide showed that the isotype distribution of anti-histone and anti-denatured DNA remained remarkably constant. However, during periods of large increases in autoantibody activity, a shift from predominantly IgG3 to predominantly IgG1 occurred, consistent with the interpretation that there might be a sequential activation of heavy chain constant regions as the immune response matures. The disproportionately high levels of IgG1 and IgG3 displayed by all the autoantibody specificities examined may indicate that a common immunogenic feature of autoantigens or a common control mechanism underlies the regulation of autoantibody expression.

Serodiagnosis of Crimean-Congo haemorrhagic fever

Several methods for demonstrating antibody to Crimean-Congo haemorrhagic fever virus were compared on serum samples taken from 101 patients during the acute stage of illness and at intervals for up to 59 months thereafter, with emphasis on early detection of the immune response. The deaths of 23 patients on days 5-14 of illness were ascribed to the effects of the disease; two patients died later from other causes. Very few of the patients who died from the acute illness mounted an antibody response detectable by the methods tested. Four patients who died and 18 who recovered were treated with immune plasma collected from recovered patients. Treated patients acquired IgG antibody from the plasma, but it was possible to discern the onset of an endogenous IgM response in those individuals who survived the disease by all of the methods tested. Indirect immunofluorescence (IF) tests detected IgM and/or IgG antibodies at the earliest on day 4 of illness in about 10% of patients who survived the disease, and by day 9 all survivors had antibodies demonstrable by IF. A biotin-streptavidin IF technique offered no advantage over the standard IF test for the early detection of IgG antibody, but demonstrated higher antibody titres and detected IgM antibody earlier in about a quarter of the patients tested. An IgM-capture enzyme-linked immunoassay (ELISA) and an IgG sandwich ELISA demonstrated higher antibody titres than did IF tests, and detected antibody responses at an earlier stage of infection than did IF tests in about one-fifth of patients, but the reverse was true in a similar proportion of instances. A competition ELISA, which detected total antibody activity, produced lower titres than did the IgM and IgG ELISAs, but yielded results which were in close agreement with the findings in IF tests. It was concluded that the IF tests were most convenient for use in making a rapid serodiagnosis of the disease.

Thiophilic adsorption: rapid purification of F(ab) 2 and Fc fragments of IgG1 antibodies from murine ascitic fluid

A thiophilic adsorption method has been developed for rapid purification and separation of mouse F(ab) 2 and Fc fragments obtained after proteolytic digestion of IgG1 monoclonal antibodies. Partially purified Mabs were digested with papain. Thiophilic chromatography was performed using stepwise elution with decreasing concentrations of ammonium sulphate. Most contaminating proteins did not react with the thiophilic adsorbent, and chromatography efficiently resolved the F(ab) 2 and Fc fragments, as judged by electrophoresis. Fractions containing the F(ab) 2 fragments retained about 90% of the total antibody activity loaded onto the column.

Enzyme-linked immunosorbent assays for the detection of antibody to Crimean-Congo haemorrhagic fever virus in the sera of livestock and wild vertebrates.

IgM antibody response to Crimean-Congo haemorrhagic fever (CCHF) virus was monitored in experimentally infected sheep and cattle by an IgM capture enzyme-linked immunoassay (ELISA). Specific binding of antigen was detected by a rabbit anti-CCHF horseradish peroxidase conjugate or a sandwich technique with hyperimmune mouse anti-CCHF ascitic fluid and commercially available anti-mouse immunoglobulin peroxidase conjugate. The persistence of IgM antibody activity was found to be of shorter duration than in humans, and this may be a function of the relative lack of susceptibility of these animals to infection with CCHF virus. IgG antibody responses in the sheep could be monitored by sandwich ELISA using commercially available anti-sheep immunoglobulin peroxidase conjugates. Total antibody activity in the sera of experimentally infected sheep, cattle and small mammals could be monitored in a competitive ELISA (CELISA) using rabbit anti-CCHF peroxidase conjugate. The CELISA was applied to the sera of 960 wild vertebrates from a nature reserve in South Africa, and the prevalence of antibody was found to be greatest in large mammals such as rhinoceros, giraffe and buffalo, which are known to be the preferred hosts of the adult tick (Hyalomma) vectors of the virus.

Kinetics and specificity of T and B cell responses in relapsing experimental allergic encephalomyelitis.

T and B cell responses to myelin basic protein (MBP) and its relevant peptide fragments were examined throughout the course of MBP-induced relapsing experimental allergic encephalomyelitis (REAE) in (SJL X PL)F1 mice. T cell reactivity, measured by the antigen-driven proliferation of lymph node T cells in vitro, was directed predominantly against the encephalitogenic MBP-P2 peptide (amino acids 1 to 37) at all stages of disease. Levels of responsiveness did not correlate with disease expression, but declined over time to a relapse level that was four- to sixfold lower than that observed during peak acute stage reactivity. Relapse responses were further distinguished by the detection of host I-E restrictions on Lyt-1+ T cell recognition of P2, P2 recognition by acute-stage T cells occurring solely in the context of host I-A molecules. These data imply an increase in the heterogeneity of relapse T cell responses to MBP to include clones restricted by additional class II glycoproteins. A role for additional CNS autoantigens in the stimulation of relapse T cells is also considered. Serum antibody responses to MBP or the P2 fragment fluctuated randomly throughout R-EAE when total antibody activity (IgM plus IgG) was measured. However, analysis of individual isotypes of IgG immunoglobulins revealed an apparent correlation between peak antigen-binding activity and disease expression which may reflect either an effector or regulatory role for humoral immunity in recurrent EAE. Patterns of early antibody reactivity also distinguished F1 mice that developed or failed to develop disease signs after immunization, the latter exhibiting a consistent drop in antigen-binding activity 4 to 5 days before the usual onset of acute-stage paralysis. The results are considered with regard to possible mechanisms of chronic disease regulation in an environment of functional T cell suppression.

IgG subclasses of autoantibodies in systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity.

The IgG subclasses displayed by autoantibodies were examined in patients with systemic rheumatic diseases. Solid-phase assays performed with purified antigens were combined with a set of four mouse monoclonal antibodies specific for each human subclass to provide quantitative data for all the major autoantibody specificities. IgG1 accounted for an average of 55% of the total antibody activity to native and denatured DNA, Sm antigen, and histone and constituted significantly more anti-SS-B and anti-nRNP (84% and 92%, respectively). The remaining antibody activity consisted largely of IgG3, and this subclass was particularly prominent with anti-histone and anti-Sm in patients with systemic lupus erythematosus. In contrast, IgG2 constituted 3 to 12% of the anti-native and anti-denatured DNA and less than 5% of the anti-SS-B/La activity in only three patients with Sjogren's syndrome. IgG2 was essentially undetectable in antibodies to Sm and RNP antigens. IgG4 was also uncommon, although this isotype was significantly more prevalent in anti-histone from patients treated with procainamide showed that the isotype distribution of anti-histone and anti-denatured DNA remained remarkably constant. However, during periods of large increases in autoantibody activity, a shift from predominantly IgG3 to predominantly IgG1 occurred, consistent with the interpretation that there might be a sequential activation of heavy chain constant regions as the immune response matures. The disproportionately high levels of IgG1 and IgG3 displayed by all the autoantibody specificities examined may indicate that a common immunogenic feature of autoantigens or a common control mechanism underlies the regulation of autoantibody expression.

Clinical and immunologic correlations in trimellitic anhydride airway syndromes

Twenty workers exposed to trimellitic anhydride (TMA) powder were evaluated to correlate respiratory symptoms with total antibody activity against trimellityl human serum albumin (TM-HSA). Further, specific IgG, IgA, and IgE against TM-HSA were compared to total specific antibody levels against that antigen. The workers were categorized clinically as having either the late respiratory systemic syndrome (LRSS), immediate rhinitis and asthma, or an irritant reaction. There were no histories compatible with the pulmonary disease-anemia syndrome. Total antibody and IgG antibody activity to TM-HSA correlated well ( R 8 = 0.75, p < 0.05). Total antibody activity and IgG discriminated between the LRSS and asymptomatic workers. Although IgA antibody activity was found in almost all TMA-exposed workers, it did not discriminate the symptomatic workers from asymptomatic workers as well as did the IgG or total antibody determinations. IgE antibody activity against TM-HSA was elevated in one worker with immediate-type asthma. Previous studies correlated symptoms caused by TMA inhalation with antibody activity. This study indicates that clinical assessment plus total antibody determination or IgG antibody and IgE antibody determination should establish a means of diagnosis of the presence or absence of immunologic respiratory disease caused by TMA powder.

Uneven distribution of antitreponema antibody acitivity in differing immunoglobulin G fractions from patients with early syphilis.

Three fractions, containing immunoglobulin G's (IgG's) of differing relative electrophoretic mobility, were isolated from sera of 33 male patients with untreated early syphilis. Serological testing, employing four different serodiagnostic procedures for these fractions at equal amounts of IgG, revealed a very constant reaction pattern for all patients. In the earliest stages of the disease, the most basic fraction was the first to show antibody activity. In progressive stages, antibody activity subsequently was also found in the two less basic fractions. However, in all stages of disease studied, the basic part of the IgG made the largest contribution of the total antibody activity within the IgG class, indicating an uneven distribution of antitreponema antibody activity over heterogeneous IgG. Several possible explanations of the observations are discussed.

Uptake of maternal antibody by the neonatal pig following intramuscular and intramammary vaccination of the preparturient sow

Administration of heat inactivated Escherichia coli antigens by intramuscular and intramammary routes induced elevated antibody levels in sow serum and colostrum, predominantly associated with IgG. Colostral IgG accounted for approximately 80 per cent of the total antibody activity, and there was a similar distribution in the sera of one-day-old piglets. The additional antibody activity was carried almost entirely by IgM following intramuscular injections and was evenly distributed between IgM and IgA following intramammary stimulation. The distribution of antibody activity and all three major immunoglobulin classes in colostrum and milk from individual mammary glands was remarkably uniform. A similar uniformity was inferred for the ingestion and absorption of colostrum by individual piglets as judged by the contents of their blood sera during the neonatal period.

A fragment comprising the last third of bovine serum albumin which accounts for almost all the antigenic reactivity of the native protein.

The fragmentation of native bovine serum albumin by trypsin has been studied in aqueous solution under various conditions with regard to the yield and size of the fragments obtained. From a partial tryptic hydrolysate at pH 8.2 (40 degrees, 1 hour), a homogeneous fragment was isolated in high yield by gel filtration on Sephadex G-100, followed by chromatography on DEAE-cellulose. The molecular weight of the fragment by gel filtration on calibrated Sephadex G-100 columns and by sodium dodecyl sulfate electrophoresis was 22,500. After reduction of the disulfide bonds followed by alkylation of the resultant thiol groups with iodoacetamide, the fragment retained homogeneity by disc electrophoresis and its molecular weight remained unchanged, indicating that it was composed of a single polypeptide chain. From its amino acid composition, sequence of the first 20 residues, and actions of carboxypeptidases A or B, it was unequivocally assigned to positions 377-571 in albumin. The inhibitory activity of the fragment was 90 to 93% towards the immune reaction of the protein with the IgG fraction of the antisera. The IgGfraction accounted for 96% of the total antibody activity in the antisera. An immunoabsorbent of fragment 377-571 removed 89 to 95% of the antibody to albumin. A fluorescent derivative of the fragment, which retained full immunochemical activity, was found to bind 2 mol of antibody/mol of peptide. The disulfides in peptide 377-571 were essential for its immunochemical reaction because the latter was entirely abolished upon reduction and S-alkylation of the disulfides. Since this fragment comprised only a third of the albumin molecule, but accounted for 90 to 95% of its antigenic reactivity, the results indicated that native albumin carries identical repeating antigenic reactive sites.

Demonstration of cytotoxic antibodies in rabbits bearing tumors induced by Shope fibroma virus.

The (51)Cr-release test was used to detect cytotoxic antibodies in adult rabbits bearing tumors induced by Shope fibroma virus. The following are the recommended experimental conditions: the infection of RK-13 cells with a multiplicity of 1 to 2 infectious units per cell for 48 hr, (51)Cr labeling of infected cells during the last 12 hr of incubation, sensitization of suspended labeled infected cells for 1 hr with immune serum, and quantitation of cell damage by the amount of (51)Cr released after 6 hr of incubation in the presence of complement. The immune sera reacted only with fibroma virus-infected cells but not with cells infected with vaccinia virus or herpesvirus type 1. Similarly, sera prepared against vaccinia virus and herpesvirus type 1 were not cytotoxic to fibroma virus-infected cells, although they were cytotoxic to cells infected with homologous viruses. The total antibody activity in sera of rabbits infected with Shope fibroma virus was detected first on day 7, gradually rose to its peak by day 23, and persisted at that level for at least 50 days. The 19S antibody was detected on day 7, reached peak titers by day 13, and disappeared by day 17. The 7S antibody was barely detectable on day 7, reached maximum titers on day 13, and remained high for at least 50 days. The tumors appeared on the 3rd day after virus inoculation, reached maximum size on day 13, and regressed completely by day 23.

Quantitative Estimation of Antibody in the Immunoglobulin Classes of the Mouse

Abstract Thymectomized mice produced a significantly lower level of antibody against human serum albumin (HSA) and the decrease in total antibody activity was due to a decrease in the IgG1 while the other immunoglobulin classes were largely unaffected. The same animals did produce a normal response in all immunoglobulin classes to a high dose of keyhole limpet hemocyanin (KLH). However, a low dose (1 µg) of the same antigen produced a poor response in thymectomized mice, and, as was found with HSA, IgG1 was the main immunoglobulin class affected. It is concluded a) that KLH is a “non-thymic-dependent” antigen only at a relatively high dose and b) that of all the immunoglobulin classes only IgG1 is thymic-dependent.

Tobacco mosaic virus specific immunoglobulins from horse serum. II. Structural specificity and association constants.

AbstractTobacco mosaic virus (TMV)‐specific IgG and IgG(T) isolated from a horse were found to contain mainly antibodies with conformational specificity. The amount of antibodies with sequential specificity for the C‐terminal amino acid sequence of the TMV protein subunit must be less than 1 × 10−4 of total antibody activity. The binding properties of both specific immunoglobulins were investigated. No heterogeneity of the association constants for either IgG or IgG(T) could be detected.