Production of monoclonal antibody against Listeria monocytogenes and its application to immunochromatography strip test

An immunochromatography (ICG) strip test based on a monoclonal antibody for the rapid detection of L. monocytogenes in meat and processed-meat samples was developed in this study. A monoclonal antibody (MAb) specific to L. monocytogenes was produced from cloned hybridoma cells (FKLM-3B12-37) and used to develop an ICG strip test. The antibody showed a stronger binding to L. monocytogenes than other Listeria species, and a weak cross-reaction to S. aureus based on an ELISA. The detection limit of the ICG strip test was 10(5) cell/ml. In total, 116 meat and processed-meat samples were collected and analyzed using both the ICG strip test and a PCR. The ICG strip test and PCR indicated L. monocytogenes contamination in 34 and 27 meat samples, respectively. The 7 meat samples not identified as L. monocytogenes positive by the PCR were also tested using an API kit and found to be contaminated by Listeria species. In conclusion, the ICG strip test results agreed well with those obtained using the PCR and API kit. Thus, the developed ICG has potential use as a primary screening tool for L. monocytogenes in various foods and agricultural products, generating results within 20 min without complicated steps.     

Production of monoclonal antibody against histamine and its application to immunohistochemical study in the stomach

A monoclonal antibody against histamine has been produced. A histamine–haemocyanin conjugate prepared using 1-ethyl-3-(3-dimethylami nopropyl) carbodiimide as a coupling agent was used for immunizing mice. Immunized mice were sacrificed to prepare monoclonal antibody using a hybridoma technique. On immunospot assay, the hybridoma culture supernatant containing a monoclonal antibody was capable of detecting 50 pmol of histamine. Using this antibody, we examined the cellular localization of histamine-like immunoreactivity in the stomach of normal or -fluoromethylhistidine-treated rats and mice. Immunoreactive cells were abundant in the gastric mucosal layer. These positive cells were often located in the basal half of the fundic gland but were rare in the pyloric gland. The cells, small or medium in size, spindle or cone in shape, were intermingled with immunonegative epithelial cells. In the cytoplasm of the positive cells, granular reaction products were densely deposited. In addition, a few positive cells, identified as mast cells by Toluidine Blue staining, were distributed mainly in the submucosal and muscular layer. The antibody preabsorbed with 10 mm histamine gave no positive immunostaining. For pharmacological study, some rats were injected six times with -fluoromethylhistidine every 8 h. In these rats, positive cells except mast cells were no longer detected. In conclusion, the monoclonal antibody produced appears to be highly specific for histamine. Its application in immunohistochemistry should provide a powerful tool for analysing the roles of histamine in enterochromaffin-like or mast cells in the stomach. © 1998 Chapman & Hall     

Production and immunocytochemical application of a highly sensitive and specific monoclonal antibody against rat dopamine-beta-hydroxylase

A highly sensitive and specific monoclonal antibody against the enzyme dopamine beta-hydroxylase (DBH) from rat was produced and coded DBH 41. The generated hybridoma secreted immunoglobulins of mouse IgG1 subtype, as determined by radial immunodiffusion. This antibody, characterized by immunoblotting against a crude rat DBH preparation, was found to specifically recognize two bands of molecular weight 70 and 75 kDa corresponding to the soluble and membrane bound forms of the enzyme, respectively. With regard to species specificity, the anti-DBH antibody recognizes only the rat DBH molecule as it exhibits no cross-reactivity with either mouse, human, rabbit, guinea pig, cat or bovine DBH. Comparative immunocytochemical localization of DBH and TOH immunoreactivity was performed in different brain regions and we found that the DBH 41 antibody specifically stained DBH-containing neurons and fibers in the rat central nervous system (CNS). The high sensitivity of the DBH 41 antibody permitted us to detect immunologically the presence of the enzyme even in areas where only scattered DBH-containing fibers were present.     

Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands

Summary A monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fragment (aa28–37) of -human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal antibody 129CD8 was corroborated by dot immunobinding experiments, enzyme-linked immunoassay and immunostaining of tissue sections. In vitro studies showed that the mAb 129CD8 readily recognized the fragment 28–37 of -human CGRP and to a slightly lesser degree whole -human CGRP and the fragments containing the C-terminal part of the molecule. The mAb 129CD8 also recognized the -human CGRP but not the -rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of -human CGRP lacking the C-terminal part of the molecule.Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigeminal ganglion, and rat thyroid gland. Our findings demonstrate, in keeping with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accordance with previous investigators, the Merkel cells were immunoreactive for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactivity was localized in the C-cells. The distribution of CGRP immunoreactivity in the guinea-pig salivary glands is different from that previously reported for rat salivary glands. In the guinea-pig trigeminal ganglion, CGRP immunoreactivity was localized mainly in small-sized neurons and fibres traversing the ganglion. Double staining with substance P performed on guinea-pig trigeminal ganglion revealed four types of sensory neurons, those containing both peptides, those containing only substance P or CGRP and those lacking both peptides. Guinea-pig parotid gland, but not the submandibular or sublingual glands, contained periacinar fibres exhibiting both immunoreactivities. Substance P-positive, CGRP-positive fibres were also seen around parotid and submandibular, but not around sublingual, gland ducts. All glands received perivascular innervation showing immunoreactivities for both peptides. The present results support the idea that in the peripheral nervous system only a subpopulation of sensory neurons contains both substance P and CGRP. Consequently, colocalization of substance P and CGRP indicates a sensory nerve, while those containing either substance P or CGRP may be sensory or parasympathetic.     

Production of monoclonal antibody against Scrippsiella trochoidea cysts and its application to analysis during cyst formation and enzyme-linked immunosorbent assay

A monoclonal antibody was produced by the fusion of mouse myeloma cells with spleen cells from a mouse immunized with the cysts of Scrippsiella trochoidea, marine phytoplankton. Immunofluorescence microscopic observation showed that the antibody reacted with the spines on the cyst but not with the vegetative cells and cyst walls. An enzyme-linked immunosorbent assay could be used to measure the cysts in muddy bottom sediments using the purified antibody conjugated with horseradish peroxidase.     

Production of cholera-like enterotoxin by Aeromonas hydrophila

A total of 249 strains of mesophilic Aeromonas including 179 A. hydrophila and 70 A. caviae were tested for production of cholera-like enterotoxin by reversed passive latex agglutination (RPLA) assay. A cholera-like enterotoxin neutralized with cholera antitoxin was demonstrated in the culture filtrates from eight (4.5%) of the 179 A. hydrophila strains, while none of A. caviae strains revealed the enterotoxin production in the test. Production of the cholera-like enterotoxin in the eight strains of A. hydrophila was also confirmed by enzyme-linked immunosorbent assay (ELISA).     

鲟源嗜水气单胞菌拮抗解淀粉芽孢杆菌微胶囊的制备工艺及其特性

【目的】优化鲟源嗜水气单胞菌拮抗解淀粉芽孢杆菌微胶囊的制备工艺,并观察其特性。【方法】以明胶为壁材,采用单因素法,考察了明胶浓度、进风温度、进料速度、空气流量等因素对解淀粉芽孢杆菌微胶囊有效含菌量的影响,并进一步通过正交试验设计优化制备解淀粉芽孢杆菌微胶囊的喷雾干燥工艺参数,观察其微胶囊颗粒形态以及对人工模拟胃液和肠液的耐受力。【结果】解淀粉芽孢杆菌微胶囊喷雾干燥的最佳制备工艺条件为:明胶浓度为3%,进风温度为155°C,进料速度为8 mL/min,空气流量为700 L/h,各因素对其喷雾干燥工艺的影响程度为:明胶浓度进料速度空气流量进风温度。此外,解淀粉芽孢杆菌微胶囊的颗粒呈球形,表面有凹陷,但没有孔和裂纹,颗粒粒径分布基本均匀,平均大小为9.22μm,对人工模拟胃液和肠液具有较好的耐受力,对鲟源嗜水气单胞菌具有良好的生长抑制效果。【结论】本研究结果为鲟源嗜水气单胞菌拮抗解淀粉芽孢杆菌微胶囊的工业化生产奠定了基础。     

Immune response of zebrafish (Danio rerio) against a newly isolated bacterial pathogen Aeromonas hydrophila

A strain of Aeromonas hydrophila associated with unusual mortalities in zebrafish (Danio rerio) culture facilities was isolated, identified and characterized. In challenge experiments, adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection with viable bacteria and its extracellular products (ECPs) reaching very high mortalities in a few hours. The infection, by the viable bacteria or the ECPs, caused cell death in kidney, due to the cytotoxic and haemolytic activities of the bacterial ECPs. Moreover, the infection affected the release of oxygen (ROS) and nitrogen (NO) reactive free radicals. To determine if this A. hydrophila infection induces an inflammatory response, mRNA expression levels of tumour necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), interferon-gamma (IFNgamma) and inducible nitric oxide (iNOS) were assessed by real time PCR. The expression levels of TNFalpha, IL-1beta and IFNgamma were upregulated in the kidneys of infected zebrafish with viable bacteria, heat-killed bacteria and ECPs. Expression levels of iNOS were upregulated by ECPs. Mortality rate (LD(50)) and histopathology were also determined.     

Production and characterization of a novel monoclonal antibody against Vibrio parahaemolyticus F0F1 ATP synthase's delta subunit and its application for rapid identification of the pathogen

We raised monoclonal antibodies (MAbs) against Vibrio parahaemolyticus cell extracts. One of the MAbs, designated MAb-VP34, reacted in enzyme-linked immunosorbent assays (ELISAs) with 140 V. parahaemolyticus strains, regardless of serotype or origin. MAb-VP34 did not detectably react with 96 strains belonging to 27 other Vibrio species (except for Vibrio natriegens) or with 29 non-Vibrio species. These results show that MAb-VP34 is highly specific for V. parahaemolyticus. Western blotting and mass spectrometry analyses revealed that MAb-VP34 recognized V. parahaemolyticus F₀F₁ ATP synthase's delta subunit.Using MAb-VP34, a rapid and simple immunodot blotting assay (VP-Dot) was developed to determine whether bacterial colonies growing on selective agar, represented V. parahaemolyticus. To evaluate VP-Dot, 20 V. parahaemolyticus strains and 19 non-related strains were tested. The results indicated that VP-Dot is a reliable tool for identification of V. parahaemolyticus colonies. The simple VP-Dot procedure took 40 min, indicating that the MAb-VP34 based immunological method will greatly reduce labor, time, and costs required to verify V. parahaemolyticus colonies as compared with the conventional biochemical test.     

Development and application of a monoclonal antibody against Thiothrix spp.

Historically, methods used to identify Thiothrix spp. in environmental samples have been inadequate because isolation and identification procedures are time-consuming and often fail to separate Thiothrix spp. from other filamentous microorganisms. We described a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) procedure which was used to identify Thiothrix spp. in wastewater, artesian springs, groundwater, and underwater subterranean samples. The ELISA utilized monoclonal antibody T3511 to a species-specific carbohydrate epitope of Thiothrix spp. No cross-reactions were observed among non-Thiothrix strains consisting of 12 species and nine genera. In field trials, the ELISA identified 100% of 20 biochemically and cytologically confirmed Thiothrix spp.-containing samples with no false positives. Indirect immunofluorescent microscopy utilizing T3511 was effective for wastewater samples but not for those from natural spring water because of background fluorescence in the latter. In addition, electron micrographs of Thiothrix spp. labeled with T3511-biotin-anti-mouse antibody-gold showed that epitope T3511 was intracellular both in laboratory strains and environmental isolates. The minimum level of detection of the ELISA was 0.10 microgram/ml.     

抗吡虫啉单克隆抗体的制备及鉴定

为制备灵敏度高,特异性强的抗吡虫啉单克隆抗体,建立经济、快速、准确的吡虫啉残留免疫学分析方法,采用分子模拟技术分析吡虫啉及其类似农药的电荷分布后,选择1-[6-(2-羧乙硫基-3-吡啶)甲基]-N-硝基-2-咪唑啉亚胺 (H1) 作为免疫半抗原,1-(6-氯-3-吡啶甲基)-3-羧丙基-N-硝基-2-咪唑啉亚胺 (H2) 作为包被半抗原,利用NHS酯法将H1和H2分别与牛血清蛋白 (BSA) 和卵清蛋白 (OVA) 偶联合成免疫原与包被原。免疫BALB/c小鼠后,采用常规杂交瘤技术共获得2株稳定分泌抗吡虫     

Production and properties of a monoclonal antibody against human epidermal growth factor

A monoclonal antibody against human epidermal growth factor (hEGF) was obtained from a mouse hybridoma cell line. The purified monoclonal antibody from the ascites fluid of a mouse injected with one of the cell lines was specific for hEGF and did not cross-react with mouse EGF (mEGF). Its Kd value for hEGF was 1.4 X 10(-9) M. This monoclonal antibody inhibited the biological activities of hEGF, including its binding to the receptor of BALB/3T3 cells and its stimulation of DNA synthesis in the cells, but did not affect the activities of mEGF. The monoclonal antibody completely inhibited DNA synthesis stimulated by human urine from a patient without a tumor, but only partially inhibited the stimulatory activity in urine from a tumor-bearing patient.     

抗人血栓调节蛋白单克隆抗体的制备与鉴定

为了制备特异性抗人血栓调节蛋白(human thrombomodulin,hTM)的单克隆抗体(monoclonal antibody,McAb),利用脂质体Lipofectamine 2000将包含hTM全长cDNA序列的重组表达质粒pThr402转染CHO细胞,经G418筛选及相关鉴定后获得高效稳定表达hTM的CHO-TM5细胞株。将CHO-TM5细胞直接免疫Balb/c小鼠,应用杂交瘤技术,通过细胞ELISA (cellular enzyme-linked immunoabsorbent assay,CELISA)筛选出阳性克隆后,将杂交瘤细胞株腹腔注射Balb/c小鼠诱生腹水。用CELISA、流式细胞术、免疫组织化学染色法及免疫印迹法对所获McAb的特异性进行鉴定。我们获得了1株可稳定分泌抗hTM的McAb的杂交瘤细胞株NH-1,其亚型为IgGl,McAb腹水效价为1×10~(-6),腹水抗体含量为20 mg/ml。NH-1对相应抗原具有较高的组织特异性,在体内与正常组织的交叉反应少,对人脐静脉内皮细胞、CHO-TM5有特异性结合反应,说明NH-1可特异性识别天然的hTM分子,为进一步应用此McAb进行hTM生物学功能及临床意义研究提供了基础。     

Production of a monoclonal antibody directed against rat liver glutathione-insulin transhydrogenase

A hybridoma cell line secreting monoclonal antibody specific for glutathione-insulin transhydrogenase has been produced by fusing mouse myeloma cells with spleen cells from mice immunized to purified rat liver glutathione-insulin transhydrogenase. The secreted antibody isotypes were found to be: Ig gamma 1 heavy chains and kappa light chains. This monoclonal antibody has been used to screen glutathione-insulin transhydrogenase in various rat tissue extracts (liver, fat, heart, testis, spleen, lung and kidney) following separation on NaDodSO4/urea polyacrylamide disc-gel electrophoresis and electrophoretic transfer to nitrocellulose. Screening with the monoclonal antibody showed the presence of one immunoreactive protein band equal in molecular weight to that of purified rat liver GIT (Mr 53,000) in extracts of all tissues studied and a second immunoreactive protein band of lower molecular weight (Mr 49,000) in spleen and lung tissue extracts. Separation of these two proteins by HPLC using a TSK-DEAE column demonstrated that both proteins exhibit insulin degrading activity. These data indicate that GIT may occur in multiple forms in some tissues.     

Production, characterisation and potential application of a novel monoclonal antibody for rapid identification of virulent Listeria monocytogenes

A panel of hybridomas was produced using intact Listeria monocytogenes serotype 1/2a cells as the immunogen. An IgG2a monoclonal antibody (mAb) 'mAb2B3' was isolated that reacted with L. monocytogenes but not with a representative panel of related Listeria spp. and non-Listeria spp. Binding activity was greatest against L. monocytogenes serotype 1/2a and was significantly enhanced when cells were prepared in Listeria enrichment broth (LEB). The reactive epitope was deduced, by immunoblot analysis, to be a surface localised protein of approximately 80 kilodaltons (kDa), putatively assumed to be internalin A (InlA). Recombinant InlA protein was subsequently expressed in Escherischia coli. When crude E. coli cell lysates were subjected to immunoblot analysis, it was demonstrated that the mAb bound specifically to the heterologously expressed recombinant InlA protein, thus confirming the specificity of the mAb. The mAb was further evaluated in a series of enzyme-linked-immunosorbent assay (ELISA)-based formats and in a surface plasmon resonance (SPR)-based biosensor platform. Both configurations were capable of differential identification of virulent L. monocytogenes at concentrations greater than or equal to 1x10(7) cells/ml. Notwithstanding the apparent insensitivity, the results indicate that InlA could be exploited as a marker for highly specific confirmatory identification of pathogenic L. monocytogenes following primary enrichment of suspect food samples, using the anti-InlA antibody 'mAb2B3', described herein.     

Production and characterisation of a monoclonal antibody to progesterone and its effect on fertility

A monoclonal antibody reacting with progesterone has been raised by fusion of mouse myeloma cells (SP20) and splenocytes of BALB/c mice hyperimmunized with 4-pregnane 3,20 dione conjugated to bovine serum albumin. Association constant of this antibody for binding with progesterone was 0.22 x 10(9) l/mole. The antibody was highly specific for progesterone. A single ip injection of this antibody brought about an antifertility effect which is influenced by genotype. Antibody treatment brought about a significant decrease in the fetal weight and a slight decrease in the plasma progesterone levels. The antifertility effect could be reversed only up to day 3 by exogenous administration of progesterone.     

Production of a monoclonal antibody strongly reacting with immature thymic T lymphocytes and its immunohistological application

A monoclonal antibody Th-5 has been produced against mouse immature thymic lymphocytes and employed to study the process of T cell differentiation in the thymus. Immunohistologically, Th-5 positive thymic T lymphocytes were first found at Day 12 of gestation. They increased in number as well as staining intensity until Day 18 of gestation and decreased thereafter. Th-5 antigen expression was not seen in lymphoid cells in the fetal liver. In the newborn thymus, lymphocytes in the subcapsular layer were still strongly positive, while other cortical lymphocytes became moderately positive for Th-5. Th-5 positiveness was more pronounced in the medulla than in the cortex in the thymus of young adult mice. The staining pattern of Th-5 in the thymus was apparently different from those with other T cell markers (Thy-1, CD3, CD4, CD5, CD8) including J11d, Pgp-1, IL-2R, and 3A10 (TCR gamma delta). Flow cytometric analyses showed that the expression of Th-5 was mostly associated with the Thy-1 antigen. However, the fluorescent intensity of Th-5 gradually declined with ontogenic development of the thymus, and the molecular size of the antigen was approximately 100 kDa, which is different from Thy-1 antigen (25-30 kDa). Considering these findings, the strong expression of Th-5 could be one of the markers of immature thymic T lymphocytes in the early phase of the ontogenic development.     

1株猪δ冠状病毒N蛋白单克隆抗体制备与鉴定

【背景】猪δ冠状病毒(porcine deltacoronavirus,PDCoV)是一种新发现的猪肠道冠状病毒,主要引起以急性水样腹泻、呕吐和脱水等特征的胃肠道疾病。在PDCo V的4个结构蛋白中,核衣壳蛋白(nucleocapsid protein,N)为高度保守的结构蛋白,在病原诊断方面具有重要意义。【目的】以纯化的重组PDCo VN蛋白为抗原,制备抗N蛋白的单克隆抗体并进行特性鉴定。【方法】利用已构建的p ET-28a-N表达菌,经IPTG诱导表达获得具有免疫原性的重组N蛋白,并将纯化的重组N蛋白免疫BALB/c小鼠制备杂交瘤细胞,经3次亚克隆筛选,选取抗体效价最高的一株杂交瘤细胞注射小鼠腹腔并制备腹水单克隆抗体,利用Protein G亲和层析柱纯化。通过腹水单克隆抗体效价测定、抗体亚型鉴定、Western blot鉴定其特性;利用细胞间接免疫荧光试验、组织石蜡切片荧光试验、免疫组化、流式细胞荧光分选技术鉴定其诊断应用价值。【结果】经筛选后得到一株杂交瘤细胞命名为4E88,其腹水单克隆抗体效价为1:105,亚型为Ig G1型,轻链为κ链,Western blot试验表明该单克隆抗体与重组N蛋白和超速离心纯化的PDCo V全病毒反应形成特异性条带。此外,单克隆抗体4E88可以用于猪δ冠状病毒检测的间接免疫荧光试验、免疫组化染色和流式细胞荧光分选技术。【结论】研究获得的单克隆抗体4E88具有较好的反应性,为后续开展PDCo V鉴定、诊断和N蛋白功能研究等奠定基础。     

Production of monoclonal antibody against protopectinase from Kluyveromyces wickerhamii

Four monoclonal antibodies (MCA 3–6, 4-2, 9-2, and 10-2) against protopectinase (an endo-polygalacturonase) from Kluyveromyces wickerhamii were prepared. MCAs 3-6 and 4-2 reacted to protopectinases produced by K. fragilis and K. marxianus as well as to the protopectinase produced by K. wickerhamii. However, 9-2 and 10-2 were specific for the protopectinase from K. wickerhamii. These MCAs did not inhibit the protopectinase reaction or the polygalacturonase reaction of protopectinases produced by these species. We used these MCAs to find protopectinases in the culture filtrates of Kluyveromyces yeasts.