Production of species-specific monoclonal antibodies that react with surface components on zoospores and cysts of Phytophthora nicotianae

1998 ◽  
Vol 44 (12) ◽  
pp. 1161-1170 ◽  
Author(s):  
A V Robold ◽  
A R Hardham
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Phytophthora Cinnamomi ◽  
Phytophthora Nicotianae ◽  
The Other ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Hybridoma Cell Lines ◽  
Species Specific

Monoclonal antibodies were generated against components on the surface of zoospores and cysts of the Oomycete, Phytophthora nicotianae, with the aim of obtaining antibodies diagnostic for this species of plant pathogen. A dipstick version of an enzyme-linked immunosorbent assay was used to screen hybridoma cell lines produced by following a coimmunization protocol in which a mouse was immunized with Phytophthora nicotianae cysts mixed with murine antisera raised against cysts of Phytophthora cinnamomi and Phytophthora cryptogea. Of the nine hybridoma cells lines which remained positive, five produced antibodies that reacted with species-specific epitopes on the surface of the spores. Immunofluorescence, immunogold, and immunoblot labelling showed that three of the five species-specific antibodies reacted with a polypeptide of relative molecular mass greater than 205 kDa which was distributed over the entire zoospore surface, including that of the two flagella. These antibodies also labelled the surface of cysts to varying degrees. The other two species-specific antibodies bound to the shaft of tubular mastigonemes that form two rows on the anterior flagellum. In immunoblots, one of these antibodies recognised a 40-kDa glycoprotein. Antibodies produced by the other four hybridoma cell lines reacted with all Phytophthora and Pythium species tested. The results (i) showed that the coimmunization technique effectively produced antibodies directed towards components specific for Phytophthora nicotianae in the presence of antigens common to many Phytophthora species, and (ii) revealed for the first time the biochemical nature of molecular constituents of flagellar mastigonemes in the Oomycetes.Key words: cell surface, flagella, immunodiagnostics, mastigonemes, monoclonal antibodies.

scholarly journals Identification and Subgrouping of Cucumber mosaic virus with Mouse Monoclonal Antibodies

2000 ◽  
Vol 90 (6) ◽  
pp. 615-620 ◽  
Author(s):  
H. T. Hsu ◽  
L. Barzuna ◽  
Y. H. Hsu ◽  
W. Bliss ◽  
K. L. Perry
Keyword(s):  
Monoclonal Antibodies ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Cell Lines ◽  
Reliable Method ◽  
Specific Antibodies ◽  
Efficient Detection ◽  
Subgroup Ii ◽  
The World ◽  
Hybridoma Cell Lines

Using a mixture of isolates of Cucumber mosaic virus (CMV) from subgroups I and II as immunogens, 20 mouse hybridoma cell lines secreting monoclonal antibodies were produced. A reliable method for efficient detection and accurate subgrouping of CMV isolates has been developed. Tests with 12 well-characterized strains of CMV and other cucumoviruses demonstrated the presence of epitopes that were virus and subgroup specific. Analyses of 109 accessions of CMV isolates collected from various parts of the world revealed 70% were subgroup I, with 20% identified as subgroup II. Seven isolates (6%) did not react with group-specific antibodies but did react with antibodies that recognized all CMV isolates. Differential reactions among isolates suggested a total of 10 epi-topes were recognized. The antigenic diversity among subgroup II CMVs was greater than for the subgroup I isolates, even though fewer subgroup II isolates were tested.

Monoclonal antibodies to isolate-, species-, and genus-specific components on the surface of zoospores and cysts of the fungus Phytophthora cinnamomi

1986 ◽  
Vol 64 (2) ◽  
pp. 311-321 ◽  
Author(s):  
A. R. Hardham ◽  
E. Suzaki ◽  
J. L. Perkin
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Fungal Pathogens ◽  
Phytophthora Cinnamomi ◽  
Pathogenic Fungus ◽  
Immunofluorescence Assay ◽  
Pythium Species ◽  
Specific Antigens ◽  
Species Specific ◽  
First Time

Monoclonal antibodies have been raised to components on the surface of glutaraldehyde-fixed zoospores and cysts of an isolate of the pathogenic fungus Phytophthora cinnamomi. Hybridoma supernatants were screened using an immunofluorescence assay, and of 35 cell lines producing antibodies that reacted with the P. cinnamomi cells, 10 have been selected and their specificities examined in detail. The monoclonal antibodies were found to possess a valuable spectrum of taxonomic specificities, and have revealed, for the first time, the presence of isolate-specific antigens on the surface of P. cinnamomi cells. The monoclonal antibodies were tested against six isolates of P. cinnamomi, six species of Phytophthora, and three species of Pythium. In addition to the isolate-specific monoclonal antibodies, species-specific and genus-specific markers which are unambiguous in tests conducted so far have been obtained. The monoclonal antibodies have also revealed the presence of spatially restricted antigens on the surface of the zoospores. Some of these segregated antigens are species-specific and others are more general, occurring in all Phytophthora and Pythium species examined. All of the monoclonal antibodies promise to be of great assistance in investigations of the biology and taxonomy of P. cinnamomi. The methods described should be readily applicable to studies of other fungal pathogens.

Development of a gold nanoparticle enhanced enzyme linked immunosorbent assay based on monoclonal antibodies for the detection of fumonisin B1, B2, and B3in maize

2018 ◽  
Vol 10 (28) ◽  
pp. 3506-3513 ◽  
Author(s):  
Zhi Li ◽  
Wei Sheng ◽  
Qi Liu ◽  
Shijie Li ◽  
Yingjie Shi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Gold Nanoparticle ◽  
Cell Lines ◽  
Heavy Chain ◽  
Light Chain ◽  
Fumonisin B1 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Kappa Light Chain ◽  
Cell Cloning ◽  
Hybridoma Cell Lines

In this paper, three hybridoma cell lines that secrete monoclonal antibodies against fumonisin B1(FB1), specifically antibody subtypes IgA (heavy-chain) and kappa (light-chain), were obtained by immunization and cell cloning procedures.

Three Highly Sensitive and High-Throughput Serological Approaches for Detecting Dickeya dadantii in Sweet Potato

Plant Disease ◽  
2021 ◽  
Vol 105 (4) ◽  
pp. 832-839
Author(s):  
Wanqin He ◽  
Deqing Huang ◽  
Jiayu Wu ◽  
Xue Li ◽  
Yajuan Qian ◽  
...  
Keyword(s):  
Sweet Potato ◽  
Cell Lines ◽  
Immunosorbent Assay ◽  
Root Rot ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dickeya Dadantii ◽  
Crude Extracts ◽  
Tissue Print ◽  
Hybridoma Cell Lines ◽  
Dot Elisa

Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using Dickeya dadantii strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for D. dadantii detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect D. dadantii in suspensions diluted to 4.89 × 104, 4.89 × 104, 9.78 × 104, and 9.78 × 104 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four D. dadantii strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of D. dadantii in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect D. dadantii in suspensions diluted up to 1.23 × 104 and 1.17 × 106 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting D. dadantii in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.

scholarly journals Development of Opsonic Mouse Monoclonal Antibodies against Multidrug-Resistant Enterococci

2019 ◽  
Vol 87 (9) ◽  
Author(s):  
Ermioni Kalfopoulou ◽  
Diana Laverde ◽  
Karmela Miklic ◽  
Felipe Romero-Saavedra ◽  
Suzana Malic ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Selection Process ◽  
Multidrug Resistant ◽  
Expression Vectors ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Strains ◽  
Variable Regions ◽  
Content Type

ABSTRACTMultidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide ofEnterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein fromEnterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing againstE. faecalistype 2, and SagA.01 showed 40% killing againstE. faecium11231/6. In addition, both MAbs showed cross-reactivity toward otherE. faecalisandE. faeciumstrains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.

Rapid, sensitive two-site ELISA for detection of cows' milk in goats' or ewes' milk using monoclonal antibodies

1994 ◽  
Vol 61 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Didier Levieux ◽  
Annie Venien
Keyword(s):  
Monoclonal Antibodies ◽  
Sandwich Elisa ◽  
High Specificity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Factors Affecting ◽  
Assay Performance ◽  
Capture Antibody ◽  
Constant Quality ◽  
Species Specific ◽  
Β Lactoglobulin

SummaryA sandwich ELISA (enzyme-linked immunosorbent assay) of the two-site type has been successfully developed for the detection of cows' milk in goats' or ewes' milk. The assay uses two monoclonal antibodies (MAb) raised in mice against cows' β-lactoglobulin (β-lg). These MAb recognize different epitopes of the β-lg, which are sufficiently distinct to allow simultaneous binding of the corresponding antibodies. One of the MAb recognizes a species-specific epitope of the bovine β-lg and was adsorbed to a plastic microtitration plate (capture antibody). The second MAb was labelled with peroxidase and used to detect the captured cows' β-lg. Factors affecting assay performance were investigated. The optimized assay is highly specific, reproducible (intra- and inter-assay CV were 8 and 13% respectively) and sensitive: as little as 5 ng β-lg/ml or 1 part cows' milk per 100000 parts goats' or ewes' milk can be detected. The technique is robust, cheap, rapid, reliable and suitable for high sample throughput, semi-automation and screening surveys. The MAb used guarantee the high specificity of the assay and indefinite reagent supply of constant quality once approved by collaborative national or international trials.

scholarly journals Hybridoma technology; advancements, clinical significance, and future aspects

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Sanchita Mitra ◽  
Pushpa Chaudhary Tomar
Keyword(s):  
Monoclonal Antibodies ◽  
Clinical Significance ◽  
Cell Lines ◽  
Tumor Antigens ◽  
Specific Antigen ◽  
Hybridoma Cells ◽  
Main Body ◽  
Highly Sensitive ◽  
Hybridoma Technology

Abstract Background Hybridoma technology is one of the most common methods used to produce monoclonal antibodies. In this process, antibody-producing B lymphocytes are isolated from mice after immunizing the mice with specific antigen and are fused with immortal myeloma cell lines to form hybrid cells, called hybridoma cell lines. These hybridoma cells are cultured in a lab to produce monoclonal antibodies, against a specific antigen. This can be achieved by an in vivo or an in vitro method. It is preferred above all the available methods to produce monoclonal antibodies because antibodies thus produced are of high purity and are highly sensitive and specific. Main body of the abstract Monoclonal antibodies are useful in diagnostic, imaging, and therapeutic purposes and have a very high clinical significance. Once hybridoma cells become stable, these cell lines offer limitless production of homogenized antibodies. This method is also cost-effective. The antibodies produced by this method are highly sensitive and specific to the targeted antigen. It is an important tool used in various fields of research such as in toxicology, animal biotechnology, medicine, pharmacology, cell, and molecular biology. Monoclonal antibodies are used extensively in the diagnosis and therapeutic applications. Radiolabeled monoclonal antibodies are used as probes to detect tumor antigens in the living system; also radioisotope coupled antibodies are used for therapeutic target specific action on oncogenic cells. Short conclusion Presently, the monoclonal antibodies used are either raised in mice or rats; this poses a risk of disease transfer from mice to humans. There is no guarantee that antibodies thus created are entirely virus-free, despite the purification process. Also, there are some immunogenic responses observed against the antibodies of mice origin. Technologically advanced techniques such as genetic engineering helped in reducing some of these limitations. Advanced methods are under development to make lab-produced monoclonal antibodies as human as possible. This review discusses the advantages and challenges associated with monoclonal antibody production, also enlightens the advancement, clinical significance, and future aspects of this technique.

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