BACKGROUND Antinuclear antibodies(ANAs)are crucial in diagnosing autoimmune diseases,mainly systemic lupus erythematosus(SLE).This study aimed to compare the performance of chemiluminescence assay(CLIA)and line immuno...BACKGROUND Antinuclear antibodies(ANAs)are crucial in diagnosing autoimmune diseases,mainly systemic lupus erythematosus(SLE).This study aimed to compare the performance of chemiluminescence assay(CLIA)and line immunoassay(LIA)in detecting ANAs in patients with autoimmune diseases,evaluate their diagnostic accuracy for SLE,and develop a novel diagnostic model using CLIA-detected antibodies for SLE.Specimens from patients with autoimmune diseases and physical examination specimens were collected to parallel detect specific antibodies.Individual antibodies'diagnostic performance and a model combining multiple antibodies were assessed.The findings provide valuable insights into improving the diagnosis of SLE through innovative approaches.AIM To compare the performance of CLIA and LIA in detecting ANAs in patients with autoimmune diseases,assess their accuracy for SLE,and develop a novel diagnostic model using CLIA-detected antibodies for SLE.METHODS Specimens have been obtained from 270 patients with clinically diagnosed autoimmune disorders,as well as 130 physical examination specimens.After that,parallel detection of anti-double-stranded DNA(dsDNA)antibody,anti-histone(Histone)antibody,anti-nucleosome(Nuc)antibody,anti-Smith(Sm)antibody,anti-ribosomal P protein(Rib-P)antibody,anti-sicca syndrome A(Ro60)antibody,anti-sicca syndrome A(Ro52)antibody,anti-sicca syndrome(SSB)antibody,anticentromere protein B(Cenp-B)antibody,anti-DNA topoisomerase 1(Scl-70)antibody,anti-histidyl tRNA synthetase(Jo-1)antibody,and anti-mitochondrial M2(AMA-M2)antibody was performed using CLIA and LIA.The detection rates,compliance rates,and diagnostic performance for SLE were compared between the two methodologies,followed by developing a novel diagnostic model for SLE.RESULTS CLIA and LIA exhibited essentially comparable detection rates for anti-dsDNA antibody,anti-Histone antibody,anti-Nuc antibody,anti-Sm antibody,anti-Rib-P antibody,anti-Ro60 antibody,anti-Ro52 antibody,anti-SSB antibody,anti-Cenp-B antibody,anti-DNAScl-70 antibody,anti-Jo-1 antibody and anti-AMA-M2 antibody(P>0.05).The two methods displayed identical results for the detection of anti-dsDNA antibody,anti-Histone antibody,anti-Nuc antibody,anti-Sm antibody,anti-Ro60 antibody,anti-Ro52 antibody,anti-SSB antibody,anti-Cenp-B antibody,anti-Scl-70 antibody,and anti-AMA-M2 antibody(Kappa>0.7,P<0.05),but showed a moderate agreement for the detection of anti-Rib-P antibody and anti-Jo-1 antibody(Kappa=0.671 and 0.665;P<0.05).In addition,the diagnostic performance of these antibodies detected by both methods was similar for SLE.The diagnostic model's area under the curve values,sensitivity,and specificity,including an anti-dsDNA antibody and an anti-Ro60 antibody detected by CLIA,were 0.997,0.962,and 0.978,respectively.These values were higher than the diagnostic performance of individual antibodies.CONCLUSION CLIA and LIA demonstrated excellent overall consistency in detecting ANA profiles.A diagnostic model based on CLIA-detected antibodies can successfully contribute to developing a novel technique for detecting SLE.展开更多
Using western immunoblotting, we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimp Fenneropenaeus chinensis during thermal and immune-challenged stresses. This is ...Using western immunoblotting, we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimp Fenneropenaeus chinensis during thermal and immune-challenged stresses. This is probably the first report of the effects of various stressors on the expression of HSP70 in shrimp. HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, when the shrimp were exposed to heat shock and Vibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10℃ above ambient temperature.展开更多
Serological tests for antibodies specific for Epstein-Barr virus(EBV) antigens are frequently used to define infection status and for the differential diagnosis of other pathogens responsible for mononucleosis syndrom...Serological tests for antibodies specific for Epstein-Barr virus(EBV) antigens are frequently used to define infection status and for the differential diagnosis of other pathogens responsible for mononucleosis syndrome. Using only three parameters [viral capsid antigen(VCA) Ig G, VCA Ig M and EBV nuclear antigen(EBNA)-1 Ig G],it is normally possible to distinguish acute from past infection: the presence of VCA Ig M and VCA Ig G without EBNA-1 Ig G indicates acute infection, whereas the presence of VCA Ig G and EBNA-1 Ig G without VCA Ig M is typical of past infection. However, serological findings may sometimes be difficult to interpret as VCA Ig G can be present without VCA Ig M or EBNA-1 Ig G in cases of acute or past infection, or all the three parameters may be detected simultaneously in the case of recent infection or during the course of reactivation. A profile of isolated EBNA-1 Ig G may also create some doubts. In order to interpret these patterns correctly, it is necessary to determine Ig G avidity, identify anti-EBV Ig G and Ig M antibodies by immunoblotting, and look for heterophile antibodies, anti-EA(D) antibodies or viral genome using molecular biology methods. These tests make it possible to define the status of the infection and solve any problems that may arise in routine laboratory practice.展开更多
Bisperoxo(1,10-phenanthroline) oxovanadate(BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases(DNMTs), which would impact the cel...Bisperoxo(1,10-phenanthroline) oxovanadate(BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases(DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells(HT_(22)) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT_(22) cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT_(22) cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT_(22) cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.展开更多
Objective:To characterize the major allergens of Macrobrachium rosenbergii.(giant freshwater prawn).Methods:Raw and cooked extracts of the giant freshwater prawn were prepared.The IgE reactivity pattern was identified...Objective:To characterize the major allergens of Macrobrachium rosenbergii.(giant freshwater prawn).Methods:Raw and cooked extracts of the giant freshwater prawn were prepared.The IgE reactivity pattern was identified by sodium dodecyl sulfate polyaerylamide gel electrophoresis(SDS-PAGE)and immunoblotting technique with the sera of 20 skin prick test(SPT)positive patients.The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional(2-DE)electrophoresis,mass spectrometry and bioinformatics tools.Results:SDS-PAGE of the raw extract showed 23 protein bands(15-250 kDa)but those ranging from 40 to 100 kDa were not found in the cooked extract.From immunoblotting experiments,raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins,respectively,with a molecular mass ranging from 15 to 155 kDa.A heat-resistant 36 kDa protein was identified as the major allergen of both extracts.In addition,a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract.The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots.Of these,10 spots showed specific:IgE reactivity with patients'sera.Matrix assisted laser desorption/ionization-lime of flight(MALDI-TOF)analysis led to identification of 2 important allergens,tropomyosin and arginine kinase.Conclusions:It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.展开更多
Merozoite surface antigens can induce protective immune responses and may becandidate antigens for malaria vaccine.Four hybridoma cell lines secreting monoclonalantibodies against Plasmodium falciparum Fcc7801/HN were...Merozoite surface antigens can induce protective immune responses and may becandidate antigens for malaria vaccine.Four hybridoma cell lines secreting monoclonalantibodies against Plasmodium falciparum Fcc7801/HN were produced.Antibodies fromthree of the four lines showed significant growth-inhibiting effect on P.falciparum invitro.One monoclonal antibody,known as C6,conjugated the antigen located exclusivelyon the merozoite surface and distributed evenly over the entire surface,as wasdemonstrated by immunoelectron microscopy.C6 also precipitated a single protein of Mr71000.展开更多
OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The...OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The cells were serum starved and treated with either human relaxin-3(H3relaxin)(10nmol·L-1),R3B1-22R(10μmol·L-1)and pertussis toxin(PTX,100ng·mL-1).The cells were lysed and the ERK1/2 activation was determined by SDS-PAGE followed by immunoblotting for phosphorylated ERK1/2(pERK1/2)and total ERK1/2(tERK1/2)for the lysates.RESULTSThe quantification of the data revealed that the peak of ERK1/2activation can be detected precisely at 10 min post stimulation with 10nmol·L-1 H3 relaxin in both HEK-RXFP3 and CHO-RXFP3 cell lines in all three trials compared to the cells treated with vehicle(P<0.05).However,HEK-RXFP3 cells demonstrated a transient activation of ERK1/2and CHORXFP3 cells demonstrated a continuous activation of ERK1/2which was inhibited by the Gi inhibitor,PTX.Activation of ERK1/2was significantly inhibited by pre-treating the cells with RXFP3 antagonist R3B1-22 Rin HEK-RXFP3 cells.ERK1/2 activation was observed neither in wild type HEK293 Tnor in CHO-K1 cells.CONCLUSION The developed assay can detect RXFP3 activation or inhibition by agonists and antagonists via the detection of pERK1/2 in multiple cell lines.This assay will also be useful to detect signaling pathways upstream to ERK1/2 activation mediated by RXFP3.Activation of ERK1/2 in CHO-RXFP3 cells was mediated by Gi proteins at 10 min as well as at 25-35 min time points.展开更多
Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and...Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. This would help to obtain definitive early diagnosis of TB. The present study was developed to determine the role of using early secreted antigenic target-6 (ESAT-6) in immunodiagnosis of Mycobacterium tuberculosis. Methods: Serum samples were obtained from TB infected patients and normal healthy controls. Two rapid immunodiagnostic assays (Enzyme-linked immunosorbent assay (ELISA) and Immunoblotting) were performed. Results: The sensitivity of immunoblotting assay was 100%;however, ESAT-6 antigen was not able to discriminate between patients and normal controls. Application of direct ELISA using ESAT-6 antigen yielded 97.6% sensitivity and 75% specificity for the diagnosis of TB infection. Conclusion: In conclusion, the detection of antibodies against ESAT-6 antigen in the sera of TB patients by direct ELISA could be used as a preliminary assay for diagnosis of human M. tuberculosis infection. A combination of the ELISA with either radiological or microscopic examination is required to overcome the low specificity of the assay for negative results.展开更多
Objective:To determine the profdes of anti-Entamoeba histolytica(E.histolytica) IgA.IgC. and IgM in sera of diarrheic and non-diarrheic individuals and partially characterize target antigens.Methods:Serum samples from...Objective:To determine the profdes of anti-Entamoeba histolytica(E.histolytica) IgA.IgC. and IgM in sera of diarrheic and non-diarrheic individuals and partially characterize target antigens.Methods:Serum samples from thirty diarrheic and thirty non-diarrheic individuals were subjected to IgA,IgG,and IgM profiling through enzyme-linked immunosorbent assay (EI.ISA),flow cytometry,and immunoblot.Results:ELBA titer results showed that both diarrheic and non-diarrheic individuals possess high levels of E.histolytica-specific IgG compared to IgA and IgM.How cytometry data showed that diarrheic serum samples had higher mean reaction percentages against E.histolytica cells compared to non-diarrheic samples.Immunoreactive E.histolytica proteins with molecular weights ranging belween 7 kDa and 292 kDa were recognized by diarrheic serum IgG,and 170 kDa and 250 kDa by non-diarrheic serum IgG. Conclusions:Our findings suggest that serum anti-E.histolytica IgG,compared with serum anti-E.histolytica IgA and IgM responses,was generally high in both diarrheic and nondiarrheic sera,indicating a past exposure to the organism both in symptomatic patients as well as in asymptomatic carriers,respectively.In addition,serum IgG from diarrheic and non-diarrheic patients were able to detect immunogenic E.histolytica proteins.展开更多
Twenty patients with chronic hepatitis c were investigated during the treatment with interferon to explore the changes of antibodies to HCV (anti-RCV). Anti-HCV was tested with recombinant immunoblot assay (RIBA) by u...Twenty patients with chronic hepatitis c were investigated during the treatment with interferon to explore the changes of antibodies to HCV (anti-RCV). Anti-HCV was tested with recombinant immunoblot assay (RIBA) by using three antigens (C22, C33c, and C100-3) encoded by different regions of HCV genome. The changes of individual anti-HCV and ALT were compared with the change of HCV RNA. The results showed that persistent disappearance of serum HCV RNA was closely related to the changes of anti-C33c (P<0. 01) and anti-C100-3 (P<0. 005), but there was no relation between persistent ALT normality and HCV viremia clearance (P<0. 05). In conclusion, monitoring anti-C33c and anti-C100-3 could indicate the changes or HCV viremia. The normalization of ALT after interferon treatment did not indicate disappearance of HCV viremia.展开更多
Forty-one patients with age-related macular degeneration(AMD) were detected for serum autoantibodies against normal humanretinal protein by means of Western immunoblot analysis.Twenty-sevenout of the 41 patients showe...Forty-one patients with age-related macular degeneration(AMD) were detected for serum autoantibodies against normal humanretinal protein by means of Western immunoblot analysis.Twenty-sevenout of the 41 patients showed positive response,with a rate of 66 percent.The positive rate of antiretinal antibody in the AMD patients wassignificantly higher than that in normal controls (18%) and in patients withother retinal diseases (24%) (p<0.0005).These antiretinal antibodies fromthe AMD patients partly reacted...展开更多
Background:Tall fescue(Festuca arundinacea[Schreb.],Lolium arundinaceum[Schreb.]Darbysh)and perennial ryegrass(Lolium perenne)are important cool-season forage and amenity grasses that have a mutualistic association wi...Background:Tall fescue(Festuca arundinacea[Schreb.],Lolium arundinaceum[Schreb.]Darbysh)and perennial ryegrass(Lolium perenne)are important cool-season forage and amenity grasses that have a mutualistic association with an endophytic fungus.Endophytes confer insect and drought resistance to plants but can produce mammalian toxins.Novel endophytes that do not produce mammalian toxins have been introduced to elite cultivars for commercial production.Seed companies need to maintain adequate levels of novel endophytes within the elite forage cultivars.Endophyte detection is performed using immunochemical and molecular techniques because of their speed and reliability.Early detection in seedlings is essential to evaluate the viability of the endophyte within seed lots.Methods:This research aimed to identify the earliest growth stage in which immunochemical and molecular methods can detect viable endophyte in seedlings of tall fescue cultivars BarOptima(e34),Texoma MaxQII(584),and Jesup MaxQ(542),as well as the perennial ryegrass cultivar Remington(NEA2).Results:Immunochemical testing detected endophytes in seedlings 14 days after germination(DAG),but the detection rate increased until 42 DAG in some cultivars tested.The molecular marker Tef1exon detected endophytes at a lower rate than the immunochemical method at 28–42 DAG.However,there was insufficient DNA to detect endophytes in 14 DAG seedlings using markers.Conclusions:We conclude that the most accurate detection of viable endophytes in seedlings was 42 DAG,at which sufficient and consistent endophyte colonization occurred.展开更多
Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sink-organ development and the sink strength modulation in crops. The experiment conducted with 'Starkrimson' apple (Malus do...Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sink-organ development and the sink strength modulation in crops. The experiment conducted with 'Starkrimson' apple (Malus domestica Borkh) fruit showed that, during the fruit development, the activity of acid invertase gradually declined concomitantly with the progressive accumulation of fructose, glucose and sucrose, while Western blotting assay of acid invertase detected a 30 ku peptide of which the immuno-signal intensity increased during the fruit development. The immuno-localization via immunogold electron microscopy showed that, on the one hand, acid invertase was mainly located on the flesh cell wall with numbers of the immunosignals present in the vacuole at the late stage of fruit development; and on the other hand, the amount of acid invertase increased during fruit development, which was consistent with the results of Western blotting. The in vivo pre-incubation of fruit discs with soluble sugars showed that the activity of extractible acid invertase was inhibited by fructose or glucose, while Western blotting did not detect any changes in apparent quantity of the enzyme nor other peptides than 30 ku one. So it is considered that fructose and glucose induced the post-translational or translocational inhibitory regulation of acid invertase in developing apple fruit. The mechanism of the post-translational inhibition was shown different from both the two previously reported ones that proposed either the inhibition by hexose products in the in vitro chemical reaction equilibrium system or the inhibition by the proteinaceous inhibitors. It was hypothesized that fructose and glucose might induce acid invertase inhibition by modulating the expression of some inhibition-related genes or some structural modification of acid invertase.展开更多
The vaccinia viral vector containing T7 promoter was used to construct the expression plasmids carrying HCV structural genes of C, E1 and E2/NS1. These genes were transiently expressed in mammalian cells in the presen...The vaccinia viral vector containing T7 promoter was used to construct the expression plasmids carrying HCV structural genes of C, E1 and E2/NS1. These genes were transiently expressed in mammalian cells in the presence of the T7 RNA polymerase which was provided by the recombinant vaccinia virus vTT7. Expression of mature core protein, envelope protein E1 and E2 was detected by Western blot using HCV patient sera as the primary antibodies. It was found that the sera from different HCV patients reacted differently with the expressed products, so did the sera collected at different times from the same patient, from whom the HCV structural genes were isolated. Among six mammalian cell lines, Vero and HeLa were the most suitable for the expression of C, E1 and E2. The recombinant vaccinia viruses have been constructed to constantly produce the C, E1 and E2 proteins for further research.展开更多
Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peri...Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to IgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T.aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye),Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.展开更多
It has been found that the fourth component of human complement （C4） is a β1E globulin with a sedimentation coefficient of 10S and its average concentration in human serum is 430 μg/ml. The molecular weight of nat...It has been found that the fourth component of human complement （C4） is a β1E globulin with a sedimentation coefficient of 10S and its average concentration in human serum is 430 μg/ml. The molecular weight of native C4 was estimated to be 202,000 and is composed of three disulfide-linked polypeptide chains, α, β and γ,展开更多
Objective To study the expression and significance of COX-2 and C/EBP-βin refractory sinusitis with nasal polyps,and to explore the relationship between them and the recurrence of sinusitis with nasal polyps.Methods ...Objective To study the expression and significance of COX-2 and C/EBP-βin refractory sinusitis with nasal polyps,and to explore the relationship between them and the recurrence of sinusitis with nasal polyps.Methods The protein expression of COX-2 and C/EBP-βin 20 cases of refractory sinusitis with nasal polyps,20 cases of sinusitis with nasal polyps and 20 cases of normal nasal mucosa were detected by western blot,and the relationship between the two was compared.Results The expression levels of COX-2 and C/EBP-βin refractory sinusitis with nasal polyps were significantly different from those in refractory sinusitis with nasal polyps(P<0.05);The expression levels of COX-2 and C/EBP-βin sinusitis tissues with nasal polyps were significantly different from those in normal nasal mucosa tissues(P<0.05);The expression levels of COX-2 and C/EBP-βin each group were significantly correlated(P<0.05).Conclusions The high expression of COX-2 and C/EBP-βmay be closely related to postoperative recurrence of sinusitis patients with nasal polyps.Both may be used as objective indicators to judge the postoperative follow-up and recurrence tendency of patients with sinusitis with nasal polyps..展开更多
文摘BACKGROUND Antinuclear antibodies(ANAs)are crucial in diagnosing autoimmune diseases,mainly systemic lupus erythematosus(SLE).This study aimed to compare the performance of chemiluminescence assay(CLIA)and line immunoassay(LIA)in detecting ANAs in patients with autoimmune diseases,evaluate their diagnostic accuracy for SLE,and develop a novel diagnostic model using CLIA-detected antibodies for SLE.Specimens from patients with autoimmune diseases and physical examination specimens were collected to parallel detect specific antibodies.Individual antibodies'diagnostic performance and a model combining multiple antibodies were assessed.The findings provide valuable insights into improving the diagnosis of SLE through innovative approaches.AIM To compare the performance of CLIA and LIA in detecting ANAs in patients with autoimmune diseases,assess their accuracy for SLE,and develop a novel diagnostic model using CLIA-detected antibodies for SLE.METHODS Specimens have been obtained from 270 patients with clinically diagnosed autoimmune disorders,as well as 130 physical examination specimens.After that,parallel detection of anti-double-stranded DNA(dsDNA)antibody,anti-histone(Histone)antibody,anti-nucleosome(Nuc)antibody,anti-Smith(Sm)antibody,anti-ribosomal P protein(Rib-P)antibody,anti-sicca syndrome A(Ro60)antibody,anti-sicca syndrome A(Ro52)antibody,anti-sicca syndrome(SSB)antibody,anticentromere protein B(Cenp-B)antibody,anti-DNA topoisomerase 1(Scl-70)antibody,anti-histidyl tRNA synthetase(Jo-1)antibody,and anti-mitochondrial M2(AMA-M2)antibody was performed using CLIA and LIA.The detection rates,compliance rates,and diagnostic performance for SLE were compared between the two methodologies,followed by developing a novel diagnostic model for SLE.RESULTS CLIA and LIA exhibited essentially comparable detection rates for anti-dsDNA antibody,anti-Histone antibody,anti-Nuc antibody,anti-Sm antibody,anti-Rib-P antibody,anti-Ro60 antibody,anti-Ro52 antibody,anti-SSB antibody,anti-Cenp-B antibody,anti-DNAScl-70 antibody,anti-Jo-1 antibody and anti-AMA-M2 antibody(P>0.05).The two methods displayed identical results for the detection of anti-dsDNA antibody,anti-Histone antibody,anti-Nuc antibody,anti-Sm antibody,anti-Ro60 antibody,anti-Ro52 antibody,anti-SSB antibody,anti-Cenp-B antibody,anti-Scl-70 antibody,and anti-AMA-M2 antibody(Kappa>0.7,P<0.05),but showed a moderate agreement for the detection of anti-Rib-P antibody and anti-Jo-1 antibody(Kappa=0.671 and 0.665;P<0.05).In addition,the diagnostic performance of these antibodies detected by both methods was similar for SLE.The diagnostic model's area under the curve values,sensitivity,and specificity,including an anti-dsDNA antibody and an anti-Ro60 antibody detected by CLIA,were 0.997,0.962,and 0.978,respectively.These values were higher than the diagnostic performance of individual antibodies.CONCLUSION CLIA and LIA demonstrated excellent overall consistency in detecting ANA profiles.A diagnostic model based on CLIA-detected antibodies can successfully contribute to developing a novel technique for detecting SLE.
基金Supported by the National Key Fundamental Research Program"Study of the Major Diseases and Resistance Mechanism of Mariculture Organisms",NSFC,No.30140017the international cooperative program"Immunaqua"by the European Commisssion,No.ICA4-CT-2001-10023
文摘Using western immunoblotting, we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimp Fenneropenaeus chinensis during thermal and immune-challenged stresses. This is probably the first report of the effects of various stressors on the expression of HSP70 in shrimp. HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, when the shrimp were exposed to heat shock and Vibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10℃ above ambient temperature.
文摘Serological tests for antibodies specific for Epstein-Barr virus(EBV) antigens are frequently used to define infection status and for the differential diagnosis of other pathogens responsible for mononucleosis syndrome. Using only three parameters [viral capsid antigen(VCA) Ig G, VCA Ig M and EBV nuclear antigen(EBNA)-1 Ig G],it is normally possible to distinguish acute from past infection: the presence of VCA Ig M and VCA Ig G without EBNA-1 Ig G indicates acute infection, whereas the presence of VCA Ig G and EBNA-1 Ig G without VCA Ig M is typical of past infection. However, serological findings may sometimes be difficult to interpret as VCA Ig G can be present without VCA Ig M or EBNA-1 Ig G in cases of acute or past infection, or all the three parameters may be detected simultaneously in the case of recent infection or during the course of reactivation. A profile of isolated EBNA-1 Ig G may also create some doubts. In order to interpret these patterns correctly, it is necessary to determine Ig G avidity, identify anti-EBV Ig G and Ig M antibodies by immunoblotting, and look for heterophile antibodies, anti-EA(D) antibodies or viral genome using molecular biology methods. These tests make it possible to define the status of the infection and solve any problems that may arise in routine laboratory practice.
基金supported by the National Natural Science Foundation of China,No.81160244,81360316,81460283,81660307(all to GS)the Inner Mongolia Science Foundation of China,No.2018LH08078(to GS),2016MS(LH)0307(to SYJ)+4 种基金the Baotou Health Foundation,China,No.WSJJ2016008(to SYJ)the Inner Mongolia Educational Research Foundation of China,No.NJZY207(to GS),NJZY17243(to SCY),NJZY17250(to XLL),NJZY17251(to SYJ)the Baotou Medical College Foundation of China,No.BYJJ-DF201602,BYJJ-YF201615,BSJJ201617,BYJJ-QM201633,BYJJ-QM201656,BYJJ201502(to GS)the Science and Technology Planning Project of Baotou of China,No.CX2017-5(to GS)the National Key R&D Program of China,No.2017YFC1308405(to GS)
文摘Bisperoxo(1,10-phenanthroline) oxovanadate(BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases(DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells(HT_(22)) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT_(22) cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT_(22) cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT_(22) cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.
基金Supported by a research grant from UPSI(grant No.UPSI2011-0018-102-01)
文摘Objective:To characterize the major allergens of Macrobrachium rosenbergii.(giant freshwater prawn).Methods:Raw and cooked extracts of the giant freshwater prawn were prepared.The IgE reactivity pattern was identified by sodium dodecyl sulfate polyaerylamide gel electrophoresis(SDS-PAGE)and immunoblotting technique with the sera of 20 skin prick test(SPT)positive patients.The major allergen identified was then characterized using the proteomics approach involving a combination of two-dimensional(2-DE)electrophoresis,mass spectrometry and bioinformatics tools.Results:SDS-PAGE of the raw extract showed 23 protein bands(15-250 kDa)but those ranging from 40 to 100 kDa were not found in the cooked extract.From immunoblotting experiments,raw and cooked extracts demonstrated 11 and 5 IgE-binding proteins,respectively,with a molecular mass ranging from 15 to 155 kDa.A heat-resistant 36 kDa protein was identified as the major allergen of both extracts.In addition,a 42 kDa heat-sensitive protein was shown to be a major allergen of the raw extract.The 2-DE gel fractionated the prawn proteins to more than 50 different protein spots.Of these,10 spots showed specific:IgE reactivity with patients'sera.Matrix assisted laser desorption/ionization-lime of flight(MALDI-TOF)analysis led to identification of 2 important allergens,tropomyosin and arginine kinase.Conclusions:It can be concluded that the availability of such allergens would help in component-based diagnosis and therapy of prawn allergies.
基金The project was supported by the National Natural Science Foundation of China,No 3860869
文摘Merozoite surface antigens can induce protective immune responses and may becandidate antigens for malaria vaccine.Four hybridoma cell lines secreting monoclonalantibodies against Plasmodium falciparum Fcc7801/HN were produced.Antibodies fromthree of the four lines showed significant growth-inhibiting effect on P.falciparum invitro.One monoclonal antibody,known as C6,conjugated the antigen located exclusivelyon the merozoite surface and distributed evenly over the entire surface,as wasdemonstrated by immunoelectron microscopy.C6 also precipitated a single protein of Mr71000.
基金The project supported by the Biomedical Research Council of Singapore(BMRC 10/1/21/19/645)the National Medical Research Council of Singapore(NMRC/1287/2011)the Ministry of Education,Singapore,Academic Research Fund Tier 1Seed Fund for Basic Science Research(T1-BSRG 2014-03)
文摘OBJECTIVE To develop ERK1/2 activation assays to detect RXFP3 activation or inhibition by its agonists or antagonists.METHODS Plated HEK-RXFP3,CHO-RXFP3,HEK293 Tand CHO-K1 cells in poly-L-lysine coated well plates.The cells were serum starved and treated with either human relaxin-3(H3relaxin)(10nmol·L-1),R3B1-22R(10μmol·L-1)and pertussis toxin(PTX,100ng·mL-1).The cells were lysed and the ERK1/2 activation was determined by SDS-PAGE followed by immunoblotting for phosphorylated ERK1/2(pERK1/2)and total ERK1/2(tERK1/2)for the lysates.RESULTSThe quantification of the data revealed that the peak of ERK1/2activation can be detected precisely at 10 min post stimulation with 10nmol·L-1 H3 relaxin in both HEK-RXFP3 and CHO-RXFP3 cell lines in all three trials compared to the cells treated with vehicle(P<0.05).However,HEK-RXFP3 cells demonstrated a transient activation of ERK1/2and CHORXFP3 cells demonstrated a continuous activation of ERK1/2which was inhibited by the Gi inhibitor,PTX.Activation of ERK1/2was significantly inhibited by pre-treating the cells with RXFP3 antagonist R3B1-22 Rin HEK-RXFP3 cells.ERK1/2 activation was observed neither in wild type HEK293 Tnor in CHO-K1 cells.CONCLUSION The developed assay can detect RXFP3 activation or inhibition by agonists and antagonists via the detection of pERK1/2 in multiple cell lines.This assay will also be useful to detect signaling pathways upstream to ERK1/2 activation mediated by RXFP3.Activation of ERK1/2 in CHO-RXFP3 cells was mediated by Gi proteins at 10 min as well as at 25-35 min time points.
文摘Purpose: There is a need for a continued effort to develop rapid immunodiagnostic assays for tuberculosis (TB) infection with greater sensitivity and specificity that can be used in the field and in the laboratory and that can be formatted for use with multiple species. This would help to obtain definitive early diagnosis of TB. The present study was developed to determine the role of using early secreted antigenic target-6 (ESAT-6) in immunodiagnosis of Mycobacterium tuberculosis. Methods: Serum samples were obtained from TB infected patients and normal healthy controls. Two rapid immunodiagnostic assays (Enzyme-linked immunosorbent assay (ELISA) and Immunoblotting) were performed. Results: The sensitivity of immunoblotting assay was 100%;however, ESAT-6 antigen was not able to discriminate between patients and normal controls. Application of direct ELISA using ESAT-6 antigen yielded 97.6% sensitivity and 75% specificity for the diagnosis of TB infection. Conclusion: In conclusion, the detection of antibodies against ESAT-6 antigen in the sera of TB patients by direct ELISA could be used as a preliminary assay for diagnosis of human M. tuberculosis infection. A combination of the ELISA with either radiological or microscopic examination is required to overcome the low specificity of the assay for negative results.
基金supported by a research grant from the Natural Sciences Research Institute of the University of the Philippines(B10-09-2-03) to W.L.R.
文摘Objective:To determine the profdes of anti-Entamoeba histolytica(E.histolytica) IgA.IgC. and IgM in sera of diarrheic and non-diarrheic individuals and partially characterize target antigens.Methods:Serum samples from thirty diarrheic and thirty non-diarrheic individuals were subjected to IgA,IgG,and IgM profiling through enzyme-linked immunosorbent assay (EI.ISA),flow cytometry,and immunoblot.Results:ELBA titer results showed that both diarrheic and non-diarrheic individuals possess high levels of E.histolytica-specific IgG compared to IgA and IgM.How cytometry data showed that diarrheic serum samples had higher mean reaction percentages against E.histolytica cells compared to non-diarrheic samples.Immunoreactive E.histolytica proteins with molecular weights ranging belween 7 kDa and 292 kDa were recognized by diarrheic serum IgG,and 170 kDa and 250 kDa by non-diarrheic serum IgG. Conclusions:Our findings suggest that serum anti-E.histolytica IgG,compared with serum anti-E.histolytica IgA and IgM responses,was generally high in both diarrheic and nondiarrheic sera,indicating a past exposure to the organism both in symptomatic patients as well as in asymptomatic carriers,respectively.In addition,serum IgG from diarrheic and non-diarrheic patients were able to detect immunogenic E.histolytica proteins.
文摘Twenty patients with chronic hepatitis c were investigated during the treatment with interferon to explore the changes of antibodies to HCV (anti-RCV). Anti-HCV was tested with recombinant immunoblot assay (RIBA) by using three antigens (C22, C33c, and C100-3) encoded by different regions of HCV genome. The changes of individual anti-HCV and ALT were compared with the change of HCV RNA. The results showed that persistent disappearance of serum HCV RNA was closely related to the changes of anti-C33c (P<0. 01) and anti-C100-3 (P<0. 005), but there was no relation between persistent ALT normality and HCV viremia clearance (P<0. 05). In conclusion, monitoring anti-C33c and anti-C100-3 could indicate the changes or HCV viremia. The normalization of ALT after interferon treatment did not indicate disappearance of HCV viremia.
基金This work was Bupported by grants from Chinese Medical Board
文摘Forty-one patients with age-related macular degeneration(AMD) were detected for serum autoantibodies against normal humanretinal protein by means of Western immunoblot analysis.Twenty-sevenout of the 41 patients showed positive response,with a rate of 66 percent.The positive rate of antiretinal antibody in the AMD patients wassignificantly higher than that in normal controls (18%) and in patients withother retinal diseases (24%) (p<0.0005).These antiretinal antibodies fromthe AMD patients partly reacted...
基金The University of Georgia Cultivar Research and Development Fund。
文摘Background:Tall fescue(Festuca arundinacea[Schreb.],Lolium arundinaceum[Schreb.]Darbysh)and perennial ryegrass(Lolium perenne)are important cool-season forage and amenity grasses that have a mutualistic association with an endophytic fungus.Endophytes confer insect and drought resistance to plants but can produce mammalian toxins.Novel endophytes that do not produce mammalian toxins have been introduced to elite cultivars for commercial production.Seed companies need to maintain adequate levels of novel endophytes within the elite forage cultivars.Endophyte detection is performed using immunochemical and molecular techniques because of their speed and reliability.Early detection in seedlings is essential to evaluate the viability of the endophyte within seed lots.Methods:This research aimed to identify the earliest growth stage in which immunochemical and molecular methods can detect viable endophyte in seedlings of tall fescue cultivars BarOptima(e34),Texoma MaxQII(584),and Jesup MaxQ(542),as well as the perennial ryegrass cultivar Remington(NEA2).Results:Immunochemical testing detected endophytes in seedlings 14 days after germination(DAG),but the detection rate increased until 42 DAG in some cultivars tested.The molecular marker Tef1exon detected endophytes at a lower rate than the immunochemical method at 28–42 DAG.However,there was insufficient DNA to detect endophytes in 14 DAG seedlings using markers.Conclusions:We conclude that the most accurate detection of viable endophytes in seedlings was 42 DAG,at which sufficient and consistent endophyte colonization occurred.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 39730340, 39870487 & 30070532) the National Key Basic Research Special Funds, P. R. China (G1999011704).
文摘Acid invertase (EC 3.2.1.26) is one of the key enzymes involved in the carbohydrate sink-organ development and the sink strength modulation in crops. The experiment conducted with 'Starkrimson' apple (Malus domestica Borkh) fruit showed that, during the fruit development, the activity of acid invertase gradually declined concomitantly with the progressive accumulation of fructose, glucose and sucrose, while Western blotting assay of acid invertase detected a 30 ku peptide of which the immuno-signal intensity increased during the fruit development. The immuno-localization via immunogold electron microscopy showed that, on the one hand, acid invertase was mainly located on the flesh cell wall with numbers of the immunosignals present in the vacuole at the late stage of fruit development; and on the other hand, the amount of acid invertase increased during fruit development, which was consistent with the results of Western blotting. The in vivo pre-incubation of fruit discs with soluble sugars showed that the activity of extractible acid invertase was inhibited by fructose or glucose, while Western blotting did not detect any changes in apparent quantity of the enzyme nor other peptides than 30 ku one. So it is considered that fructose and glucose induced the post-translational or translocational inhibitory regulation of acid invertase in developing apple fruit. The mechanism of the post-translational inhibition was shown different from both the two previously reported ones that proposed either the inhibition by hexose products in the in vitro chemical reaction equilibrium system or the inhibition by the proteinaceous inhibitors. It was hypothesized that fructose and glucose might induce acid invertase inhibition by modulating the expression of some inhibition-related genes or some structural modification of acid invertase.
文摘The vaccinia viral vector containing T7 promoter was used to construct the expression plasmids carrying HCV structural genes of C, E1 and E2/NS1. These genes were transiently expressed in mammalian cells in the presence of the T7 RNA polymerase which was provided by the recombinant vaccinia virus vTT7. Expression of mature core protein, envelope protein E1 and E2 was detected by Western blot using HCV patient sera as the primary antibodies. It was found that the sera from different HCV patients reacted differently with the expressed products, so did the sera collected at different times from the same patient, from whom the HCV structural genes were isolated. Among six mammalian cell lines, Vero and HeLa were the most suitable for the expression of C, E1 and E2. The recombinant vaccinia viruses have been constructed to constantly produce the C, E1 and E2 proteins for further research.
文摘Wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 isolated by preparative SDS-PAGE are used as antigen to immunize BALB/c mice. Subcutaneous inoculation of the antigen is performed. The intra-peritoneal injection is completed 3 days before fusion with myeloma cell (SP2/0) via PEG-1500. The fusion cells are selected by indirect enzyme-linked immuno-sorbent assay (ELISA). Positive hybrid cells are further verified three times by limit dilution of the culture cells. A hybridoma cell line is successfully obtained. The monoclonal antibody belongs to IgG1 subclass. In immunoblotting, the antibody binds to all HMW-GS of T.aestivum cultivars, but does not bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences among the HMW glutenin subunits in wheat. The antibody also binds to HMW-GS storage proteins in Aegilops squarrosa and T. durum (durum wheat). Furthermore, it also binds to HMW storage proteins in Secale cereale (rye),Hordeum vulgare (barley). However, it never binds seed storage proteins in other cereals such as maize, oat, rice, foxtail millet, sorghum etc. The antigen determinant recognized by the antibody has been located within hexapeptide [PGQGQQ] or / and nonapeptide [GYYPTSPQQ] in the central repetitive region of HMW-GS.
文摘It has been found that the fourth component of human complement （C4） is a β1E globulin with a sedimentation coefficient of 10S and its average concentration in human serum is 430 μg/ml. The molecular weight of native C4 was estimated to be 202,000 and is composed of three disulfide-linked polypeptide chains, α, β and γ,
基金Health Science and Technology Program of Yunnan Province(No.2010NS034)Joint Special Project of Yunnan Provincial Science and Technology Department/Kunming Medical University Applied Basic Research(No.201901C07041)。
文摘Objective To study the expression and significance of COX-2 and C/EBP-βin refractory sinusitis with nasal polyps,and to explore the relationship between them and the recurrence of sinusitis with nasal polyps.Methods The protein expression of COX-2 and C/EBP-βin 20 cases of refractory sinusitis with nasal polyps,20 cases of sinusitis with nasal polyps and 20 cases of normal nasal mucosa were detected by western blot,and the relationship between the two was compared.Results The expression levels of COX-2 and C/EBP-βin refractory sinusitis with nasal polyps were significantly different from those in refractory sinusitis with nasal polyps(P<0.05);The expression levels of COX-2 and C/EBP-βin sinusitis tissues with nasal polyps were significantly different from those in normal nasal mucosa tissues(P<0.05);The expression levels of COX-2 and C/EBP-βin each group were significantly correlated(P<0.05).Conclusions The high expression of COX-2 and C/EBP-βmay be closely related to postoperative recurrence of sinusitis patients with nasal polyps.Both may be used as objective indicators to judge the postoperative follow-up and recurrence tendency of patients with sinusitis with nasal polyps..
