Production and characterization of a monoclonal antibody against 28.5 kDa tegument antigen of Fasciola gigantica

A monoclonal antibody (MoAb) against the 28.5 kDa tegumental antigen of Fasciola gigantica was produced by the hybridoma technique using spleen cells from BALB/c mice immunized with the tegumental extract from adult F. gigantica. This MoAb was found to be of the isotype IgG(1), kappa-light chain, and shown by immunoblotting to specifically react with the 28.5 kDa antigen present in the tegument, excretion-secretion material of the adult, whole-body extracts of newly excysted juveniles, 5-week-old juvenile and adult parasites. It did not cross-react with antigens from other trematode parasites, including Schistosoma mansoni, Eurytrema pancreaticum and Paramphistomum spp. Immunolocalization of this antigen by indirect immunofluorescence indicated that it was present as a major component of the adult tegument, particularly in its outer rim, tegumental cells, and their processes. Furthermore, the epithelium linings of the oral sucker, buccal tube, pharynx, caecal bifurcation, both male and female genital canals, which were the continuation of the tegumental-type epithelium, were also positively stained with this MoAb. A similar pattern of immunolocalization, but with weaker staining intensity, was observed in newly excysted, 5- and 7-week-old juveniles. Thus this antigen is expressed in all developmental stages of the parasite, and it could be a strong candidate for immunodiagnosis and vaccine development.     

Preparation of monoclonal antibody against Angiostrongylus cantonensis antigen

Monoclonal antibodies (MAb) against A cantonensis were produced through fusion of immunised spleen cells from BALB/c mice with NS-1 myeloma cells at a ratio of 10:1. The successful fusion rate on the 3rd day of fusion was 90.1%. Ten MAb were characterised, six of which were IgG1 and the remaining four were IgG2a, IgG2b, IgM and IgA respectively. Among 6 IgG1 MAb, four were A. cantonensis-specific, of which three reacted to adult worm antigen only and one reacted to both adult worm and juvenile worm antigens. Two other IgG1 MAb showed cross-reaction with other helminthic antigens of Toxocara canis. Ascaris suum. Paragonimus westermani, Dirofilaria immitis, Anisakis Spp, Gnatostoma Spinigerum and Clonorchis sinensis.     

嗜人按蚊中肠单克隆抗体的制备及初步鉴定

目的 制备并鉴定抗嗜人按蚊中肠单克隆抗体杂交瘤细胞株。方法 用嗜人按蚊中肠做抗原，用细胞融合技术制备单克隆抗体，并采用ELISA法对单克隆抗体进行筛选和鉴定。结果 建立了1株持续分泌抗嗜人按蚊中肠单克隆抗体的杂交瘤细胞株。BALB／c小鼠腹水单克隆抗体的最高稀释度达1：12800；单克隆抗体的重链属IgG1亚类；Western blot证实单抗能与嗜人按蚊中肠抗原特异性结合。结论 成功制备了抗嗜人按蚊中肠单克隆抗体。     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the diagnosis of Penicillium marneffei infection

A monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay was developed for the detection of Penicillium antigen in clinical specimens from patients with Penicillium marneffei infection. The IgM from clone 8C3, reactive with both yeast and mycelial antigens, was immobilized on a microtiter plate. The antigen in serum or urine was captured and detected with biotinylated polyclonal rabbit anti-P. marneffei antibody. The test was sensitive in detecting soluble yeast exoantigen at a concentration as low as 4.88 ng/mL. The reliability of the assay method was evaluated using sera at a dilution of 1:2 from 18 patients with culture-proven penicilliosis, 23 patients with other fungal and bacterial infections, and 125 healthy volunteers. The method exhibited a sensitivity of 72%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 97%, and an accuracy of 97%. However, the sensitivity could be increased to 90% when the specimens were used undiluted. The method was also useful for the detection of antigen secreted in the urine of the patients. The results showed that the newly described assay method can be used in the diagnosis of P. marneffei infection with a high degree of reliability.     

Platelet activation induced by a murine monoclonal antibody directed against a novel tetra-span antigen

MAb 14A2.H1 identifies a novel low-abundance platelet surface antigen, PETA-3, which is a member of the tetra-span (TM4) family. This MAb brings about platelet aggregation and mediator release, which is completely inhibitable by prostaglandin E₁, and partially inhibitable by aspirin and ketanserin. Platelet activation by MAb 14A2.H1 is dependent on interaction with both the platelet Fc receptor, FcγRII, and the specific antigen as it was prevented by either a blocking MAb to FcγRII (IV.3) or F(ab')₂ fragments of 14A2.H1. The extent of platelet activation by the antibody varied considerably between donors, and is believed to reflect the polymorphism of FcγRII. Subaggregating concentrations of 14A2.H1 synergized with other platelet agonists, ADP, adrenaline, collagen and serotonin, indicating signalling via a pathway distinct from these activators. Synergy was also blocked by MAb rv.3, or F(ab')₂ fragments of 14A2.H1. The similar low copy number of PETA-3 and FcγRII in the platelet membrane (approximately 1000/platelet). together with the dependence on FcγRII for activation by MAb 14A2.H1, suggests that PETA-3 may be a component of the FcγRII signal transducing complex in platelets.     

Production and characterization of a monoclonal antibody against the beta-subunit of bullfrog lutropin

We generated a high-affinity and highly specific monoclonal antibody (BL4B11)-producing hybridoma against bullfrog lutropin (LH) beta by fusing mouse myeloma, X63.Ag8.653, with spleen lymphocytes obtained from BALB/c mice immunized with bullfrog LH-IV (pI 9.3) beta-subunit. The resultant antibody-secreting hybridoma was injected into intraperitoneally pristane-primed BALB/c mice to obtain a large amount of antibody. Noncompetitive binding tests revealed that the ascitic fluid (BL4B11) could be diluted up to 1:12,000 for 50% binding to 125I-labeled bullfrog LH beta and also bound strongly to bullfrog intact LH, but not to LH alpha, follitropin (FSH), FSH alpha, FSH beta, and rat glycoprotein hormones (LH, FSH, and thyrotropin (TSH) significantly. The immunoblotting results also showed a similar immunological specificity of BL4B11. Cross-reactivities of bullfrog LH, FSH beta, FSH, LH alpha, and FSH alpha against BL4B11 were 9.69, 3.76, 2.40, 1, and 1%, respectively, when compared with bullfrog LH beta in the competitive inhibition assay system. The affinity constant (Ka) of the BL4B11 was 1.09 X 10(9) M-1. In the sexually mature bullfrog pituitary, immunoreactive LH cells which were revealed by this BL4B11 were distributed throughout the pars distalis except the rostral region. They were especially large, numerous, and polygonal, with well-developed cytoplasm. In the rostral region, immunoreactive LH cells were larger and more intense than those in the central region. In the case of young bullfrog, several immunoreactive LH cells were found only in the dorsocaudal region of the pars distalis. The distribution and histological characteristics of immunoreactive LH cells were different from those of immunoreactive TSH cells revealed by anti-human TSH beta serum.     

Systemic mycosis due to Penicillium marneffei in a patient with antibody to human immunodeficiency virus

Systemic mycosis due to Penicillium marneffei is described in a man infected with human immunodeficiency virus and who had travelled in S.W. China. He responded completely to treatment with amphotericin B and a prolonged course of ketoconazole. Problems of diagnosis are discussed and all previously reported cases reviewed.     

Penicillium marneffei infection in a lung transplant recipient

Penicillium marneffei is a thermally dimorphic fungus that can cause severe opportunistic infections in endemic regions of Southeast Asia, particularly in individuals infected with human immunodeficiency virus‐1, but has rarely been reported in solid organ transplant recipients. Herein, we report the first case, to our knowledge, of P. marneffei infection in a lung transplant recipient, occurring in a 41‐year‐old woman 28 months post lung transplantation, after recent travel to Vietnam. We have reviewed the literature to derive some management principles for this rare infection in this clinical context. The number of P. marneffei infections in transplant recipients may increase, as a result of increasing rates of transplantation and travel to endemic areas.     

Cutaneous manifestations of Penicillium marneffei infection

From an almost unknown disease 15 years ago, Penicillium marneffei has emerged to become one of the most common opportunistic fungal pathogens among HIV-infected patients in the endemic area of southern China and northern Thailand. The mode of infection is primarily airborne, with the reticuloendothelial system as the main target. Penicilliosis is a fatal disease and systemic antifungals are the mainstay of therapy. Direct and mycological examinations are sufficient to make a diagnosis and to differentiate P. marneffei from other opportunistic fungi, although advances in serodiagnosis may potentially enhance understanding of the pathogenesis and identification of early asymptomatic cases.     

五种抗真菌药物对马尔尼菲青霉菌的体外抗菌活性观察

目的 了解两性霉素B(AMB)、酮康唑(KET)、氟康唑(FCA)、5-氟胞嘧啶(5-Fc)和伊曲康唑(ICA)5种抗真菌药物对马尔尼菲青霉菌体外抗菌活性,为临床用药提供参考依据.方法 采用浓度梯度法(E-test)测试AMB、KET、FCA、5-Fc、ICA 5种抗真菌药物对52株从不同AIDS患者骨髓、血液、皮肤损害标本中分离的马尔尼菲青霉菌酵母相和菌丝相的体外抗菌活性.数据采用U检验.结暴 AMB、KET、FCA、5-Fc、ICA的90%酵母相马尔尼菲青霉菌的最低抑制浓度(MIC90)分别为0.250、0.160、24.000、4.000和0.006 mg/L,最低抑制浓度(MIC)范围分别为0.004～0.500、00002～0.016、1.000～256.000、0.002～32.000和0.002～0.008 mg/L;对90%菌丝相马尔尼菲青霉菌的MIC90分别为1.500、0.125、256.000、24.000和0.012 mg/L,MIC范围分别为0.064～4.000、0.006～0.940、1.000～256.000、0.125～32.000和0.002～0.064 mg/L.不同抗真菌药对双相马尔尼菲青霉菌的体外抗菌活性不同,以ICA最强,其次为KET.酵母相和菌丝相的马尔尼菲青霉菌对同一药物的MIC比较差异有统计学意义(AMB、KET、FCA、5-Fc和ICA的U值分别为4.221 9、1.912、28.798、6.43、7.21,均P＜0.05).结论 进行马尔尼菲青霉菌体外抗真菌药物敏感实验对临床有重要参考意义.     

Synthesis and evaluation of monoclonal antibody against Plasmodium falciparum merozoite surface antigen 2

ObjectiveTo assess the quality of expressed MSP-2 and also to confirm the immune response against different domains of these proteins.MethodsMice were immunized with a schizont extract to stimulate the immune system to make antibodies against different antigens of the late stage parasite including production of antibodies against different domains of Plasmodium falciparum (P. falciparum) MSP-2. B lymphocytes of immunized mice were extracted from the spleen and the fusion was performed using NS-1 myeloma cells and the hybridoma cells were assayed by ELISA either with a schizont extract or different domains of MSP-2 and/or by IFAT with whole schizont preparation. Fusion of NS-1 and spleen cells was performed. The positive hybrids were cloned and ELISA was applied against different dilutions. The positive clones were transferred to a small tissue culture flask and after developing they were assayed against schizont extract and the different MSP-2 domains. The positive clones were expanded to large (75 cm²) flask and cultured under the same conditions, checking them using both ELISA and IFAT and the positive cells were frozen as soon as possible.ResultsA total number of 7 fusions including 26 plates (2 496 wells) were performed, of which 1 336 hybrids were produced and the overall efficiency (1 336/2496 × 100) was about 53%. ELISA was performed to detect the positive hybrids against crude schizont extract by which the highest frequency to crude schizont extract was found for the supernatant of the hybrids produced in fusion number 3 (66 out of 315 hybrids). The supernatant of both B5 and F1 hybridoma cells were more positive against domain 2 of the MSP-2 recombinant protein in Western blotting test. Western blotting results also showed that different domains of the MSP-2 recombinant protein and also the MSP-2 of the P. falciparum parasite were recognized by some of the positive clones and also immune sera.ConclusionsBringing together all the results of this study it has been confirmed that some clones have recognized both schizont extract and different domains of the MSP-2 recombinant protein and therefore confirming the quality of the MSP-2 domains.     

Production and characterization of a monoclonal antibody against recombinant saposin-like protein 2 of Fasciola gigantica

A monoclonal antibody (MoAb) against recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with rFgSAP-2. This MoAb is an IgG1, κ light chain isotype. By immunoblotting and indirect ELISA, the MoAb reacted specifically with rFgSAP-2, the natural FgSAP-2 at 10 kDa in whole body (WB) and excretory–secretory (ES) fractions of F. gigantica. It did not cross react with antigens in WB fractions from other parasites, including Opisthorchis viverrini, Schistosoma mansoni which are human parasites, Haemonchus placei, Setaria labiato-papillosa, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Paramphistomum cervi which are ruminant parasites. By immunohistochemistry, the FgSAP-2 protein was localized only in the cytoplasm of caecal epithelial cells of 4-week-old juvenile and adult stages, but not in metacercariae, newly excysted juvenile (NEJ), 2- and 3-week-old juveniles. This finding indicated that FgSAP-2 is an abundantly expressed parasite protein that is released into the ES, hence SAP-2 and its MoAb may be used for immunodiagnosis of ruminant and human fasciolosis.     

Characterization of a monoclonal antibody against a differentiation antigen of human natural killer cells (NK cells)]

The new murine monoclonal antibody BL-(H5) reacts with a novel surface molecule which is mainly expressed on human NK and B cells. The antigen is not expressed on peripheral T lymphocytes, thymocytes and different human T-cell lines. BL-(H5) does not bind to erythrocytes and platelets. The monoclonal antibody reacts in western blotting experiments with an antigen of 78kDa.     

Antigen analyses of serotypes of streptococcus mutans using a monoclonal antibody elaborated against serotype g polysaccharide antigen

A hybridoma (F4B) which produced a monoclonal antibody (mAb) specific for serotype g carbohydrate antigen (RRg) of Streptococcus mutans 6715 was obtained. The F4B mAb cross-reacted with purified carbohydrate antigens of serotype d (RRd) and serotype h (TCAh). In immunodiffusion tests, F4B mAb produced a stable precipitin band with RRg, while the band developed between the mAb and RRd/TChh in the cold disappeared when incubated at room temperature. The immunoprecipitin reaction between F4B mAb and RRg was strongly inhibited upon addition of lactose.     

HSP70基因在马内菲青霉酵母相和菌丝相中的差异表达

目的初步探讨马内菲青霉双相转化中HSP70基因转录、蛋白表达变化情况。方法对马内菲青霉临床分离株SUMS0152进行双相性诱导,收集菌丝相和酵母相菌体。使用RT-PCR技术半定量测定HSP70基因转录水平,Western blot技术半定量测定HSP70蛋白水平,分别在两相间进行比较。结果马内菲青霉SUMS0152酵母相HSP70基因转录较菌丝相升高;酵母相的HSP70蛋白表达较菌丝相升高。结论马内菲青霉SUMS0152HSP70基因在两相间存在明显表达差异,其基因转录和表达水平在酵母相均表现为一致的上调。