Pair Of mAbs Research Articles

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric “RCR-complex”. We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called “R78C”, combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena

BackgroundAntigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA.MethodsWe report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland–Altman (BA) analysis and Lin’s concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA.ResultsThe TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin’s CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin’s CCC: 0.24) but increased on days 28 and 90 PI (Lins’ CCC: 0.88 and 0.98, P < 0.001).Conclusions/significanceThe TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.Graphical

Rapid detection of varicella-zoster virus based on an immunochromatographic strip

Varicella-zoster virus (VZV) is a highly infectious DNA virus that can cause varicella (chickenpox) and herpes zoster (HZ). A simple, sensitive and specific detection method is desirable for the VZV infection. In this study, VZV gE protein, expressed in CHO cells, was used to immunize BALB/c mice for the generation of monoclonal antibodies (mAbs). For the first time, we developed a colloidal gold-based immunochromatographic strip for rapid detection of VZV using a pair of mAbs against gE protein. The limit of detection (LOD) of the strip was 30 ng mL−1 of purified VZV gE antigen, and it could specifically test VZV without cross-reactivity with Enterovirus 71 (EV-71), Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2). The coincidence rate between the strip and commercial real-time PCR diagnostic kit was 100% using vesicle as the clinical sample. Our strip provided a technical support for rapid and specific detection of VZV.

Development of a new immunodiagnostic tool for poultry coccidiosis using an antigen-capture sandwich assay based on monoclonal antibodies detecting an immunodominant antigen of Eimeria

This study was conducted to develop an antigen-capture ELISA that detects an immunodominant antigen of Eimeria, 3-1E which is present in all Eimeria species, using a set of 3-1E-specific mouse monoclonal antibodies (mAbs). Highly sensitive 3-1E-specific antigen-capture ELISA was established using compatible mAb pairs (#318 and #320) selected from 6 mAbs (#312, #317, #318, #319, #320, and #323) with high binding activity against recombinant 3-1E protein. These anti-3-1E mAbs specifically recognized E. tenella sporozoites and a higher level of 3-1E was detected in the lysate of sporozoites than in sporocysts. Immunofluorescence assay (IFA) using 2 mAbs (#318 and #320) showed specific staining around the membrane of E. tenella sporozoites. In order to measure the changes in the 3-1E level during in coccidiosis, serum, feces, jejunal, and cecal contents were individually collected daily for 7-days postinfection (dpi) with E. maxima and E. tenella. The new ELISA was sensitive and specific for 3-1E detection in all samples collected daily from E. maxima- and E. tenella-infected chickens for a week, and the detection sensitivity ranges were 2 to 5 ng/mL and 1 to 5 ng/mL in serum, 4 to 25 ng/mL and 4 to 30 ng/mL in feces, 1 to 3 ng/mL and 1 to 10 ng/mL in cecal contents, and 3 to 65 ng/mL and 4 to 22 ng/mL in jejunal contents. Following coccidiosis, the overall 3-1E levels started to increase from 4 dpi, and the highest production was shown on 5 dpi. Among the samples collected from Eimeria-infected chickens, the highest detection level was found in the jejunal contents of E. maxima-infected chickens. Furthermore, the level of IFN-γ in serum was significantly (P < 0.05) increased from 3 dpi and peaked on 5 dpi post E. maxima infection. Post E. tenella infection, the level of IFN-γ in serum gradually (P < 0.05) increased from 2 to 5 dpi and plateaued at 7 dpi. The level of TNF-α in serum was rapidly (P < 0.05) increased from 4 dpi and those levels were kept until 7 dpi post both Eimeria infections (E. maxima and E. tenella). More importantly, the daily changes in the 3-1E levels in different samples from E. maxima- and E. tenella-infected chickens were effectively monitored with this new antigen-capture ELISA. Therefore, this new immunoassay is a sensitive diagnostic tool to monitor coccidiosis in a large field population in the commercial poultry farms before clinical symptoms develop using serum, feces, and gut samples during the entire period of infection cycle starting from 1 d after infection.

Development of swine immune reagents for swine immunity and biomedical research

Abstract The USDA-NIFA Swine Immune Toolkit Initiative has been involved in generating priority immune reagents for swine immunity and biomedical research and pipelining them for marketing. With the help of commercial partners we expressed each target immune molecule as a soluble protein using yeast expression systems and then produced panels of monoclonal antibodies (mAbs) against each target. Most recently we generated panels of mAbs reactive with porcine IL-5, IL-6, IL-13, IL-21, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. We determined mAb reactivity with orthologous proteins for most panels of mAbs. A sensitive sandwich ELISA is now available for IL-13 and CXCL10; other targets are being screened for best mAb pairs. Reactivity tests of labeled α-CXCL10, α-IL-6 and α-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assays are underway using different cell stimulation conditions. Immunohistochemistry analyses for binding of α-CXCL10 and other mAbs on formalin fixed pig lymph nodes and spleen tissues are in progress. Further, analysis of a large panel of commercial α-human CD antigen mAbs for cross-reactivity with porcine cells is ongoing. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Finally, efforts are proceeding to develop SLA tetramers for antigen presentation studies. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease and vaccine and use for biomedical research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815 and USDA ARS project 8042-32000-117.

Collagen adhesin protein and necrotic enteritis B-like toxin as biomarkers for early diagnosis of necrotic enteritis in commercial broiler chickens

Mouse monoclonal antibodies (mAbs) reactive with Clostridium perfringens collagen adhesin protein (CNA) and necrotic enteritis B-like toxin (NetB) were developed. The best capture/detection mAb pairs for CNA and NetB were selected based on their affinity and specificity to develop sandwich enzyme-linked immunosorbent assays (ELISAs) to detect CNA and NetB proteins, respectively, in jejunal digesta samples from commercial broiler farms in the United States. Prior to the analysis of samples from commercial broiler flocks, the specificity and sensitivity of the CNA and NetB ELISAs were validated using sera, jejunal digesta, and fecal samples from chickens coinfected with Eimeria maxima and CNA+/NetB+C. perfringens in an animal model of necrotic enteritis (NE). Subsequently, a total of 251 field samples were collected from 74 commercial poultry farms. Among these, 18 samples were from 6 broiler farms that used certified organics (CO), and 155 samples were from 42 farms with nonantibiotics (NA). In jejunal digesta samples, CNA levels ranged from 0.02 to 0.59 ng/mL and NetB levels ranged from 0.09 to 1.91 ng/mL. CNA and NetB levels showed a positive correlation with each other (Pearson correlation coefficient r=0.772, P < 0.001). CNA and NetB levels in jejunal digesta were significantly decreased in CO farms compared with those from NA farms (P < 0.001). In conclusion, these new C. perfringens antigen-specific sandwich ELISAs offer a sensitive and specific means to detect C. perfringens CNA and NetB proteins as biomarkers of early NE occurrence in field samples from commercial broiler chickens.

Development and characterization of mouse monoclonal antibodies to canine morbillivirus

Canine morbillivirus is a highly contagious multi-host pathogen with high morbidity and mortality. Timely diagnosis is of utmost importance to effectively control such a dreadful disease. Monoclonal antibodies (mAbs) serve as a high throughput diagnostics and applied tools for research and development (R&D). In the present study, a total of six mouse monoclonal antibodies were developed. All the mAbs generated belonged to IgG class. Of the six mAbs, two of them, namely CD-2F8 and CD-3D8 were directed against the nucleocapsid protein of CDV as determined in western blotting. The reactivity of all the mAbs was checked in indirect-ELISA and cell-ELISA using different morbilliviruses. The mAbs could broadly be categorized as; CDV specific (CD-3D8 and CD-2F8), cross-reactive to PPR virus (CD-AB3 and CD-4D6) and cross-reactive to both PPR virus and measles virus (CD-5D10 and CD-6E5). The characterized mAbs were used for antigenic profiling of CDV, PPR virus and measles virus. Based on the reactivity pattern; a close antigenic relationship was found among CDV and PPR virus as compared to measles virus. A pair of CDV specific mAbs namely CD-2F8 and CD-3D8 were identified which did not cross-react with measles and PPR viruses and thus could be used for diagnostic applications.

Investigation of the location and secretion features of Candida albicans enolase with monoclonal antibodies

PurposeThe glycolytic enzyme enolase plays important role in the pathogenesis of Candida albicans infection and has been also considered as a promising molecular marker for the diagnosis of invasive candidiasis. This study aimed to investigate the location and secretion features of Candida albicans enolase (CaEno) with a couple of specific monoclonal antibodies (mAbs).MethodsTwo mAbs named 9H8 and 10H8 against CaEno were generated by fusing SP2/0 myeloma cell with the spleen lymphocytes from CaEno immunized mice. The specificity of the mAbs was then validated by Western blot and liquid chromatography-mass spectrometry (LC–MS/MS). A diverse set of experiments were conducted based on the pair of mAbs which involved immunohistochemical staining analysis, whole cell enzyme-linked immunosorbent assay (ELISA), double antibody sandwich ELISA, and confocal microscopy to analyze the possible location and secretion features of CaEno.ResultsCaEno is abundantly expressed in the cytoplasm of C. albicans blastospores and is distributed in a ring-shaped pattern along the cell wall. CaEno appeared in the hyphal C. albicans as just a “mushroom” form. CaEno was found to be weakly expressed on the surface of blastospores but constantly expressed at various stages of growth. CaEno concentrations in C. albicans blastospores culture supernatant are considerably higher than in C. albicans hyphae culture supernatant. The dynamic changes of supernatant CaEno concentration in blastospores and hyphal C. albicans exhibit distinct features, although both appear to be associated with the C. albicans growth state. When cultivated under normal circumstances, however, no apparent CaEno degradation was seen in the cell-free supernatant.ConclusionOur results implied that CaEno was constantly expressed on the cell surface and its secretion features varied according to the growth stage of C. albicans. However, further experimental and theoretical studies are needed in future to identify the specific mechanisms by which this phenomenon can arise.

Development of swine immune reagents for analysis of immune correlates for vaccines, infection, and in biomedical research

Abstract The goal of the USDA-NIFA Swine Immune Toolkit Initiative has been to generate priority immune reagents based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. So far in our efforts we express soluble proteins in yeast system and then produce panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production. So far we generated several new panels of mAbs reactive to porcine IL-6, IL-13, IFN-γ, IL-17A, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. Reactivity tests of labeled anti-IL-6 and anti-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assay are in progress. Our results have confirmed the reactivity of porcine IL-17A, IFN-γ and CXCL10 mAbs. A sensitive sandwich ELISA is now available for IL-17A, IL-13 and CXCL10; other targets are being screened for best mAb pairs for ELISA. Planning has been initiated for the generation of IL-5 and IL-21 mAbs; and use of SLA-I &amp; -II tetramers to identify swine CD4 and CD8 T cells specific for influenza virus peptides. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease, vaccine and biomedical research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815

Development of a sandwich ELISA for the detection of chicken colony-stimulating factor 1

Macrophage colony-stimulating factor-1 (M-CSF-1 or CSF-1) is a hematopoietic growth factor that stimulates the survival, proliferation, and differentiation of the mononuclear phagocyte lineage and is involved in bone metabolism, fertility, pregnancy, inflammatory processes, and homeostasis. CSF-1-activated macrophages display unique features, such as distinguishable cell surface antigens, enhanced Fc-γ-receptor-mediated phagocytosis, intensified reactive oxygen species activity, enhanced proliferation, and enhanced chemotaxis. Five mouse monoclonal antibodies (mAbs) for the detection of chicken CSF-1 were developed and characterized using western blot, indirect ELISA, and in vitro functional assays. One of the anti-chCSF-1 mAbs, 8A12, showed neutralization of chicken macrophage cell line (HD11) proliferation and CSF-induced nitric oxide release, whereas mAb 1G4 inhibited the phagocytosis of fluorescent-labeled E. coli by HD11 cells in vitro. For the quantitative assessment of native chCSF-1 in biological samples from chickens, a sensitive sandwich ELISA was developed using the best capture and detection pair of mAbs that were selected from newly developed anti-chCSF-1 mAbs. Chickens that were challenged with Eimeria acervulina, E. maxima, and E. tenella showed a steady increase in the circulating levels of serum CSF-1, starting from day 1 to 7 postchallenge reaching their peak levels at day 10 postchallenge infection. The CSF-1 synthesis induced by 3 different species of Eimeria was quite similar, even though these they are reported to be phenotypically and immunologically different. Therefore, this mAb-based sandwich ELISA will be a valuable tool for the detection of CSF-1 production during various poultry infections, and these new anti-chCSF-1 mAbs will facilitate the fundamental and applied research related to CSF-1 function in normal and disease states in chickens.

Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect.

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.

Monoclonal antibody pairs against SARS-CoV-2 for rapid antigen test development.

BackgroundThe focus on laboratory-based diagnosis of coronavirus disease 2019 (COVID-19) warrants alternative public health tools such as rapid antigen tests. While there are a number of commercially available antigen tests to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), all cross-react with the genetically similar SARS-CoV-1 or require an instrument for results interpretation.Methodology/Principal findingsWe developed and validated rapid antigen tests that use pairs of murine-derived monoclonal antibodies (mAbs), along with gold nanoparticles, to detect SARS-CoV-2 with or without cross-reaction to SARS-CoV-1 and other coronaviruses. In this development, we demonstrate a robust antibody screening methodology for the selection of mAb pairs that can recognize SARS-CoV-2 spike (S) and nucleocapsid (N) proteins. Linear epitope mapping of the mAbs helped elucidate SARS-CoV-2 S and N interactions in lateral flow chromatography. A candidate rapid antigen test for SARS-CoV-2 N was validated using nasal swab specimens that were confirmed positive or negative by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Test results were image-captured using a mobile phone and normalized signal pixel intensities were calculated; signal intensities were inversely correlated to RT-PCR cycle threshold (Ct) value.Conclusion/SignificanceOverall, our results suggest that the rapid antigen test is optimized to detect SARS-CoV-2 N during the acute phase of COVID-19. The rapid antigen tests developed in this study are alternative tools for wide scale public health surveillance of COVID-19.

Development of Novel Dengue NS1 Multiplex Lateral Flow Immunoassay to Differentiate Serotypes in Serum of Acute Phase Patients and Infected Mosquitoes

Dengue is among the most rapidly spreading arboviral disease in the world. A low-cost, easy to use point-of-care diagnostic tool for the detection and differentiation of dengue virus serotypes could improve clinical management, disease prevention, epidemiological surveillance, and outbreak monitoring, particularly in regions where multiple serotypes co-circulate. Despite widespread deployment, no commercial dengue antigen diagnostic test has proven effective in differentiating among dengue virus serotypes. In the current study, we first established mAb pairs and developed a multiplex lateral flow immunoassay for the simultaneous detection of the dengue viral NS1 antigen and identification of serotype. The proposed system, called Dengue serotype NS1 Multiplex LFIA, provides high sensitivity and specificity. In testing for JEV, ZIKV, YFV, WNV, and CHIKV, the multiplex LFIA gave no indication of cross- reactivity with cell culture supernatants of other flaviviruses or chikungunya virus. In analyzing 187 samples from patients suspected of dengue infection, the detection sensitivity for serotype D1 to D4 was 90.0%, 88.24%, 82.61%, and 83.33% and serotype specificity was 98.74%, 96.13%, 99.39%, and 97.04%, respectively. Our multiplex LFIA can also identify mono- and co-infection of different serotype of dengue viruses in mosquitoes. The proposed Multiplex LFIA provides a simple tool for the rapid detection of dengue serotypes and in the differential diagnosis of fever patients in regions where medical resources are limited and/or multiple DENVs co-circulate.

R346K Mutation in the Mu Variant of SARS-CoV-2 Alters the Interactions with Monoclonal Antibodies from Class 2: A Free Energy Perturbation Study.

The Mu variant of SARS-CoV-2 has been recently classified as a variant of interest (VOI) by the World Health Organization (WHO) but limited data are available at the moment. In particular, special attention was given to the R346K mutation located in the receptor binding domain (RBD). In the current study, we performed free energy perturbation (FEP) calculations to elucidate its possible impact on a set of neutralizing monoclonal antibodies (mAbs) that have been shown to be strong inhibitors of the most other known COVID-19 variants. Our results show that R346K affects class 2 antibodies but its effect is not so significant (0.66 kcal/mol), i.e., it reduces the binding with antibodies by about 3-fold. An identical value was also calculated in the presence of both class 1 and class 2 antibodies (BD-812/836). Further, a similar reduction in the binding (0.4 kcal/mol) was obtained for the BD-821/771 pair of mAbs. For comparison, the addition of the K417N mutation, present in the newly registered Mu variant in July 2021 in the U.K., affected the class 1 mAbs by strongly reducing the binding by 1.29 kcal/mol or about 10-fold. Thus, the resistance effect of the R346K mutation on the Mu variant is possible but not so significant and is due to the additional decrease of antibody neutralization based on the reduced binding of class 2 antibodies.

A novel gold nanosensor and ‘Blockade-of-Binding’ based immunochromatographic rapid antigen test kit for Zika virus

The present study relates to the development of an immunochromatographic, antigen test kit for rapid detection of Zika virus using stable gold nanoparticles (AuNPs). Herein, we have developed an alternate viral NS1 antigen (Ag) based rapid test that uses monoclonal antibody (Mab) pairs to detect ZIKV without cross-reactivity in 5 min. To mask the non-specific results, the “Blockade of Binding' assay was employed in lateral flow kits. Also, for better performance of kits, a sample pad having an absorption capacity of 20 µl/cm2 was utilized. Herein, AuNPs-ZIKA NS1 Mab is capable of binding to specific sites of the Zika antigen. Anti-DENV-MAb is capable of binding to a specific site for Dengue, anti-CHKV-Mab is capable of binding to a specific site for Chikungunya, ZIKA NS1 Mab is capable of binding to anti-ZIKA NS1 Mab and GAM Mab is capable of binding to leftover anti-ZIKA NS1 MAb. The kit was found to be stable for 12 months while its specificity and sensitivity were found to be 95.83% and 100%, respectively.

An Antigen Capture Assay for the Detection of Mycolactone, the Polyketide Toxin of Mycobacterium ulcerans.

Mycolactone is a cytotoxin responsible for most of the chronic necrotizing pathology of Mycobacterium ulcerans disease (Buruli ulcer). The polyketide toxin consists of a 12-membered lactone ring with a lower O-linked polyunsaturated acyl side chain and an upper C-linked side chain. Mycolactone is unique to M. ulcerans and an immunological Ag capture assay would represent an important tool for the study of Buruli ulcer pathogenesis and for laboratory diagnosis. When testing sets of mycolactone-specific mouse mAbs, we found that Abs against the hydrophobic lower side chain only bind mycolactone immobilized on a solid support but not when present in solution. This observation supports previous findings that mycolactone forms micellar structures in aqueous solution with the hydrophobic region sequestered into the inner core of the aggregates. Although an Ag capture assay typically requires two Abs that recognize nonoverlapping epitopes, our search for matching pairs of mAbs showed that the same mAb could be used both as capture and as detecting reagent for the detection of the mycolactone aggregates. However, the combination of a core-specific and a core/upper side chain-specific mAb constituted the most sensitive ELISA with a sensitivity in the low nanogram range. The results of a pilot experiment showed that the sensitivity of the assay is sufficient to detect mycolactone in swab samples from Buruli ulcer lesions. Although the described capture ELISA can serve as a tool for research on the biology of mycolactone, the assay system will have to be adapted for use as a diagnostic tool.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken

Abstract Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed a sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast was used to immunize the mice. Five mAbs which specifically recognize chIFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing assay to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were identified as optimum pairs of antibodies for detection of chIFN-α. The sandwich ELISA effectively detected native chicken IFN-α in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus (VSV) was evaluated in chicken embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize the activity of chicken IFN-α against VSV were identified. The newly developed antigen capture sandwich ELISA for the detection of chIFN-α will be a useful tool to monitor IFN-α production in chickens during viral infections.

Development and characterization of new swine immune reagents to understand immune correlates for vaccines, infection, and biomedical research outcomes

Abstract The USDA-NIFA Swine Immune Toolkit Initiative has a goal to generate priority immune reagents, based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. Our efforts are aimed at expression of soluble proteins and production of panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production. Generation of new panels of mAbs reactive with porcine IL-5 and IL-21 have been initiated. Panels of mAbs to IL-6, IL-13, IL-17A, IL-28B, CXCL10, and BAFF are being screened for their reactivity in multiple immune assays. Reactivity tests of labeled anti-IL-6, -IL-17A, -IL-13 and -CXCL10 mAbs for intracellular staining of porcine myeloid and T cells using flow cytometry-based assays are in progress. Sensitive sandwich ELISA assays are now available for IL-17A, IL-13 and CXCL10; other targets are being screened for best mAb pairs for such assays. Planning for the generation of SLA-I &amp; -II tetramers to identify swine CD4 and CD8 T cells specific for influenza virus peptides has been initiated. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized techniques using these reagents for their research efforts. Tools and reagents generated by this project will undoubtedly advance swine immune, disease, vaccine, and biomedical research efforts.

Novel monoclonal antibody-sandwich immunochromatographic assay based on Fe3O4/Au nanoparticles for rapid detection of fish allergen parvalbumin

In this study, a rapid sandwich immunochromatographic assay (ICA) was developed to detect parvalbumin (PV). Firstly, two optimum primary monoclonal antibody (mAb) against PV had been screened out: mAb1 was used as the capture antibody, and mAb2 conjugated to Fe3O4/Au nanoparticles (Fe3O4/AuNPs) that served as a detection reagent. Using this pair of mAbs, a sandwich ICA strip based on Fe3O4/AuNPs was developed. The results showed that the color intensity of test line positively correlated with the PV concentration in the standard or spiked sample. The limit of detection for qualitative (LOD) and quantitative detection (LOQ) were 2 ng/mL and 0.691 ng/mL, respectively. Besides, the detection time of this ICA strip was within 15 min. The recovery rates ranged from 104.0% to 117.4%, within an acceptable level (80–120%). Moreover, the developed assay also showed high cross reaction in different fish species. These results demonstrated that the established test strip has the potential to be used as a rapid screening tool for large scale determination of PV in foodstuffs.

Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8

The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10−10 M to 10−11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.