Immunodiagnosis of bancroftian filariasis—Problems and progress

The immunodiagnosis of bancroftian filariasis is a major challenge to the immunoparasitologist. Significant progress is yet to be made in developing convenient laboratory animal model and inin vitro cultivation of filarial parasites making it very difficult to obtain required amount of parasite material for research. Parasitological examination techniques are not useful in low microfilaraemia, occult or chronic.filarial infections. A precise and accurate immunodiagnostic technique is very much needed for successful filaria control programmes. Such a test will also avoid the need for laborious night blood examination in bancroftian filariasis. Due to comparatively easy availability, a good amount of work has been done to explore immunodiagnostic potential of heterologous filarial antigens isolated fromLitomosoide carinii, Dirofilaria immitis, Brugia malayi, Setaria digitata, Setaria cervi and number of other filarial species. However, there has been limited or no significant success due to number of false negative and false positive reactions. Extensive study has also been made with antigens isolated fromWuchereria bancrofti microfilariae. Soluble antigens of microfilariae have been used in different immunological techniques such as skin test, counter immuno electrophoresis, indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay. Fractionation of Wuchereria bancrofti microfilarial soluble antigens yielded mfS3e antigen fraction which was found to be highly reactive in microfilaraemia by enzyme linked immunosorbent assay, but the yield of the purified antigen was not sufficient enough to make it a practical proposition for large scale isolation of antigen. Wuchereria bancrofti microfilarial excretory-secretory antigens were found to be specific and highly sensitive requiring as little as 0.35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. One ml of culture fluid was found to be sufficient for 400,000 tests. Field evaluation of this test showed that it can replace laborious night blood examination. Assay systems have been developed for detection of filarial antigen in serum, urine, hydrocele fluid and immune complexes using immunoglobulins from chronic filarial sera and antisera to excretory filarial antigens. Further purification of excretory-secretory antigens by affinity chromatography and production of monoclonal antibodies should hopefully give suitable reagents for use in sensitive assays such as enzyme immuno assay and immuno radiometric assay, providing an ideal assay system for detection of active filarial infection in the not too distant future.     

Stick enzyme-linked immunosorbent assay using the avidin-biotin system for detection of circulating antigen in bancroftian filariasis

Detection of filarial antigen in different groups of sera was carried out by sandwich as well as inhibition enzyme-linked immunosorbent assays using antibody-coated sticks. Both systems were found to be equally sensitive in detecting antigen in 90% of microfilariae carriers. Incorporation of avidin-biotin in the sandwich assay system increased the sensitivity of antigen detection from 10⁻⁶ to 10⁻¹⁶ pg. A 67% decrease in the number of false negative results was observed when the sensitive avidin-biotin inhibition enzyme-linked immunosorbent assay system was used for analysis of filaria blood samples.     

Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.     

Development and evaluation of a rapid flow-through immuno filtration test using recombinant filarial antigen for diagnosis of brugian and bancroftian filariasis

There is an imperative need to develop a rapid antibody test that can be used for diagnosis of clinical cases in travelers and expatriates, primary surveillance in areas of unknown endemicity, detection of early infection in childhood and for monitoring chemotherapeutic programs. A rapid-format, simple and qualitative flow through immuno filtration test has been developed for the identification of total IgG antibodies to recombinant filarial antigen WbSXP-1. This test system employs colloidal gold-protein A reagent as the antibody capture reagent. The sensitivity and specificity of the test was evaluated in a total of 1,230 serum samples. The sensitivity of the test was found to be 90.8% with brugian (n = 70) and 91.4% with bancroftian (n = 140) microfilaraemic subjects. The test showed minimum reactivity (4/10) with Loa loa microfilaria (MF) positive sera and no reactivity (0/20) with Onchocerca MF positive sera. This rapid diagnosis is found to be non-reactive with individuals having other parasitic diseases including schistosomiasis (n = 10), soil-transmitted helminthiases (n = 34) and protozoan infections (n = 33) indicating the potential of this test as a prospective method of diagnosis for both brugian and bancroftian lymphatic filariasis. Stability kinetics was studied at different temperatures and different time periods. The rapid flow-through immuno filtration test is advantageous since it can be stored at room temperature, is user friendly and is particularly applicable in the field as an initial screening method, for epidemiological monitoring of filarial infections in bancroftian and brugian endemic regions of the world.     

Detection of filarial infection usingWuchereria bancrofti microfilariae culture antigen and filter paper blood samples in enzyme linked immunosorbent assay

Blood collected on filter paper by finger-prick gave results comparable to intravenous serum samples when analysed by enzyme-linked immunosorbent assay (ELISA). All the 100 microfilaraemia, 5 out of 100 endemic normals and none of the 10 nonendemic normal filter paper blood samples showed the presence of filarial antibody when tested by this method,using culture antigen and anti-immunoglubulins, class G, M and A — penicillinase conjugate. When the same samples were screened for the presence of IgM antibody, 91 out of 100 microfilaraemia, 13 out of 100 endemic normal and none of the 10 nonendemic normal samples showed a positive reaction. Enzyme linked immunosorbent assay, using culture antigen and filter paper blood samples, appears to work in large field studies for detection of filarial infection.     

酶联免疫法和胶体金法在胃镜检查前检测乙肝表面抗原的结果观察

目的:比较酶联免疫法和胶体金法在胃镜检查前检测乙肝表面抗原的结果,以探讨酶联免疫法和胶体金法在检验与病理科乙肝表面抗原检测中的应用价值.方法:选择2017年1月～2019年12月我院进行胃镜检查的100例上消化道疾病患者,均在胃镜检查前检测乙肝表面抗原,对照组使用胶体金法进行检测,观察组使用酶联免疫法进行检测.比较酶联...     

The cell surface receptor G6b, a member of the immunoglobulin superfamily, binds heparin

The G6b gene, located in the human Major Histocompatibility Complex, encodes a receptor of the immunoglobulin (Ig) superfamily. In this study, we show using a variety of techniques that the extracellular domain of the G6b protein, containing a single Ig-like domain, binds to heparin with high affinity. In an ELISA assay, this binding was displaceable with soluble heparin with an IC50 value of approximately 0.5 microg/ml. Other sulfated glycans showed weaker or no competition. The observed interaction between G6b and heparin is strongly salt dependent suggesting a mainly electrostatic interaction. Heparin might modulate the interaction of G6b with its as yet unidentified protein ligand.     

Evidence for the occurrence of calmodulin in the yeasts Candida albicans and Saccharomyces cerevisiae

The lectin extracted from Vicia graminea seeds has been purified by conventional techniques but such procedures did not give a satisfactory yield. We describe a new purification which involves 3 steps after obtention of the crude extract. The first step is based on affinity chromatography on con A—Sepharose. Further purification steps were performed on DEAE-Sephacel chromatography and ultrogel AcA₄₄ gel filtration. The homogeneity of the lectin was demonstrated by polyacrylamide gel electrophoresis. Purification of the lectin by this new method was less time consuming, the yield was higher and the specific activity increased.     

ERK phosphorylation and tumor necrosis factor-alpha production by monocytes are persistent in response to immobilized IgG

Engagement of Fcγ receptors on leukocytes by immune complexes induces both cytokine production and immune complex internalization. The relationship between these processes is unclear. In many disease states, Fcγ receptors encounter their ligands in deposited forms that cannot be readily internalized. In this study, we examined the kinetics of ERK1/2 phosphorylation and TNF-α secretion in primary human monocytes in response to soluble heat-aggregated IgG or surface-bound IgG, to mimic soluble immune complexes and tissue-deposited IgG, respectively. Soluble aggregated IgG induced transient signaling, leading to peak phosphorylation of ERK1/2 by 15 min and peak TNF-α levels by 1 h, whereas surface-bound IgG caused sustained responses over the course of several hours. Treatment with the vacuolar ATPase inhibitor bafilomycin led to increased persistence of ERK1/2 phosphorylation in response to aggregated IgG. When monocytes were incubated with both soluble aggregated IgG and surface-bound IgG simultaneously, ERK1/2 phosphorylation was transient. These results suggest that Fcγ receptor internalization is an important step in termination of inflammatory signaling, and that small immune complexes can exert an overall down-modulatory effect when encountered in the presence of immobilized IgG.     

A reducing and denaturing step maximizes the immunoprecipitations of m-calpain and I-2(PP2A)/SET: an approach toward antibodies that do not work well in immunoprecipitation

Immunoprecipitation is an elegant method to isolate a specific protein of interest from a complex protein mixture such as cell lysate. We tried to increase the efficiency of m-calpain immunoprecipitation with anti-m-calpain antibodies directed toward denatured antigens that only work for immunoblotting and immunohistochemistry. We found that a reducing and denaturing step prior to immunoprecipitation greatly potentiates the efficiency of the immunoreaction. This improved method is also applicable for the immunoprecipitation of oncoprotein I-2(PP2A)/SET with antibodies directed toward a synthetic peptide that only work for immunoblotting. Thus, our improved method provides a way to maximize immunoprecipitation when using antibodies that do not work well under conventional immunoprecipitation conditions. Furthermore, the improved method is also suitable for decreasing the contaminating proteins during immunoprecipitation.     

Autolysis of a novel multidomain subtilase-cold-adapted deseasin MCP-01 is pH-dependent and the surface loops in its catalytic domain, the linker, and the P_proprotein domain are susceptible to proteolytic attack

Cold-adapted deseasin MCP-01 is a novel type subtilase with a multidomain structure containing a catalytic domain, a linker, a P_proprotein domain, and a PKD domain. Its autolysis was pH-dependent due to its flexible structure. N-terminal sequence analysis of the autolytic peptides revealed four autolytic sites in the catalytic domain. Three of these are in the same loops as mesophilic subtilases and one is unlike anything previously reported. Two autolytic sites were deduced in its linker and three in its P_proprotein domain, indicating the linker and the P_proprotein domain are flexible and susceptible to proteolytic attacks. Therefore, during MCP-01 autolysis, the linker and the P_proprotein domain of MCP-01 were easily attacked by proteolysis, resulting in cleavage of the C-terminal region. At the same time, some autolytic sites in the surface loops of the catalytic domain were cleaved. This is the first report describing the autolytic mechanism of a multidomain subtilase.     

Actin protein up-regulated upon infection and development of the filarial parasite, Wuchereria bancrofti (Spirurida: Onchocercidae), in the vector mosquito, Culex quinquefasciatus (Diptera: Culicidae)

Detection and identification of humoral proteins, which are up-regulated in Culex quinquefasciatus upon infection by Wuchereria bancrofti, is important in tracing out the biochemical consequences of the filarial parasite development in the vector mosquito. Analysis of the haemolymph of infected mosquitoes through SDS-PAGE and RP-HPLC showed up-regulation of five proteins of molecular weights 40, 66, 22, 14, and 7-kDa. Among these, only the 40-kDa was unknown and the others were comparable with those already reported as transferrin, attacin, lysozyme, and defensin, respectively. In the present study, the 40-kDa protein up-regulated upon infection was identified as actin through nano-LC-MS/MS analysis. Actin is known to be one of the cytoskeletal proteins up-regulated in the haemolymph, as part of the innate immune system, of Escherichia coli challenged Drosophila melanogaster larvae. For the first time, we have observed an increased level of actin in the haemolymph of W. bancrofti-infected Cx. quinquefasciatus. However, the exact mechanism of actin involvement in the immune system of this mosquito is yet to be studied.     

Cell surface of the fish pathogenic bacterium Aeromonas salmonicida: II. Purification and characterization of a major cell envelope protein related to autoagglutination,adhesion and virulence

The purification of the major protein of the membrane fraction of an autoagglutinating strain of Aeromonas salmonicida is described. This protein, designated as additional cell envelope protein, is water-insoluble, has a molecular weight of about 54 000 and its amino terminal sequence is H₂N-Asp-Val-Leu-Leu. Neither sulphur-containing amino acids nor sugar residues were detected. Its amino acid composition, which shows that the additional cell envelope protein is hydrophobic in nature, is remarkably similar to those of various proteins known to be present in additional surface layers of other bacteria, to the adhesive K88 fimbriae of enteropathogenic Escherichia coli and to a pore protein of the outer membrane of E. coli K12.     

Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes

(³H)Cytochalasin B has been photoincorporated into membrane fractions of the human erythrocyte, Rous sarcoma virus-transformed chicken embryo fibroblast and rat adipocyte. Identification of D-glucose sensitive cytochalasin B binding sites was achieved by photolyzing membranes with radioligand in the presence of 0.5–0.7M D- or L-glucose. In the erythrocyte the major labeled bands on SDS-polyacrylamide gels were at 55,000 and 46,000 daltons. In the virus-transformed fibroblasts a major labeled band was at 55,000 daltons, and in adipocyte microsomal membranes, peaks at 50,000 and 45,000 daltons were observed. Binding characteristics of these polypeptides suggest that they are the putative glucose transport proteins in these three cell types.     

Purification and immunochemical characterization of human adrenal tyrosine hydroxylase

Tyrosine hydroxylase (TH) was purified from the soluble fraction of human adrenal glands. The enzyme in human adrenal glands that was purified to apparent homogeneity had an apparent M_r of about 280,000. Sodium dodecyl sulfate (SDS) gel electrophoresis gave a single band with a M_r of 60,000 similar to the M_r of bovine adrenal enzyme. The enzyme is considered to be composed of four identical subunits. The specific activity of the final preparation was approximately 310 nmol 3,4-dihydroxyphenylalanine (DOPA) formed/min/mg protein. The use of the “Western Blot” method showed that human adrenal TH did not aggregate as rapidly as bovine adrenal TH.     

Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1s, C1r2-C1s2 and C1

Photosynthetic membranes contain considerable regions of high surface curvature, notably at their margins, where the average radius of curvature is about 10 nm. The proportion of total membrane lipid in the outer and inner thylakoid margin monolayers is estimated at 21% and 13%, respectively. The major thylakoid lipid, monogalactosyldiacylglycerol, is roughly cone-shaped and will not form complete lamellar bilayer phases, even in combination with other thylakoid lipids. It is proposed that this galactolipid plays a role in: (a) stabilising regions of concave curvature in thylakoids; and (b) packaging hydrophobic proteins in planar bilayer regions by means of inverted micelles. This model predicts substantial asymmetries in the distribution of lipids both across and along the thylakoid bilayer plane.