Relative Functional Binding Activity of IgGl and IgG3 Anti-D in IgG Preparations

The intention to replace polyclonal IgG anti-D with human monoclonal antibody in the prophylaxis of haemolytic disease of the newborn requires knowledge concerning the relative content of IgG1 and IgG3 anti-D in prophylactic IgG preparations that are in present use. This has been carried out using a functional assay in which the absolute amount of IgG1 and IgG3 anti-D present on red cells was determined after incubation with IgG preparations. The assay was carried out by flow cytometry on 17 samples; expressed as a percentage of the total, the average value for the amount of IgG3 anti-D on the cells was 8% (range 1-18%). Similar measurements were also made on the anti-D present in 18 samples of antisera; IgG3 anti-D formed a larger fraction of the total, the average value being 17% (range 0-60%) confirming previously reported estimates. It is suggested that some of the low values found for IgG3 in IgG preparations may be due do preferential loss during production.     

Use of the DAF Assay to Assess the Functional Properties of Polyclonal and Monoclonal Rh D Antibodies

The mechanism whereby passive Rh (D) immunoglobulins suppress the feto-maternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developped to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.     

Differential binding of IgG anti-D and IgG autoantibodies to reticulocytes and red blood cells

125I IgG anti-D binding to reticulocytes obtained by density fractionation is reduced relative to that bound to all other red cell (RBC) fractions. Maximum D antigen reactivity occurs following reticulocyte maturation with no detectable change in D reactivity of mature RBC throughout their life span. Reticulocytes have in the range of about 60% of the content of mature RBC. Previously reported increased anti-D agglutinability and binding to old RBC it is not due to an intrinsic increase in D antigen with age, but results from an 'apparent' decrease in anti-D binding to young RBC fractions due to reticulocyte enrichment. IgG RBC autoantibodies obtained by elution from the RBC of eight Coombs-positive blood donors, probably associated with alpha-methyldopa (alpha-MD) administration, showed decreased binding to reticulocytes as determined by 125I protein A (PA). Reticulocytes bound about 70% of the IgG bound to mature RBC, indicating that the membrane antigenic determinant defined by these autoantibodies was incompletely expressed in the reticulocyte. This difference in IgG autoantibody binding between reticulocytes and mature RBC is similar to the decreased D antigen content of reticulocytes and consistent with an autoantibody determinant associated with the Rh complex. Direct testing of density fractionated Coombs-positive RBC in four out of five patients with autoimmune haemolytic anaemia (AIHA) showed reduced quantities of IgG on reticulocytes. The distribution of IgG between reticulocytes and mature RBC may be useful in serologically characterizing patients with AIHA and in identifying subpopulations of patients with this disorder.     

Lymphosarcoma,Cold Urticaria,IgG1 Monoclonal Cryoglobulin and Complement Abnormalities

A patient with lymphosarcoma and cold urticaria showed evidence of complement activation by the classical pathway with low levels of the early complement components, normal levels of late acting components, normal functioning of the alternate pathway and reduction of the C1-inhibitor level. The serum contained an IgG₁ monoclonal cryoglobulin responsible for the complement activation. In vitro tests demonstrated a high capacity of the serum to activate C1.     

Comparisons of Pooled Polyclonal Rabbit Anti-Human C3d with Four Monoclonal Mouse Anti-Human C3ds

Performance characteristics of pooled rabbit IgG polyclonal anti-C3d are compared with one mouse IgM and three mouse IgG monoclonal anti-C3d antibodies (MAs). IgG MA,s employed singly or in combination, failed to precipitate C3d; by contrast, IgM MA and polyclonal anti-C3d precipitated C3d. Measurements of polyclonal anti-C3d concentration by chemical means and by 125I-C3d radioimmunoassay (RIA) agreed closely. RIA values were 50% of chemical measurement values for three of the four MAs. Use of sucrose density gradient ultracentrifugation to assess MA C3d/anti-C3d molar combining ratios for soluble anti-C3d/C3d was not possible because fast-sedimenting multimeric C3d/anti-C3d complexes did not form. Dissociation and competitive binding studies indicate that (1) two MAs had substantially lower affinities than the other anti-C3d antibodies, and (2) polyclonal anti-C3d recognizes more C3d epitopes than are recognized by individual MAs. The results demonstrate antigenic complexity of C3d fragment and illustrate the difficulties of predicting individual MA performance based on prior experience with polyclonal antibodies.     

Comparisons of Pooled Polyclonal Rabbit Anti-Human C3d with Four Monoclonal Mouse Anti-Human C3ds

Labelled polyclonal IgG anti-C3d and monoclonal IgM and IgG anti-C3d antibodies (MAs) were employed at increasing antibody excess to measure the number of C3d molecules on human red blood cells (RBC) coated by complement in vitro and in vivo. Values for the number of C3d sites per cell determined with polyclonal anti-C3d were at least 4-fold higher than when MAs were used. The results suggest that the molar combining ratio for polyclonal anti-C3d with a single RBC-bound C3d fragment is more likely greater than 4.0 than 1.0 as previously assumed. Antiglobulin agglutination studies compared polyclonal and monoclonal anti-C3d antibodies against C3d-coated RBC from 27 patients with autoimmune hemolytic anemia. All four MAs showed striking prozones, requiring their use over a 25-fold higher range of dilutions than polyclonal anti-C3d. Polyclonal anti-C3d produced stronger agglutination reactions than any of the IgG MAs. Only the IgM MA produced agglutination as strong as, or stronger than, polyclonal anti-C3d. While IgM MA always gave the strongest MA agglutination reactions, no consistent ranking of the three IgG MAs was observed. Agglutination was not enhanced when all IgG MAs were combined; addition of IgG MAs to IGM MA reduced the strength of agglutination seen with IgM alone, suggesting blocking of IgM binding by competing IgG anti-C3d.     

IgG抗-D、抗-E致新生儿溶血病1例

<正>新生儿溶血病(hemolytic disease of the newborn,HDN)是由于孕妇和新生儿之间血型不合而产生的免疫性疾病,是造成新生儿病理性黄疸的常见原因之一,最常见的是由IgG抗-D引起,IgG抗-E次之。本例Rh(D)阴性待产妇产前血型血清学检测出血清中存在IgM和IgG性质混合的抗-D抗体、IgG性质抗-E抗体,其中IgG抗-D、抗-E抗体导致新生儿发生Rh系统HDN,现报告如     

Influence of IgG subclasses on automated anti-D (Rho) quantification

Anti-D quantification by both an automated Polybrene method and an automated trypsin-albumin-dextran (TAD) method gave discrepant results in certain cases. These discrepancies, expressed as the polybrene TAD ratio of reactivity (PTR), were related to the IgG subclass of anti-D. Anti-D of the IgG3 subclass showed a higher PTR than IgG1 (0.94 vs 1.65). No difference was shown between G1m(1) and G1m(3) (0.93 and 0.95, respectively) or between G3m(11) and G3m(21) (1.40 and 1.81, respectively) allotypes. The simultaneous use of our automated Polybrene and TAD methods provides information about the anti-D subclass composition.     

Differential production in vitro of antigen specific IgG1, IgG3 and IgA: a study in Schistosoma haematobium infected individuals

This study has evaluated the individual control of isotype production and the influence of external signals that can be experimentally provided in vitro, in antibody responses to two different recombinant Schistosoma antigens (Sh28GST and TPx-1). Peripheral blood mononuclear cells or enriched B cell fractions obtained from S. haematobium infected Senegalese adults were induced to terminal differentiation in vitro. The production of antibody to either antigen was donor-dependent and for each donor it was antigen-dependent. Differentiation to IgG1 and IgG3 production, and possibly IgA, specific to these conserved parasite antigens could be regulated differentially in vitro. Exogenous IL-2 and IL-10 or IL-10 and TGF-beta led to the production of specific IgG3 or IgG1 and/or IgA, respectively. This is the first report on such experimentally induced differential regulation of antigen-specific IgG1 and IgG3. This may have implications in designing protocols for protein based-vaccinations aiming at eliciting antibody responses of certain protective-type isotypes.     

In vitro Glycation of Human IgG and Its Effect on Interaction with Anti-IgG

Non-enzymatic glycosylation of proteins is one of the key mechanisms in the pathogenesis of diabetic complications. Glycation of IgG is of special interest due to its possible influence on the functionality of immunoglobulins and overall immuno-competence. The aim of this study was to clarify more details of in vitro glycation of IgG and to study the effect of this modification on its interation with anti-IgG. Purified human IgG was glycated in the presence of 50 and 100 mM glucose. Glycation was measured using spectrophotometric thiobarbituric acid method. To study the effect of glycation on interaction with anti IgG the Single Radial Immunodiffusion (SRID) was used and the diameters of precipitation rings of glycated IgG and non-glycated IgG were measured and compared. The results showed that IgG was glycated in presence of 50 and 100 mM glucose at 27 degrees and 37 degrees C and the extent of glycation was dependent on glucose concentration and time of incubation. In higher concentration of glucose and longer period of incubation glycation was higher at 27 degrees C (p<0.01). Similar results were obtained at 37 degrees C.The results of SRID indicated that glycated IgG showed reduced interaction with anti-IgG. The diameters of precipitated rings for glycated IgG were significantly lower than those of non-glycated IgG (p < 0.01). It can be concluded that modification that occurred in IgG structure due to glycation can be the reason of the reduction of its interaction with anti-IgG.     

Detection of IgG-Associated Determinants in Reduced and Alkylated Preparations of Human IgG3 by Monoclonal Antibodies

Abstract. Using classical typing antisera, previous experiments have failed to demonstrate IgG3 in partially reduced and alkylated preparations of human IgG intended for intravenous application (IGIV). To establish that IgG3 is actually present in such preparations, we designed an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies as solid-phase reagents and protein A-purified IgG3 as antigen. Three different samples of reduced and alkylated antigen were used: (1) IgG3 isolated from a ready-for-infusion IGIV; (2) IgG3 which was purified from an intramuscular (Cohn fraction II) IgG solution before being subjected to a mild reduction and alkylation procedure, and (3) completely reduced and alkylated IgG3. The reduction and alkylation procedure did not affect the solubility of IgG3, indicating that IGIV prepared in this manner should contain normal quantities of IgG3. In the ELISA, solid-phase monoclonals which were cross-reactive with multiple IgG subclasses clearly reacted with reduced and alkylated IgG3. Furthermore, there was no substantial difference between the quantities of modified and native antigen required for 50% maximal ELISA signal. In contrast, solid-phase monoclonals with IgG3-restricted specificity did not recognize reduced and alkylated material. These results indicate that IGIV prepared by reduction and alkylation has a normal IgG3 content and confirm that some IgG3-specific determinants are altered by the modification procedure.     

In vitro Antimicrobial Activity of Citrus aurantifolia and its Phytochemical screening

ObjectiveTo evaluate the antimicrobial efficacy of Citrus aurantifolia Linn (CA) against some microorganisms – bacteria and fungus were Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Pseudomonas spp, Aspergillus niger, Aspergillus fumigates, Mucor spp and Pencillium.Methods100 μl of 10 mg CA were assessed against eight test microorganisms by agar well Diffusion Method. Gentamicin and Ketoconazole 10 mg/ml were used as standards. A different solvent was used to obtain CA leaf extract by using maceration technique.Results%yield obtained for dried leaf extract of CA with chloroform, ethanol, acetone, petroleum ether and aqueous ethanol was approximately 15%, 18%, 09%, 11% and 24% respectively. Due to its high yield value hydroalcoholic extract of CA was used for estimating the antimicrobial activity and its phytochemical screening. Phytochemical screening of CA plant reveals the presence of Alkaloids, carbohydrates, flavonoids, steroids and tannins.ConclusionsThe study demonstrates that the hydroalcoholic extract of CA leaf exhibit antibacterial activity on Klebsiella pneumonia, Pseudomonas sp, Staphylococcus aureus and antifungal activity among Aspergillus niger, Aspergillus fumigates, Mucor species. These recognized a good support to the use of this plant in herbal medicine and as base for the development of new drugs and phytomedicine.     

Functional in vivo characterization of human monoclonal anti-D in NOD-scid mice

The prophylaxis of the hemolytic disease of the newborn requires significant amounts of plasma-derived polyclonal human anti-D. Because of procurement problems, there is a growing interest in replacing plasma-derived anti-D by in vitro-produced human monoclonal anti-D. Hundreds of monoclonal anti-D have been prepared, but the selection of the most potent for in vivo use is difficult because it cannot be predicted by in vitro characterization. This study evaluated the possibility of using nonobese diabetic/severe combined immunodeficient (NOD-scid) mice for the in vivo evaluation of human monoclonal anti-D. Human red blood cells (RBCs) were found to circulate normally in the blood of NOD-scid mice previously injected with a physiologic amount of human immunoglobulin G (10 mg). The addition of a small amount of anti-D (1 to 5 microg) resulted in the clearance of Rh D(+) RBCs within 4 hours. The comparative testing of 8 monoclonal anti-Ds showed a wide range of potency (15% to 87%) relative to plasma-derived polyclonal anti-D. There was no strong correlation between the in vivo potency index and the immunoglobulin G isotype, affinity, or fine specificity of the antibodies. These results show the usefulness of NOD-scid mice for the initial in vivo screening of human monoclonal anti-D before testing the most active antibodies in clinical trials done in human volunteers.     

In vitro and In vivo Antimalarial Activity of Amphiphilic Naphthothiazolium Salts with Amine-Bearing Side Chains

Because of emerging resistance to existing drugs, new chemical classes of antimalarial drugs are urgently needed. We have rationally designed a library of compounds that were predicted to accumulate in the digestive vacuole and then decrystallize hemozoin by breaking the iron carboxylate bond in hemozoin. We report the synthesis of 16 naphthothiazolium salts with amine-bearing side chains and their activities against the erythrocytic stage of Plasmodium falciparum in vitro. KSWI-855, the compound with the highest efficacy against the asexual stages of P. falciparum in vitro, also had in vitro activity against P. falciparum gametocytes and in vivo activity against P. berghei in a murine malaria model.     

In vitro stimulation of naive mouse lymphocytes by Heligmosomoides polygyrus adult worm antigens induces the production of IgG1

The production of high levels of IgG1, by mice chronically infected with the parasitic nematode Heligmosomoides polygyrus , has been documented for a number of years. In order to investigate this phenomenon, naive lymphocytes from B10.D2 mice were incubated in vitro with H. polygyrus adult worm homogenate (AWH) and the culture supernatants examined for immunoglobulin production. Stimulation of pooled naive splenocytes by AWH was found to produce IgG1, but not IgM, in an antigen dose dependent manner. Identical stimulation of splenocytes of individual inbred mice, indicated that this effect was reproducible but with considerable variation between mice. When the IgG1 produced was tested for specificity, it was found that there was little evidence that the immunoglobulin produced was able to bind to the inducing parasite antigens. Analysis of purified T cells reconstituted with splenocytes, demonstrated that T cells were the target lymphocytes of the stimulating molecule, contained within AWH. These results show that H. polygyrus AWH can induce the production of non-parasite specific IgG1 from naive splenocytes and that this production is crucially dependent upon the cell content of the in vitro culture. Furthermore, the production of IgG1 is not proportional to the degree of lymphocyte proliferation. It is suggested that at least part of the hypergammaglobulinaemia produced during a primary H. polygyrus infection, is due to this non-specific stimulation of mouse lymphocytes by the parasite.