Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

Identification of cocoonase and cocoonase like protein using polyclonal antibody of Antheraea mylitta cocoonase.

BACKGROUND: Cocoonase is a proteolytic enzyme released by silk moths during pupal adult emergence. Without damaging the silk fibroin, this enzyme dissolves the shell of the tasar cocoon by exclusively targeting the protein sericin. Prior to this study, there was no available antibody against Antheraea mylitta cocoonase to identify or screen out similar variants or cocoonase like protein. RESULTS: In the present study, naturally secreted A. mylitta cocoonase was purified and used to immunize New Zealand white rabbits. The developed polyclonal antibody of cocoonase was purified and its specific interaction with cocoonase was determined using Indirect ELISA. The confirmation of its specificity and immuno-reactivity was evaluated by western blot using native cocoonase of tasar silkworm A. mylitta. The efficacy and specificity of the polyclonal antibody were further verified and confirmed by western blot which was performed to detect ten different ecotypes of A. mylitta cocoonase. CONCLUSION: The developed antibody successfully detected the cocoonase of different ecotypes. Thus, in future this antibody can serve as one of the molecular detection method for cocoonase and cocoonase-like proteins.

Development of a label-free photoelectrochemical immunosensor for novel astrovirus detection.

Since 2017, an infectious goose gout disease characterized by urate precipitation in viscera, mainly caused by novel goose astrovirus (GoAstV) infection, has emerged in the main goose-producing region of China. The current challenge in managing goose gout disease is largely due to the absence of a rapid and efficient detection method for the GoAstV pathogen. Notably, the potential application of immunosensors in detecting GoAstV has not yet been explored. Herein, a label-free PEC immunosensor was fabricated by using purchased TiO2 as the photoactive material and antibody against GoAstV P2 proteins as the specific recognition element. First, we successfully expressed the capsid spike domain P2 protein of ORF2 from GoAstV CHSH01 by using the pET prokaryotic expression system. Meanwhile, the polyclonal antibody against GoAstV capsid P2 protein was produced by purified protein. To our knowledge, this is the first establishment and preliminary application of the label-free photoelectrochemical immunosensor method in the detection of AstV. The PEC immunosensor had a linear range of 1.83 fg mL-1 to 3.02 ng mL-1, and the limit of detection (LOD) was as low as 0.61 fg mL-1. This immunosensor exhibited high sensitivity, great specificity, and good stability in detecting GoAstV P2 proteins. To evaluate the practical application of the immunosensor in real-world sample detection, allantoic fluid from goose embryos was collected as test samples. The results indicated that of the eight positive samples, one false negative result was detected, while both negative samples were accurately detected, suggesting that the constructed PEC immunosensor had good applicability and practical application value, providing a platform for the qualitative detection of GoAstV.

Cloning, expression of porcine GSDME and identification of its site cleaved by caspase-3.

As a member of the gasdermin family, gasdermin E (GSDME) is specifically cleaved by caspase-3, resulting in pyroptosis. To date, the biological characteristics and functions of human and mouse GSDME have been extensively studied; however, little is known of porcine GSDME (pGSDME). In this study, the full-length pGSDME-FL was cloned, which encodes 495 amino acids (aa) that have closely evolutionary relationships to the homolog of camelus, aquatic mammals, cattle and goat. Moreover, pGSDME was detected at different levels of expression in 21 tissues and 5 pig-derived cell lines tested by qRT-PCR, with the highest expression levels in mesenteric lymph nodes and PK-15 cell lines. Anti-pGSDME polyclonal antibody (pAb) with good specificity was generated by expressing the truncated recombinant protein pGSDME-1-208 and immunizing the rabbits. By western blot analysis using highly specific anti-pGSDME polyclonal antibody (pAb) prepared as primary antibody, it was not only confirmed that paclitaxel and cisplatin were positive stimuli to pGSDME cleavage and caspase-3 activation, but also identified the aspartate (D268) at position 268th of pGSDME as a cleavage site of caspase-3, and the overexpressed pGSDME-1-268 possesses cytotoxicity to HEK-293T cells, indicating that pGSDME-1-268 may contain active domains and involve pGSDME-mediated pyroptosis. These results lay a foundation for further investigating the function of pGSDME, especially its role in pyroptosis and its interaction with pathogens.

Effect of duck interferon-α and an anti-cap protein polyclonal antibody against duck circovirus.

Duck circovirus (DuCV) is one of the most prevalent viruses in the duck breeding industry, and causes persistent infection and severe immunosuppression. Currently, there is a serious lack of prevention and control measures and no commercial vaccine against DuCV. Therefore, effective antiviral drugs are important for treating DuCV infection. Interferon (IFN) is an important component of antiviral innate immunity, but it remains unclear whether duck IFN-α has a clinical effect against DuCV. Antibody therapy is an important way to treat viral infections. The DuCV structural protein (cap) is immunogenic, and it remains to be determined whether an anti-cap protein antibody can effectively block DuCV infection. In this study, the duck IFN-α gene and the DuCV structural protein cap gene were cloned, expressed and purified in Escherichia coli to prepare duck recombinant IFN-α and the cap protein. Then, rabbits were immunized with the recombinant cap protein to prepare a rabbit polyclonal antibody. This study investigated the antiviral effect of duck recombinant IFN-α and the anti-cap protein antibody and their combined effect on Cherry Valley ducks infected with DuCV. The results showed that the treatment significantly alleviated the clinical symptoms of immune organ atrophy and immunosuppression compared with the control. The histopathological damage of the target organs was alleviated, and replication of DuCV in the immune organs was significantly inhibited. The treatment also reduced the damage caused by DuCV to the liver and immune function, and increased the level of the DuCV antibody in the blood, thereby improving antiviral activity. Notably, the combination of duck IFN-α and the polyclonal antibody completely blocked DuCV infection after 13 days under the experimental conditions, showing a better inhibitory effect on DuCV infection than single treatments. These results showed that duck recombinant IFN-α and the anti-cap protein antibody can be used as antiviral drugs to clinically treat and control DuCV infection, particularly the vertical transmission of the virus in breeding ducks.

Preparation of polyclonal antibodies against the Drosophila deacetylases SIRT 6 and SIRT 7.

SIRT6 and SIRT7, as members of the Sirtuins family, are indispensable for the growth and development of Drosophila. They play crucial roles in maintaining genome stability, regulating metabolic senescence, and controlling tumorigenesis. To investigate their involvement in the Drosophila life cycle, we focused on describing the expression and purification of recombinant Drosophila SIRT6 and SIRT7 proteins. Subsequently, these proteins were utilized for generating polyclonal antibodies against Drosophila SIRT6 and SIRT7. The recombinant expression plasmid was introduced into E. coli cells to enable the production of SIRT6 and SIRT7 proteins. Following immunizations of New Zealand white rabbits and guinea pigs with the recombinant proteins as antigens, specific polyclonal antisera against both proteins were obtained. After purification, the specificity of SIRT6 and SIRT7 was confirmed using ELISA and western blot analyses, demonstrating strong specificity. These antibodies hold promise for the development of detection assays required for further research.

Preparation of polyclonal anti-Schistosoma mansoni cysteine protease antibodies for early diagnosis.

In many parts of the tropics, schistosomiasis is a major parasitic disease second only to malaria as a cause of morbidity and mortality. Diagnostic approaches include microscopic sampling of excreta such as the Kato-Katz method, radiography, and serology. Due to their vital role in many stages of the parasitic life cycle, proteases have been under investigation as targets of immunological or chemotherapeutic anti-Schistosoma agents. Five major classes of protease have been identified on the basis of the peptide hydrolysis mechanism: serine, cysteine, aspartic, threonine, and metalloproteases. Proteases of all five catalytic classes have been identified from S. mansoni through proteomic or genetic analysis. The study aimed to produce polyclonal antibodies (pAbs) against schistosomal cysteine proteases (CP) to be used in the diagnosis of schistosomiasis. This study was conducted on S. mansoni-infected patients from highly endemic areas and from outpatients' clinic and hospitals and other patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids). In this study, the produced polyclonal antibodies against S. mansoni cysteine protease antigens were labeled with horseradish peroxidase (HRP) conjugate and used to detect CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA. The study involved 200 S. mansoni-infected patients (diagnosed by finding characteristic eggs in the collected stool samples), 100 patients infected with other parasites (Fasciola, hookworm, hydatid, and trichostrongyloids), and 100 individuals who served as parasite-free healthy negative control. The prepared pAb succeeded in detecting CP antigens in stool and serum samples of S. mansoni-infected patients by sandwich ELISA with a sensitivity of 98.5% and 98.0% respectively. A positive correlation was observed between S. mansoni egg counts and both stool and serum antigen concentrations. Purified 27.5 kDa CP could be introduced as a suitable candidate antigen for early immunodiagnosis using sandwich ELISA for antigen detection. KEY POINTS: • Detection of cysteine protease antigens can replace parasitological examination. • Sandwich ELISA has a higher sensitivity than microscopic examination of eggs. • Identification of antigens is important for the goal of obtaining diagnostic tools.

PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')2.

Therapeutic antibodies-F(ab')2 obtained from hyperimmune equine plasma could treat emerging infectious diseases rapidly because of their high neutralization activity and high output. However, the small-sized F(ab')2 is rapidly eliminated by blood circulation. This study explored PEGylation strategies to maximize the half-life of equine anti-SARS-CoV-2 specific F(ab')2. Equine anti-SARS-CoV-2 specific F(ab')2 were combined with 10 KDa MAL-PEG-MAL in optimum conditions. Specifically, there were two strategies: Fab-PEG and Fab-PEG-Fab, F(ab')2 bind to a PEG or two PEG, respectively. A single ion exchange chromatography step accomplished the purification of the products. Finally, the affinity and neutralizing activity was evaluated by ELISA and pseudovirus neutralization assay, and ELISA detected the pharmacokinetic parameters. The results displayed that equine anti-SARS-CoV-2 specific F(ab')2 has high specificity. Furthermore, PEGylation F(ab')2-Fab-PEG-Fab had a longer half-life than specific F(ab')2. The serum half-life of Fab-PEG-Fab, Fab-PEG, and specific F(ab')2 were 71.41 h, 26.73 h, and 38.32 h, respectively. The half-life of Fab-PEG-Fab was approximately two times as long as the specific F(ab')2. Thus far, PEGylated F(ab')2 has been prepared with high safety, high specificity, and a longer half-life, which could be used as a potential treatment for COVID-19.

Dengue Virus Serotype 1 Conformational Dynamics Confers Virus Strain-Dependent Patterns of Neutralization by Polyclonal Sera.

Dengue virus cocirculates globally as four serotypes (DENV1 to -4) that vary up to 40% at the amino acid level. Viral strains within a serotype further cluster into multiple genotypes. Eliciting a protective tetravalent neutralizing antibody response is a major goal of vaccine design, and efforts to characterize epitopes targeted by polyclonal mixtures of antibodies are ongoing. Previously, we identified two E protein residues (126 and 157) that defined the serotype-specific antibody response to DENV1 genotype 4 strain West Pac-74. DENV1 and DENV2 human vaccine sera neutralized DENV1 viruses incorporating these substitutions equivalently. In this study, we explored the contribution of these residues to the neutralization of DENV1 strains representing distinct genotypes. While neutralization of the genotype 1 strain TVP2130 was similarly impacted by mutation at E residues 126 and 157, mutation of these residues in the genotype 2 strain 16007 did not markedly change neutralization sensitivity, indicating the existence of additional DENV1 type-specific antibody targets. The accessibility of antibody epitopes can be strongly influenced by the conformational dynamics of virions and modified allosterically by amino acid variation. We found that changes at E domain II residue 204, shown previously to impact access to a poorly accessible E domain III epitope, impacted sensitivity of DENV1 16007 to neutralization by vaccine immune sera. Our data identify a role for minor sequence variation in changes to the antigenic structure that impacts antibody recognition by polyclonal immune sera. Understanding how the many structures sampled by flaviviruses influence antibody recognition will inform the design and evaluation of DENV immunogens. IMPORTANCE Dengue virus (DENV) is an important human pathogen that cocirculates globally as four serotypes. Because sequential infection by different DENV serotypes is associated with more severe disease, eliciting a protective neutralizing antibody response against all four serotypes is a major goal of vaccine efforts. Here, we report that neutralization of DENV serotype 1 by polyclonal antibody is impacted by minor sequence variation among virus strains. Our data suggest that mechanisms that control neutralization sensitivity extend beyond variation within antibody epitopes but also include the influence of single amino acids on the ensemble of structural states sampled by structurally dynamic virions. A more detailed understanding of the antibody targets of DENV-specific polyclonal sera and factors that govern their access to antibody has important implications for flavivirus antigen design and evaluation.

Promising targets for immunotherapeutic approaches against Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen associated with the occurrence of outbreaks in hospital settings, especially in Intensive Care Units (ICUs). Pneumonia, septicemias, urinary tract infections, and recurrent infections associated with the use of hospital devices are some of the most frequent complications. Due to the increase of A. baumannii strains resistant to multiple antimicrobials, associated with their high capacity to form biofilms on biotic and abiotic surfaces, the therapeutic strategies routinely applied have become increasingly restricted and ineffective, resulting in high mortality rates. Many studies have focused on the development of new therapeutic approaches, mainly non-antibiotic-based. Polyclonal (pAb) and monoclonal (mAb) antibodies are increasingly cited in the literature as alternative strategies to the use of antimicrobial drugs and are evaluated for the prevention and treatment of these infections. In this context, this review aimed to synthesize the main strategies based on the use of antibodies that are being evaluated for the control of A. baumannii infections. Here, we describe the main A. baumannii antigenic targets used in studies to assess their potential to induce the formation of antibodies and their possible application in immunotherapy strategies.

Production and characterization of polyclonal and monoclonal antibodies of lamprey pore-forming protein.

In the most primitive jawless vertebrate lamprey, the complement-dependent cytotoxicity regulated by variable lymphocyte receptors (VLRs) plays an important role in the adaptive immunity. Our previous studies have shown that the lamprey pore-forming protein (LPFP) acted as the terminal effector of VLR to lyse and kill the target cells. Here, the recombinant GST-LPFP protein was expressed and purified in prokaryotic expression system, and then used as the immunogen to produce mouse monoclonal antibody and rabbit polyclonal antibody. With these antibodies, we proved that LPFP existed as homodimers in the lamprey serum, and could be recruited to the membrane of target cells after stimulation. In conclusion, the antibodies we produced could specifically recognize the LPFP protein, which could be the useful tools to further study the pore-forming mechanism of LPFP.

Preparation and characterization of a polyclonal antibody against PTEN-Long.

Phosphatase and tensin homolog-long (PTEN-L) is a translational isoform of PTEN, which exists in both intracellular and extracellular locations. Previous studies demonstrated that PTEN-L could inhibit oncogenesis due to its lipid phosphatase activity. However, recent studies found that PTEN-L could promote the proliferation of some types of cancer cells. Moreover, as a protein phosphatase, PTEN-L can suppress mitophagy by counteracting PTEN-induced putative kinase protein 1 (PINK1)-Parkin-mediated ubiquitin phosphorylation, namely, PTEN-L is critical for exploring the mitophagy progression and the treatment of mitochondrial diseases. Accounting for the critical functions of PTEN-L, its antibody can be used for the treatment or prognosis of tumors and mitochondrial diseases. Currently, the commercial antibody of PTEN-L is not available. In our study, the recombinant PTEN-L protein was expressed in Escherichia coli BL21 and used as an antigen to immunize Japan's big-eared white rabbit for the preparation of polyclonal antibody. The PTEN-L protein can be captured by PTEN-L antibody specifically and effectively. Taken together, a PTEN_L antibody is a valuable tool for further exploring the function of PTEN-L in oncogenesis and mitochondrial diseases, and it would be a new choice for the prognosis or treatment of cancer and mitochondrial diseases.

Development of gold Immunochromatographic assay strip based on specific polyclonal antibodies against capsid protein for rapid detection of porcine circovirus 2 in Zhejiang province, China.

BACKGROUND: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. RESULTS: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. CONCLUSIONS: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs.

Voltammetric immunoassay based on MWCNTs@Nd(OH)3-BSA-antibody platform for sensitive BSA detection.

An electrochemical approach is presented based on multiwall carbon nanotubes (MWCNTs) and neodymium(III) hydroxide (Nd(OH)3) nanoflakes for detection of bovine serum albumin (BSA). The materials were characterized morphologically (XRPD, SEM, and HR-TEM) and electrochemically (DPV, EIS). The MWCNTs@Nd(OH)3 composite was used as support for bovine serum albumin polyclonal antibody (anti-BSA). After the antibody immobilization on the electrochemical platform and antigen/antibody binding time (optimum 60 min), the proposed approach shows a linear voltammetric response toward BSA concentration in the range 0.066 to 6.010 ng mL-1 at maximum peak potential of 0.13 V (vs. Ag/AgCl). Limit of detection (LOD) and limit of quantification (LOQ) were 18 pg mL-1 and 61 pg mL-1, respectively. The precision of the method calculated as relative standard deviation (RSD) of five independent measurements was better 3%. The selectivity of the optimized method regarding structurally similar proteins (human serum albumin and human hemoglobin), ions (Na+, K+, Ca2+, and NO2-), or compounds (glucose, ascorbic acid, dopamine, uric acid, paracetamol, and glycine) was found to be satisfactory, with the current changes of less than 5% in the presence of up to 1 × 105 times higher concentrations (depending on the compound) of the listed potential interfering compounds. Practical applicability of immunosensor for BSA determination in cow whey sample, with recovery values in the range 97 to 103%, shows that the developed method has high potential for precise and accurate detection of BSA, as well as exceptional miniaturization possibilities for on-site and equipment-free sensing.

The preparation of polyclonal antibody against chlordimeform and establishment of detection by indirect competitive ELISA.

Objective: Chlordimeform is a chemical pesticide that is highly carcinogenic and toxic. The purpose of this study was to establish an enzyme-linked immunosorbent assay (ELISA) method for the detection of chlordimeform in aquaculture and fish farming. METHODS: Chlordimeform was coupled with bovine serum albumin (BSA) and ovalbumin (OVA) as carrier proteins. A chlordimeform-BSA conjugate was used as an immunogen, and chlordimeform-OVA was used as a coating antigen. Chlordimeform-BSA was used to immunize rabbits, and a polyclonal antibody was prepared. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was established to detect chlordimeform. RESULTS: The working range of the established IC-ELISA method for chlordimeform detection was 1-20 ng/mL. The IC50 was 3.126 ng/mL, and the lower limit of detection (LOD) of chlordimeform was 0.637 ng/mL. The recovery of chlordimeform from spiked water samples ranged from 81% to 107%. CONCLUSION: An anti-chlordimeform polyclonal antibody was successfully developed, and a novel IC-ELISA was established to detect chlordimeform in aquaculture.

Designing of immunodiagnostic assay using polyclonal antibodies for detection of Shiga toxin producing pathogenic E. coli (STEC) strains.

Shiga toxin-producing Escherichia coli (STEC) are the leading causes of diarrhea and death of humans worldwide. Many diagnostic assays have been developed to aid for the diagnosis of STEC strain; however, they have limitations. Thus, this study was aimed at designing rapid, effective, sensitive and specific immunodiagnostic assay for STEC strain detection. Thus, a STEC isolate from Ethiopia was processed for LPS extraction and the LPS was used to immunize mice.. The produced antibody showed positive agglutination both on the purified LPS as well as the STEC isolate carrying LPS on their surface; however, agglutination of STEC was more pronounced. Mice immunized with LPS produced highest agglutination on tertiary immunization showing the progressive buildup of the antibody response against the antigen. Cultures from tryptone soya agar and when they refresh showed better agglutination than cultures on EMB as well as tryptone soya broth. Immunodiagnostic assay developed in this study could detect STEC strains including STEC in human feces rapidly (1-2 min), with high sensitivity (90.2%), specificity (89.5%) and accuracy (90.6%). However, further studies are still required to improve the sensitivity, specificity and reproducibility. Overall this diagnostic assay provided promising results that may curb current problem with detection methods in clinical health care and research laboratories.

Preparation of rabbit polyclonal antibody against porcine gasdermin D protein and determination of the expression of gasdermin D in cultured cells and porcine tissues.

Gasdermin-D (GSDMD) is a member of the gasdermin (Gsdm) protein family, and its cleavage by inflammatory cysteine proteases (caspases, CASPs) is a critical event in cell pyroptosis. The role and functions of GSDMD on mice and humans are widely studied, but its expression, structure, and function in other species are less known. In the present work, rabbit anti-porcine GSDMD (pGSDMD) polyclonal antibody was prepared by immunizing New Zealand white rabbits with prokaryotic expressed recombinant pGSDMD (rpGSDMD). The prepared polyclonal antibody showed good specificity in Western blot and indirect immunofluorescence (IIF) assays. Western blot results showed that the polyclonal antibody could recognize overexpressed pGSDMD in human embryonic kidney cells (HEK293T) and endogenously expressed pGSDMD in cultured intestinal porcine enterocytes (IPEC-J2) and porcine kidney cells (PK-15). Western blot also revealed that pGSDMD was expressed in the heart, liver, lung, kidney, gallbladder, and jejunum of pigs. HEK293T cells overexpressing GSDMD showed green fluorescence in the IIF assay only after being treated with 0.3% Triton-X 100, which indicated that the full-length pGSDMD was located in the plasma but not on the cell membrane. This work provides a useful tool and basic information for further studies on pGSDMD.

Preparation of a polyclonal antibody against the non-structural protein, NSs of SFTSV.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is newly discovered virus which is the member of the order Bunyavirales, family phenuiviridae, phlebovirus genus. Its genome is composed of 3 segments of negative-sense RNA L, M and S. NSs is a non structure protein encoded by S segment which is important for viral replication and virulence. NSs protein of SFTSV is only involved in the regulation of host innate immune responses and suppression of IFN-promoter activities. So, the exact functions of this protein need to be studied deeply. To understand the exact role of NSs from SFTSV in viral replication and host immune response, a qualified antibody against this protein is required. In this study, NSs gene of SFTSV, was cloned into a bacterial expression vector (pGEX-6P-1) and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. The SFTSV NSs fusion protein was purified using Glutathione Sepharose 4B and utilized as an antigen to immunize rabbits and obtain an anti-SFTSV NSs polyclonal antibody. Proper expression of the fusion protein and polyclonal antibody specificity were confirmed by western blotting and immunofluorescence analyses. The polyclonal antibody recognized NSs from SFTSV specifically. This is the first report that NSs can form viroplasm-like structures not only in infected cells but also in transfected cells with NSs plasmids. This polyclonal antibody will be useful for future studies of NSs functions.

Development of species-specific IgM antibodies and elevation of mucosal immune response in Labeo rohita using recombinant bicistronic nano DNA vaccine priming.

Immunoglobulin (IgM) is the primary immunoglobulin essential for defense mechanisms in fish. It is difficult to reliably quantify IgM because a lack of standardization in methodology and limited availability of commercially reagents. In the present study, a polyclonal antibody was developed for the specific detection and quantification of IgM in Labeo rohita. Recombinant bicistronic NanoDNA plasmid (RBND Vac) encoding the glyceraldehyde-3-phosphate dehydrogenase gene of Edwarsiella tarda conjugated with poly (lactic-co-glycolic acid) - Chitosan (PLGA-Chit) was developed and its potential as a DNA vaccine, to prevent the infection of E. tarda in L. rohita was investigated. Two treatment groups [T1 - (PLGA-Chit-NPs-pDNA), T2 - (PLGA-NPs-pDNA) and one control group (T0 - 1 × PBS)] were utilized. Polyclonal antibody was developed to estimate IgM titers in the serum and mucosal associated tissues (MAT) using Enzyme-linked Immunosorbent Assay (ELISA) technique. Additionally, immune gene expression was studied using qRT-PCR. Vaccinated groups also exhibited a significant increase in the total serum protein, globulin concentration and relatively less mortality was observed in T1 group. IgM level in serum and mucosal tissues (skin, gill and gut) increased significantly days post vaccination compared to control group, also non-specific immune parameters (myeloperoxidase and lysozyme levels) showed significant improvement in vaccinated fish. Furthermore, histopathological examination confirmed minor damage in physiological structure of kidney and liver tissues in vaccinated fish. Knowledge of the immunoglobulin in L. rohita primed with RBND Vac complex provides the better protection against E. tarda. The normal physiology findings of this study will aid in monitoring changes in the health status of fish, when the animals undergo vaccination by immersion method.

Fluorescence polarization immunoassay for rapid determination of dehydroepiandrosterone in human urine.

In this paper, five fluorescein-labeled dehydroepiandrosterone (DHEA) derivatives (tracers) with different chain lengths between the fluorescein and hapten were synthesized and featured so as to establish a fluorescence polarization immunoassay (FPIA) for DHEA detection in human urine samples with previously prepared polyclonal antibody against DHEA. The outcomes of the structure of tracer on FPIA sensitivity were investigated. Under the optimal condition, the fluorescence polarization value (FP) decreases linearly in DHEA concentration, ranging from 1.6 to 243.3 ng mL-1, with the limit of detection of 1.1 ng mL-1 and IC50 value of 25.1 ng mL-1. Moreover, the developed FPIA was time-saving as it could complete the detection within 3 min. FPIA and commercial enzyme-linked immunosorbent assay kit were both applied to analyze the spiked human urine samples with DHEA. Excellent recoveries (92.1-108.0%) and satisfactory correlation coefficient (R2 = 0.98) were acquired with the two methods, indicating that the developed FPIA was a fast and efficient screening immunoassay with accuracy and sensitivity for DHEA detection in human urine samples. Graphical abstract.