Production of Monoclonal Anti-Idiotype Antibodies Which Mimic an M-Like Protein of Streptococcus equi

Rat monoclonal anti-idiotype antibodies (mAb2) were raised against two mouse monoclonal antibodies (mAb1), 1D10 and 2A6, with specificity for the M-like protein of Streptococcus equi. The capacity of the mAb2 to inhibit the binding between the corresponding mouse mAbl against which the mAb2 were raised and the M-like protein was investigated in an inhibition EIA. One of the ten mAb2 examined, namely 5D1 (anti-mAb1 1D10), was able to inhibit this binding. The mAb2 5D1 bound to the mAb1 1D10 in such a way as to completely inhibit the subsequent binding of the M-like protein antigen to the paratope of the mAb1 1D10. The mAb2 5D1 is likely to represent a true image of the M-like protein antigen and may thus be described as an Ab2β anti-idiotype antibody.     

猪源马链球菌兽疫亚种表达的类M蛋白黏附抑制性单抗的获得

根据马链球菌兽疫亚种(Streptococcus equi subsp.zooepidemicus)猪源株ATCC35246株的类M蛋白基因序列,通过PCR技术,扩增出无信号肽的类M蛋白基因并定向克隆至表达载体pET-32a( )中。重组质粒经酶切鉴定和测序后,在大肠杆菌BL21中表达,获得60kDa产物,Western blot显示,其与ATCC35246株多克隆抗血清反应,抗原性良好。以His亲和层析柱纯化重组类M蛋白作为抗原免疫8周龄的BALB/c小鼠,采用淋巴细胞杂交瘤技术制备了12株稳定分泌抗类M蛋白单抗的细胞株,特异性检测显示其与A群链球菌、猪链球菌2型以及马链球菌马亚种等没有交叉反应。单抗亚类鉴定显示,其中6株为IgG1,3株为IgG2,另外3株为IgM。从腹水中纯化的单抗效价为2.56×104~1.01×105。黏附抑制实验显示,其中一株单抗能阻断类M蛋白黏附HEp-2细胞。     

Trichinella spiralis: murine strain variation in response to monoclonally defined, protective, nonstage-specific antigens

Nine hybridoma cell lines secreting monoclonal antibodies (mAbs) against Trichinella spiralis muscle larvae (ML) excretory/secretory antigens (ESA) were developed. Two mAbs, 6-D8-E3 (6D8) and 6-B1-G10 (6B1), were studied in detail. Western blot analysis using ML ESA showed that 6D8 recognized 35- and 40-kDa constituents whereas 6B1 identified a doublet of 33 kDa. However, Western blots of SDS-PAGE of crude ML homogenate showed that 6D8 identified proteins of approximately 35 and 43-60 kDa, whereas 6B1 recognized bands of 42-50 kDa. These results indicated substantial apparent MW differences between secreted and nonsecreted proteins recognized by both mAbs. Neither 6D8 nor 6B1 reacted with adult worm ESA, but both recognized antigens in aqueous extracts of homogenates of whole adult worms. Competitive inhibition experiments using ML ESA as a target demonstrated that the antigen epitopes recognized by monoclonals 6D8, 6B1, a rat mAb, 9D4, and a 37-kDa antigen previously defined were noncross-reactive. MAbs 6D8, 6B1, and 9D4 were used to isolate proteins possessing target determinants by affinity chromatography from crude ML homogenates. Each mAb isolated distinct protein species as determined by SDS-PAGE (6B1, approximately 42 kDa; 6D8, approximately 28, 37, and 61 kDa; 9D4, approximately 29, 33, 38-57, 80, and 86 kDa). NFS mice responded in a dose-dependent manner to affinity-purified antigens and were 25-fold more effective (by weight of antigen) than either C3Heb/Fe(C3H) or B10.BR mice. Immunization of mice with 6D8, 6B1, or 9D4 antigens induced strong protection against a subsequent challenge infection in NFS mice as indicated by accelerated intestinal adult worm expulsion, reduced fecundity of the female worms, and reduction of ML burden. Affinity-isolated antigens stimulated in vitro proliferation of spleen and MLN cells from immune mice; however, the mitogenic response to these antigens barely varied among NFS, C3H, and B10.BR strains.     

Leukocyte integrin-dependent and antibody-independent cytotoxicity of macrophage against allografts

Macrophages (Mphis), but not T cells, infiltrating into the rejection site of either i.p. allografted Meth A (H-2d) fibrosarcoma cells in C57BL/6 (B6) (H-2b) mice or BALB/c (H-2d) skin onto B6 mice are cytotoxic against allografts with H-2d specificity. To determine the mechanisms of specific killing of allografts by allograft-induced Mphi (AIM), we raised approximately 5,000 rat monoclonal antibodies (mAbs) against AIM and selected three of them (R1-73, R2-40 and R1-34), each of which inhibited cytotoxic activity against allografts in a dose-dependent manner. The antigens recognized by R1-73, R2-40 and R1-34 mAbs were defined by immunoprecipitation and Western blot analyses as CD11a, CD18 and CD11b, respectively; and the allografts expressed CD54, a ligand of CD11a or CD11b, suggesting leukocyte integrin-dependent killing. Although Ab-dependent cellular cytotoxicity has been recognized as a mechanism of specific killing by Mphis, the infiltration of AIM into the rejection site of allografts far (approximately 6 days) preceded the appearance of serum IgG Ab specific for the allograft. AIM exhibiting full cytotoxic activity against allografts was also induced in the transplantation site of Fcgamma receptor knockout [(B6x129) F1] mice as well as B10.D2 (H-2 compatible with allograft) and B6-xid (X-linked immunodeficiency with B cell-specific defect) strains of mice. In the latter two strains of mice, the levels of serum IgG Ab to the allograft were negligible. Moreover, the cytotoxic activity of AIM against allografts was not affected by pretreatment of the cells with anti-mouse IgG serum, suggesting Ab-independent cytotoxicity.     

Generation of monoclonal antibodies against non-structural protein 3AB of foot-and-mouth disease virus

To identify linear epitopes on the non-structural protein 3AB of foot-and-mouth disease virus (FMDV), BABL/c mice were immunized with the 3AB protein and splenocytes of BALB/c mice were fused with myeloma Sp2/0 cells. Two hybridoma monoclonal antibodies (mAbs) cell lines against the 3AB protein of foot-and-mouth disease virus (FMDV) were obtained, named C6 and E7 respectively. The microneutralization titer was 1:1024 for mAb C6, and 1:512 for E7. Both mAbs contain kappa light chains, and were of subclass IgG2b. In order to define the mAbs binding epitopes, the reactivity of these mAbs against FMDV were examined by indirect ELISA. The results showed that both mAbs can react with FMDV, but had no cross-reactivity with Swine Vesicular Disease (SVD) antigens. The titers in abdomen liquor were 1:5×10⁶ for C6 and 1:2×10⁶ for E7. In conclusion, the mAbs obtained from this study are specific for the detection of FMDV, can be used for etiological and immunological researches on FMDV, and have potential use in diagnosis and future vaccine designs.     

A rapid subtractive immunization method to prepare discriminatory monoclonal antibodies for food E. coli O157:H7 contamination

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1:10(6) with E. coli O157:H7 and affinity constants ranged from 1.57 × 10(10) to 2.79 × 10(10) L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food.     

Identification of protective outer membrane antigens of Brucella ovis by passive immunization of mice with monoclonal antibodies

Outer membrane proteins (OMPs) and rough lipopolysaccharide (R-LPS), the main surface antigens of Brucella ovis, display surface-exposed epitopes. Mixtures of monoclonal antibodies (mAbs) to both antigens were previously shown to protect mice against a B. ovis challenge. To further identify the antigens involved, seven mAbs against Brucella OMPs (Omp10, Omp16, Omp19, Omp25, Omp31, Omp2b and Omp1) and three to R-LPS were tested for protection either individually or in combinations. Significant reduction in spleen infection in challenged mice, relative to controls, was used as the protection criteri. Controls included nonimmunized mice and mice given an irrelevant, anti-O-polysaccharide (OPS), mAb. For comparison, a group received a mouse serum containing antibodies to both OMPs and R-LPS; this serum was prepared by immunization with a B. ovis hot-saline extract which, as described previously, induces protective immunity in mice and rams. Significant protection was observed with both mAbs to OMPs and R-LPS. mAbs to Omp16, Omp19 and Omp31 afforded the highest protection and prevented the development of splenomegaly. The protective effect of mAb to Omp31 was not interfered with by nonprotective mAbs in different mixtures. The data presented confirm the protective role of antibodies to OMPs and R-LPS against B. ovis, and identify several OMPs, especially Omp31, which are promising candidates for a subunit vaccine against ram epididymitis.     

空肠弯曲菌FlaA单克隆抗体的制备与鉴定

【目的】原核表达空肠弯曲菌鞭毛蛋白FlaA,并制备其单克隆抗体。【方法】克隆目的基因并将其构建到pET30a(+)和pGEX-6p-1表达载体,分别以变复性纯化后的rHis-FlaA、rGST-FlaA蛋白为免疫原和检测原进行杂交瘤细胞的筛选。采用间接ELISA法测定细胞上清和单抗腹水效价,Dot-ELISA、Western blot分析单抗特异性。【结果】成功构建pET30a(+)-flaA和pGEX-6p-1-flaA重组原核表达质粒,并融合表达rHis-FlaA和rGST-FlaA蛋白,Western blot试验显示天然蛋白多抗血清能与体外表达的蛋白呈现特异性反应,表明表达蛋白具有免疫原性。筛选获得3株稳定分泌抗FlaA的单克隆杂交瘤细胞株,分别命名为2D12、5E12、6A9,其Ig亚类分别为IgG2a、IgG1、IgG1,腹水效价分别为1∶102400,1∶102400和1∶51200;Western blot试验显示,3株单抗均能与表达rHis-FlaA重组蛋白的细菌发生特异性反应;Dot-ELISA试验表明,3株单抗均能与不同来源的空肠弯曲菌分离株发生特异性反应。【结论】本研究制备的单克隆抗体有较高特异性,具有良好的应用价值。为进一步研究空肠弯曲菌鞭毛蛋白的生物学特性、致病机理,以及建立快速检测技术奠定基础。     

鼠冠状病毒核衣壳蛋白的抗体制备与抗原决定簇分析

核衣壳(nucleocapsid,N)蛋白有稳定病毒基因组、调控病毒复制及细胞状态的特殊作用。鼠肝炎病毒(murine hepatitis virus,MHV)为乙型冠状病毒属的原型病毒,是研究冠状病毒N蛋白功能的经典模型。本研究用去污剂处理鼠冠状病毒粒子暴露N蛋白,另用原核表达纯化的重组N蛋白分别免疫小鼠,制备多克隆及单克隆抗体。酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和蛋白免疫印迹分析结果显示两类抗体均具有高灵敏度和特异度,与甲型冠状病毒猪传染性胃肠炎病毒(porcine transmissible gastroenteritis virus,TGEV)的N蛋白无交叉反应。原核表达缺失突变的N蛋白分析结果显示,多克隆抗体与单克隆抗体2E6识别的鼠冠状病毒N蛋白抗原决定簇完全一致,位于N端结构域(N-terminal domain,NTD)C端与SR之间的58个氨基酸残基内。此外,基于单克隆抗体2E6的ELISA及免疫荧光法能检测到感染细胞中和培养上清液中的N蛋白组分,且其含量与病毒复制的滴度一致。这些结果表明,鼠冠状病毒复制过程中粒子与细胞中的N蛋白可能维持相似的结构,使NTD与SR之间的部分氨基酸残基一直暴露在表面,从而形成了优势抗原决定簇。     

Immunization method for multi-pass membrane proteins using highly metastatic cell lines

A novel method using metastatic breast cancer cell lines was established for producing monoclonal antibodies (mAbs) against multi-span membrane proteins. Grafting of metastatic cells (MCF7-14) into the mammary gland of BALB/cJ/nu/nu mice induced splenic hypertrophy (1.6–3.0 × 10⁸ cells/spleen [n = 6]). More than half of the mAbs against MCF7-14 cells reacted with the cell membrane. Inducing production of antibodies against the extracellular domain of multi-pass membrane proteins is difficult. Because the protein structure becomes more complex as the number of transmembrane domains increases, preparing antigens for immunization in which the original structure is maintained is challenging. Using highly metastatic MDA-MB231 cells as the host cell line, we produced mAbs against a 12 transmembrane protein, solute carrier family 6 member 6 (SLC6A6), as a model antigen. When SLC6A6-overexpressing MDA-MB231 cells were grafted into nude mice, the number of splenocytes increased to 2.7–11.4 × 10⁸ cells/spleen (n = 10). Seven mAb-producing clones that not only recognized the extracellular domain of SLC6A6 but also were of the IgG subclass were obtained. Immunocytochemistry and flow cytometry analyses revealed that these mAbs recognized the native form of the extracellular domain of SLC6A6 on the cell surface. Our novel immunization method involving highly metastatic cells could be used to develop therapeutic mAbs against other multi-pass membrane proteins.     

Non-specific immunoprecipitation of rotavirus Vp6 protein with Staphylococcus aureus

The specificity of Staphylococcus aureus and protein A-Sepharose (PA-S) were compared in the radioimmunoprecipitation assay for the characterization of monoclonal antibodies (mAbs) against rotavirus proteins. Five mAbs directed against bovine rotavirus Q17 proteins Vp6 and Vp7 and one mAb directed against human rotavirus protein Vp4 were used in this study. mAbs directed against other viruses, NS-1 culture supernatant and ascitic fluid, were used as control reagents. A non-specific immunoprecipitation of the viral protein Vp6 was always found with S. aureus, but not with PA-S. mAb 74 reacted with rotavirus antigens in ELISA and in indirect immunofluorescence assay but did not immunoprecipitate a viral protein with PA-S. This mAb immunoprecipitated the viral protein Vp6 when S. aureus reagent was used. This false positive reaction was always present and could lead to confusing results in the analysis and characterization of mAbs against rotavirus.     

Immunoprotective human monoclonal antibodies against five major serotypes of Pseudomonas aeruginosa

Human monoclonal antibodies (Mabs) against the O antigens of Pseudomonas aeruginosa lipopolysaccharides (LPS) were produced by cell fusion between human tonsillar lymphocytes and P3-X63-Ag8-U1 (P3U1) mouse myeloma cells. To obtain human Mabs efficiently, 6 d culture supernatants of pokeweed-mitogen-stimulated lymphocytes (21 cultures from peripheral blood and 76 from tonsils) were assayed by ELISA. Five tonsillar lymphocytes which produced IgG antibody specific for P. aeruginosa LPS were preselected for fusion. The human Mabs, named P1-1 (IgG2, kappa), P5-1 (IgG2, lambda), P7-1 (IgG2, lambda), P8-1 (IgG2, lambda) and P10-1 (IgG2, kappa), bound with high specificity to Homma standard serotype strains A, E, B, G and I, respectively, and recognized O antigens. Each Mab showed opsonophagocytic killing activity of the corresponding serotype strain. Four of the Mabs caused agglutination at a very low concentration; a rather higher concentration of P7-1 was required for this effect. Although all the Mabs conferred type-specific protection against peritoneal infection, the strongly agglutinating Mabs provided better protection than the moderately agglutinating P7-1. The protective activity of P8-1 was estimated in compromised mice. A low dose (PD50 0.5-0.6 microgram per mouse) of P8-1 prevented subcutaneous infection in burned mice and peritoneal infection in leucopenic mice. All the hybridomas described here could be cultured in serum-free medium, and they have continued to secrete human Mabs for more than 14 months at rates of 10-20 micrograms per 10(6) cells in 24 h. These results suggested that these five human Mabs specific for O antigens might be useful in the prophylaxis and treatment of P. aeruginosa infections.     

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.     

Development,Characterization and Application of Monoclonal Antibodies against Brazilian Dengue Virus Isolates

Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV) detection through the production and characterization of 22 monoclonal antibodies (mAbs) against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some mAbs cross-reacted with yellow fever virus (YFV), West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV). None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV). Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA) for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.     

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae

Abstract Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognize serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG_2a, IgG_2b, and IgG₃ were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group I (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.     

Development of a humanized antibody with high therapeutic potential against dengue virus type 2

Background

Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control.

Methodology/Principal Findings

We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials.

Conclusions/Significance

We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.     

Development and characterization of monoclonal antibodies specific for the genus Listeria

Abstract Monoclonal antibodies were obtained by the classic hybridoma technique with lymphocytes of BALB/c mice immunized with formalin killed Listeria monocytogenes cells. Among 1000 hybridomas issued from the fusion, four monoclonal antibodies (mAbs A6 A E4, C10 A F7, G4 A D6, G7 A D5) gave interesting results. By Western-blot analysis with various soluble extracts of different Listeria species, the four mAbs reacted with two major antigens of 38 and 41 kDa, with all Listeria species tested. The mAb A6 A E4 is an IgG2b with κ light chains and reacted only with Listeria antigens without any cross reaction with other organisms tested by ELISA, dot-blotting and Western-blotting. With the same conditions, the three other mAbs reacted with Listeria and with other genus extracts, particularly with Streptococcus and Enterococcus . mAb A6 A E4-reactive antigens are proteins, and glycoprotein immunoassay indicated that the epitope is devoid of carbohydrate moiety. This mAb A6 A E4-reactive protein was neither expressed on cell surface nor released outside the bacteria; immunogold electron microscopy showed that these antigens were localized in the cytoplasma area.     

Characterization of two monoclonal antibodies, 38F10 and 44D11,against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor(F_0) and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies(mAbs) against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus(HearNPV)(Ha F) were generated. Two kinds of mAbs were obtained according to their different recognition epitopes: one kind of mAbs, as represented by 38F10,recognizes amino acid(aa) 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process.     

Development and Evaluation of a DAS-ELISA for Rapid Detection of Tembusu Virus Using Monoclonal Antibodies against the Envelope Protein

Since April 2010, Tembusu virus (TMUV) which is a contagious pathogen of waterfowls, causing symptoms of high fever, loss of appetite and fall in egg production, has been reported in east of China. A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) which detects for TMUV was developed, using two monoclonal antibodies (mAbs) against the TMUV envelope (E) protein. BALB/c mice were immunized with purified recombinant E protein expressed in E. coli. Three hybridoma cell lines designated as 12B1, 10C6 and 2D2, were screened by cell fusion and indirect ELISA for their ability to recognize different linear epitopes on the E protein, and were characterized subsequently. High-affinity mAbs 12B1 and 2D2 were used as capture and detection antibodies, respectively. The reaction conditions for the DAS-ELISA were optimized for TMUV detection. The cross-reactivity of the DAS-ELISA was determined using TMUV, duck plague virus, avian influenza virus subtype H9, Newcastle disease virus, duck hepatitis A virus type 1 and duck reovirus samples. A total of 191 homogenized tissues of field samples were simultaneously detected by DAS-ELISA and by RT-PCR. The former was found to have a high specificity of 99.1% and a sensitivity of 93.1%. These results reveal a positive coincidence between DAS-ELISA and RT-PCR at a coincidence rate of 95.8%. The method developed in this study can be used for the diagnosis of TMUV infection of duck origin.     

New developments in malaria diagnostics: Monoclonal antibodies against Plasmodium dihydrofolate reductase-thymidylate synthase,heme detoxification protein and glutamate rich protein

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, K_D) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have K_D values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR-TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (K_D of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13), making them promising reagents for further test development.Key words: plasmodium, Plasmodium falciparum, malaria, diagnostics, rapid diagnostic test, monoclonal antibodies, glutamate rich protein, dihydrofolate reductase-thymidylate synthase, heme detoxification protein