Clonorchis sinensis excretory–secretory products regulate migration and invasion in cholangiocarcinoma cells via extracellular signal-regulated kinase 1/2/nuclear factor-κB-dependent matrix metalloproteinase-9 expression

Matrix metalloproteinase-9 plays an important role in the invasion and metastasis of various types of cancer cells. We have previously reported that excretory–secretory products from Clonorchis sinensis increases matrix metalloproteinase-9 expression. However, the regulatory mechanisms through which matrix metalloproteinase-9 expression affects cholangiocarcinoma development remain unclear. In the current study, we examined the potential role of excretory–secretory products in regulating the migration and invasion of various cholangiocarcinoma cell lines. We demonstrated that excretory–secretory products significantly induced matrix metalloproteinase-9 expression and activity in a concentration-dependent manner. Reporter gene and chromatin immunoprecipitation assays showed that excretory–secretory products induced matrix metalloproteinase-9 expression by enhancing the activity of nuclear factor-kappa B. Moreover, excretory–secretory products induced the degradation and phosphorylation of IκBα and stimulated nuclear factor-kappa B p65 nuclear translocation, which was regulated by extracellular signal-regulated kinase 1/2. Taken together, our findings indicated that the excretory–secretory product-dependent enhancement of matrix metalloproteinase-9 activity and subsequent induction of IκBα and nuclear factor-kappa B activities may contribute to the progression of cholangiocarcinoma.     

应用双抗体夹心ELISA法定量检测人源破伤风毒素单克隆抗体中的IgG含量

采用山羊抗人IgG作为包被抗体,辣根过氧化物酶标记的山羊抗人IgG作为标记抗体,建立一种双抗体夹心法用于定量检测人源破伤风毒素单克隆抗体的IgG含量。以纯化的IgG作标准,用平行线法测得亲和层析纯化的人源破伤风毒素单克隆抗体G2的含量为0．512μg／ml,与分光光度法测得的结果基本一致。因而样品检测采用纯化G2作参考标准,制作标准曲线,测定了已知样品和未知样品的抗体含量。结果表明本法重复性好,特异性强,可定量测定培养及纯化样品中人源单克隆抗体的含量。     

Identification of proteins in excretory/secretory extracts of Echinostoma friedi (Trematoda) from chronic and acute infections

In the present study, we describe the investigation of Echinostoma friedi excretory/secretory products using a proteomic approach combined with the use of heterologous antibodies. We have identified 18 protein spots corresponding to ten proteins, including cytoskeletal proteins like actin, tropomyosin, and paramyosin; glycolytic enzymes like enolase, glyceraldehyde 3P dehydrogenase, and aldolase; detoxifying enzymes like GSTs; and stress proteins like heat shock protein (Hsp) 70. Among these proteins, both actin and, to a lesser extent, Hsp70, exhibited differential expression patterns between chronic and acute infections in the Echinostoma-rodent model, suggesting that these proteins may play a role in the survival within the host.     

小鼠17β-hsd10的克隆、表达及双抗夹心ELISA方法的建立

原核表达小鼠17β-羟基类固醇脱氢酶(17β-hsd10),制备抗17β-hsd10多克隆抗体,建立17β-hsd10双抗夹心酶联免疫检测方法 (Enzyme linked immunosorbent assay,ELISA)。采用RT-PCR技术克隆得到小鼠肝脏17β-hsd 10基因,构建原核表达体系p ET15b-17β-hsd10/Escherichia coli BL21(DE3),IPTG诱导重组蛋白表达,并优化表达条件;目的蛋白用His融合蛋白纯化柱(His-Binding-resin)纯化并免疫BALB/c小鼠和新西兰大耳白兔,免疫血清总Ig G采用硫酸铵沉淀法纯化,用获得的高效价鼠源和兔源两种多克隆抗体,建立17β-hsd10双抗夹心酶联免疫检测方法。表达蛋白约为29.5 k Da,纯化后浓度为1.5 mg/m L,鼠源和兔源多克隆抗体效价分别为1.25×104和2.5×104,建立的检测方法对于多种羟基类固醇脱氢酶无交叉反应,特异性良好,可检出含量约为0.05-0.1μg/m L的阳性血清。建立的17β-hsd10双抗夹心ELISA方法,简便、快速、敏感,适合实验室研究科研广泛应用。并可能为检测阿尔茨海默病提供新思路和方法。     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

单克隆抗体与多克隆抗体配对ELISA方法比较

以人绒毛膜促性腺激素(HCG)为抗原,制备出对HCG的多克隆抗体和特异性单克隆抗体,并进行抗体纯化和特性分析,利用辣根过氧化物酶(HRP)分别对其进行了标记.采用双抗夹心ELISA试验,探讨了多克隆抗体与单克隆抗体配对的若干事项.结果表明,利用单克隆抗体和酶标多克隆抗体配对,并用含动物血清的稀释液稀释酶标抗体,可实现对检测原的高特异性和高灵敏度检测.     

Generation and characterization of a lipopolysaccharide-specific murine monoclonal antibody to <Emphasis Type="Italic">Proteus vulgaris</Emphasis> OX19

The three strains of non-pathogenic Proteus species namely, Proteus vulgaris OX2, P. vulgaris OX19 and Proteus mirabilis OXK used in the Weil–Felix test are the group-specific cross-reactive antigens for Rickettsia and Orientia species. Earlier studies have revealed that the group specific and cross-reactive antigens responsible for the Weil–Felix test lie mostly in the lipopolysaccharide (LPS) moiety of the bacterial cell wall [Amano et al. (1993a) Infect Immun 61:4350–4355, (1993b) Microbiol Immunol 37:927–933, (1998) Infect Immun 66:923–926]. The three Proteus strains (OX2, OX19 and OXK) were used to raise murine monoclonal antibodies (MAbs) by hybridoma technology. Several MAb-producing hybridomas could be stabilized following limiting dilution. Affinity and specificity of these MAbs were checked by indirect ELISA using a battery of homologous and heterologous antigens including LPS. Amongst these, one MAb was found to be specific for P. vulgaris OX19 LPS. Since the Weil–Felix reaction is based on the cross-reactivity between the LPS based epitopes, this MAb could be of potential use in mapping of epitopes on the cross-reactive LPS and may also be useful as a potential diagnostic reagent.     

Biochemical characterization of activation-associated bovine class I major histocompatibility complex antigens

Summary. Utilizing a 'sandwich' ELISA assay we have been able to demonstrate that mAb W6/32, B1G6 and IL-A19 are reactive with three different monomorphic determinants on bovine class I major histocompatibility complex (MHC) molecules. Sequential immunoprecipitations performed with the mAb revealed that class I molecules on PBM comprise a single population with respect to reactivity with the mAb in that the β2m-associated proteins bear all three epitopes. By contrast, TCGF-driven lymphoblasts and cells transformed by Theileria parva (Tp) additionally express molecules of Mr 45000 bound to β2m which are recognized by mAb B1G6 and IL-A19 but not by W6/32. These two subclasses of molecules were further distinguished on the basis that, when tunicamycin was added to cultures in the preparation of cells for analysis, mAb W6/32 precipitated class I heavy chains of Mr 39000 while the extra molecules detected only by mAb B1G6 and IL-A19 were of Mr 37000 and 39000. On thymocytes, the mAb W6/32-non-reactive class I molecules are present in low amounts and are expressed by cells in the medulla area, unlike BoT1 (analogous to human CD1) molecules which are expressed by the cortical cells. Our studies also revealed that the supposed β2m-specific mAb B1G6 does not recognize the β2m-associated molecules (BoT1) precipitated by mAb TH97A and thus the specificity of mAb B1G6 in cattle is for an epitope on bovine β2m which is strongly influenced by the nature of the heavy chain with which the β2m is associated.     

Quantification of human tissue transglutaminase by a luminescence sandwich enzyme-linked immunosorbent assay

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes crosslinking of peptidic glutamine residues with primary amines via isopeptide bonds and hydrolysis of ATP or GTP. The enzyme exerts a variety of functions at the cellular and tissue levels that may be disturbed in disease. Its role in pathoprocesses is poorly understood. For investigation of the involvement of tTG in disease, sensitive and specific assays should be available. We have developed the first sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mabs) against human tTG. tTG is captured by mab 3C10 and detected by biotinylated mab 10F3. After incubation with peroxidase-conjugated streptavidin, bound tTG is visualized by peroxidase reaction applying a luminescence substrate. The detection limit was 40 pg/ml. The assay was highly reproducible. Recovery of spiked tTG in crude samples was greater than 92%. The enzyme could be detected in cellular lysates and tissue homogenates of humans. The effect of typical effectors (retinoic acid and interferon-γ) on tTG expression could be demonstrated. A low signal was also obtained in mice samples, suggesting cross-reactivity of the mabs with murine tTG. The new sandwich ELISA may be successfully applied for investigation of physiological functions of tTG and of disorders associated with inadequate tTG expression.     

双抗体夹心ELISA检测人C蛋白的研究

本文建立了检测人Ｃ蛋白的双抗夹心ＥＬＩＳＡ方法。该方法的最佳实验条件为：－抗浓度５－８．５μｇ／ｍｌ;二抗密度为１：２００－１：１０００倍稀释。制作了Ｃ蛋白浓度与ＯＤ值的关系的工作曲线,指出了用内插法测定Ｃ蛋白的最佳浓度范围为０－１００ｍｇ／ｍｌ之间。     

Paratope Duality and Gullying are Among the Atypical Recognition Mechanisms Used by a Trio of Nanobodies to Differentiate Ebolavirus Nucleoproteins

We had previously shown that three anti–Marburg virus nanobodies (VHH or single-domain antibody [sdAb]) targeted a cryptotope within an alpha-helical assembly at the nucleoprotein (NP) C-terminus that was conserved through half a century of viral evolution. Here, we wished to determine whether an anti–Ebola virus sdAb, that was cross-reactive within the Ebolavirus genus, recognized a similar structural feature upstream of the ebolavirus NP C-terminus. In addition, we sought to determine whether the specificities of a less cross-reactive anti–Zaire ebolavirus sdAb and a totally specific anti–Sudan ebolavirus sdAb were the result of exclusion from this region. Binding and X-ray crystallographic studies revealed that the primary determinant of cross-reactivity did indeed appear to be a preference for the helical feature. Specificity, in the case of the Zaire ebolavirus–specific sdAb, arose from the footprint shifting away from the helices to engage more variable residues. While both sdAbs used CDRs, they also had atypical side-on approaches, with framework 2 helping to accommodate parts of the epitope in sizeable paratope gullies. The Sudan ebolavirus–specific sdAb was more remarkable and appeared to bind two C-terminal domains simultaneously via nonoverlapping epitopes—“paratope duality.” One mode involved paratope gullying, whereas the other involved only CDRs, with CDR3 restructuring to wedge in between opposing walls of an interdomain crevice. The varied routes used by sdAbs to engage antigens discovered here deepen our appreciation of the small scaffold's architectural versatility and also reveal lucrative opportunities within the ebolavirus NP C-termini that might be leveraged for diagnostics and novel therapeutic targeting.     

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8 ± 5.2 ng/ml in proximal small intestine to 54.3 ± 10.3 ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.     

抗c-Myc标签纳米抗体的筛选与应用

目的: 以c-Myc-GST蛋白为靶分子,从纳米抗体噬菌体展示免疫文库中筛选能够特异性识别c-Myc标签(EQKLISEEDL)的纳米抗体。方法: 采用固相淘选技术,筛选出能与c-Myc标签特异性结合的噬菌体,phage-ELISA鉴定阳性克隆并测序,通过基因重组技术将阳性噬菌体编码的纳米抗体基因克隆至原核表达载体pET25b(+),再转化至大肠杆菌Rosetta(DE3),IPTG诱导表达,SDS-PAGE分析重组蛋白表达情况。采用间接ELISA和量子点免疫荧光法验证纳米抗体的结合活性和特异性。结果: 通过4轮固相淘选,具有结合活性的噬菌体克隆得到了有效富集,回收率提高了145倍,阳性率从20.83%提高至85.4%。将phage-ELISA鉴定显色值高的两个纳米抗体A25和A26分别进行了重组表达,SDS-PAGE结果显示均为可溶性表达,表达量为60 mg/L。间接ELISA结果表明重组蛋白A25和A26都能够识别c-Myc标签,量子点免疫荧光法验证得到纳米抗体A25能够对SP2/0细胞内的c-Myc蛋白进行检测。结论: 成功地筛选出与c-Myc标签结合的纳米抗体噬菌体克隆,构建了两个抗c-Myc标签纳米抗体的原核表达载体并实现了可溶性表达,为检测胞内c-Myc蛋白奠定了基础。     

Detection of Bluetongue Virus Group-specific Antigen Using Monoclonal Antibody Based Sandwich ELISA

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA.The MAb,designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study.The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein.The sELISA is based on capturing of BTV antigen with VP7 spec...     

Host cell binding of GRA10, a novel, constitutively secreted dense granular protein from Toxoplasma gondii

Monoclonal antibodies (mAbs) against Toxoplasma gondii, Tg378 and Tg556 clones, are specifically observed to localize to the dense granules of tachyzoites by immunofluorescence microscopy. mAb Tg556 is directed against GRA3, a previously described 30kDa dense granular protein. mAb Tg378 is directed against a novel 36kDa dense granular protein, which we refer to as GRA10. These are major proteins in the excretory/secretory proteins from T. gondii before the parasite's entry into host cells, and they are released into the parasitophorous vacuole (PV) during or shortly after invasion to be associated with the PV membrane. GRA10 binds to the membrane of the host cells regardless of its anchorage-dependence or -independence. The cDNA sequence encoding GRA10 was determined by screening a T. gondii cDNA expression library with mAb Tg378. The deduced amino acid sequence of GRA10 consists of a polypeptide of 364 amino acids, and it has no significant homology to any other known proteins. The sequence contains amino terminal signal peptides and two potential transmembrane domains in the middle of sequence that are not near the carboxy terminus. GRA10 has a RGD motif between the two potential transmembrane domains.