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Pepsin-solubilized collagen (PSC) was prepared from the dermis of sea cucumber Apostichopus japonicus (green type) by performing pepsin digestion to collagen fiber pretreated with disaggregating solution (0.1 M Tris–HCl, pH 8.0, containing 0.5 M NaCl, 0.05 M ethylenediaminetetraacetic acid (EDTA), and 0.2 M 2-mercaptoethanol) and 0.1 M NaOH. On sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the PSC clearly showed two alpha bands under phosphate buffer system in the presence of 3.5 M urea. An antiserum was raised against chromatographically purified major molecular species in the PSC, and immunoblot analyses were performed for the soluble fractions at 4 M guanidine hydrochloride treatment and disaggregation as well as the collagen fiber before and after treatment. These fractions and collagen fibers showed quite similar band patterns to that of the PSC, showing mainly two alpha bands. These combined results suggest that the major molecular species of collagen contains at least two distinct alpha components and that the effect of pepsin digestion is relatively small on the structure of this collagen type.

Foot-and-mouth disease (FMD) is highly infectious, limits live animal trade, and affects ranchers owing to the loss of animal yield. The present study was designed to perform vaccine matching for field FMD virus isolates from clinically diseased cattle and assess the antigenic properties of the field isolates against the current vaccine strains used for vaccine production at the National Veterinary Institute, Ethiopia. Both sequencing and reverse transcription-polymerase chain reactions were used for distinguishing between the viral strains. To evaluate the serological relationship of the vaccine strain with these field isolates (r1 value), in vitro cross-neutralization was performed using ETH/6/2000 and ETH/38/2005 antisera. Infectious field FMD viral samples represented serotypes A and O. Sequence analysis showed that serotype A VP1/1D possessed amino acid variability at positions 28 and 42 to 48, 138, 141, 142, 148, 156, 173, and 197 compared with the ETH/6/2000 vaccine strain, whereas serotype O possessed amino acid variability at positions 45, 48, 138, 139, 140, 141, and 197 compared with the ETH/38/2005 vaccine strain. Based on the one-dimensional virus neutralization test, serotypes A and O demonstrated antigenic matching of up to 13/17 (76.47%) with the vaccine strain, except for the isolates ETH/40/2018, ETH/48/2018, ETH/55/2018, and ETH/61/2018, which had r-values less than 0.3. Therefore, the currently used vaccine strains ETH/38/2005 for serotype O and ETH/6/2000 for serotype A protected against all and most field viruses characterized as serotypes O and A, respectively, and amino acid residue variation was observed in different FMD virus B-C loops, G-H loops, and C-termini of VP1 at sites 1 and 3 in both serotypes.

Here, we report the development of an indirect enzyme-linked immunosorbent assay (ELISA) method that involves using multiepitope recombinant S protein (rSP) as the coating antigen to detect antibodies against canine coronavirus (CCoV). rSP was designed by arranging its four S fragments (91–135 aa, S1 gene; 377–434 aa, S2 gene; 647–671 aa, S3 gene; 951–971 aa, S4 gene; 207–227 aa) and two T-cell epitopes in tandem: T–E1–E2–E3–E4–T. This multiepitope antigen, which has a molecular weight of approximately 25 kDa and contains a His-tag, was recognized by a CCoV-positive serum in a Western blot assay. The optimal concentration of rSP as a coating antigen in the ELISA was 2 μg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:10,000. The cutoff OD450 value was established at 0.2395. No reactivity was observed with antisera against canine distemper virus, canine parvovirus, or feline calicivirus, indicating that this assay is highly specific. We also tested 64 clinical serum samples using our newly established method, and the positive rate was found to be 82.8%. In conclusion, our assay was found to be highly sensitive and specific for the detection of antibodies against CCoV, and it can therefore serve as a new, efficient diagnostic method.

Tomato chlorosis virus (ToCV) is a whitefly-transmitted crinivirus that causes yield losses, mainly in tomato (Solanum lycopersicum) and potato (S. tuberosum) crops. In this work, a polyclonal antiserum for detecting the virus using a dot-blot immunoassay was developed and the responses of crinivirus-infected potato genotypes were evaluated. The virus was purified using infected tomato leaves and the antiserum was obtained by intramuscular injection of purified viral preparations in rabbit. Tomato and potato tissue samples were used to validate the polyclonal antiserum for detection of ToCV, previously analyzed by RT-PCR. A total of 81 tomato and potato samples were analyzed by RT-PCR and dot-blot immunoassay (DBIA). All samples were positive in RT-PCR, but three of them did not react in DBIA, showing of 96.3% efficiency compared with that in RT-PCR. All potato genotypes inoculated with ToCV by Bemisia tabaci MEAM1 were susceptible to infection. Potato plants of cv. Camila infected by ToCV showed the lowest virus titer and were asymptomatic.

Measuring corticosterone (Cort) in bird feathers has become increasingly popular as a non-invasive method of obtaining an integrated profile of Cort exposure during the period of feather replacement. Most studies use antibody-based assays to assess Cort levels in feathers [radioimmunoassays (RIA) or enzyme immunoassays (EIA)]. However, it is still unclear whether differences in Cort can be reliably and consistently detected in feathers using antibody-based assays, in part because it is not known how much Cort is present in feathers and antibodies can differ in their ability to detect their antigens. In this study, we tested six commercially available polyclonal Cort antibodies in a feather Cort RIA in nine species. We found that different antisera detected very different levels of Cort in feathers. Additionally, we found that the broad patterns of Cort across species were not the same when measured with different antibodies. Further analysis by mass spectrometry indicated the presence of very little Cort in the feathers of any of the five species tested, suggesting that antibodies were instead binding with Cort metabolites or other substances. These data indicate a potential hidden source of variability when measuring feather Cort with antibody-based tests. The data further suggest caution in cross-species comparisons because patterns seen in feather Cort may reflect artifacts of the specific antibody used in the assay.

Ninety-six symptomatic orchids representing 36 genera from seven orchid collections, including two conservatories, in Minnesota, USA were tested for cymbidium mosaic virus (CymMV), odontoglossum ringspot virus (ORSV), and orchid fleck virus (OFV) using dipsticks (CymMV and ORSV), RT-PCR (OFV), and transmission electron microscopy (all viruses). CymMV was identified in 22% of the samples, OFV was identified in 17% of the samples, and ORSV was detected in 6% of the samples. Five percent of samples were infected with both CymMV and ORSV and 1% of samples were infected with both OFV and ORSV. Characteristic orchid virus symptoms of chlorotic and necrotic patterns were observed for the majority of infected orchids. Polyclonal antibodies were produced against Escherichia coli expressed OFV phosphoprotein (OFV P) and evaluated for use in plate trapped antigen-enzyme linked immunosorbent assay (PTA-ELISA). After an overnight incubation with PNPP the OFV P polyclonal antisera diluted from 1:1000–1:20,000 (v/v) readily differentiated between fresh healthy and OFV infected orchid (Phalaenopsis hybrid) tissue diluted from 1:5–1:20 (w/v). The high incidence of viruses detected in orchids (50%) suggests limited use of certified virus-free propagation stock and highlights the importance of sanitation to prevent transmission between plants.

Peanut allergies are among the most severe food allergies, and several allergenic proteins referred to as Ara h 1-Ara h 17 have been identified from peanut seeds. The molecular characterization of Ara h 1 (63 kDa), a glycosylated allergen, has almost been completed, and the occurrence of two homologous genes (clone 41B and clone P17) has been identified. In this study, we found a new variant of Ara h 1 i.e. 54 kDa, in which the N-terminal amino acid sequence was EGREGEQ-, indicating that the N-terminal domain of 63 kDa Ara h 1 had been removed. This new isoform was obtained from the run-through fraction of hydrophobic interaction chromatography while 63 kDa Ara h 1 was tightly bound to the hydrophobic resins, suggesting that the removal of the N-terminal domain resulted in extreme hydrophilic properties. We found that 63 kDa Ara h 1 occurs as higher order homo-oligomeric conformations such as decamer or nonamer, while 54 kDa Ara h 1 occurs exclusively as a homotrimer, indicating that the N-terminal domain of the 63 kDa molecule may be involved in higher order oligomerization. When antisera from peanut-allergic patients were treated with both the Ara h 1 molecules, the immunoglobulin E (IgE) antibodies in these sera reacted with each Ara h 1 molecule, suggesting that the C-terminal as well as the N-terminal domains of Ara h 1 contribute significantly to the epitope formations of this peanut glycoallergen. Furthermore, the glycoform analyses of N-glycans linked to 63 kDa and 54 kDa Ara h 1 subunits revealed that both typical high-mannose type and β-xylosylated type N-glycans are linked to the molecules. The cross-reactivity of IgE against Ara h 1 in the serum of one peanut allergy patient was completely lost by de-N-glycosylation, indicating the N-glycan of Ara h 1 was the sole epitope for the Ara h 1- specific IgE in the patient.

Apple stem pitting virus (ASPV) and Apple mosaic virus (ApMV) are considered to be among the major viral pathogens infecting apple. These viruses have high disease incidence in the apple orchards of Jammu & Kashmir (J&K) and Himachal Pradesh (HP), so a method was required for easy virus detection. Here, we have attempted to develop a polyclonal antiserum against the coat proteins of both these viruses expressed in bacterial systems for serological based diagnostics. The purified proteins were used for rabbit immunizations and subsequently for raising antibodies. These antibodies were conjugated with alkaline phosphatase and an ELISA based protocol was standardized for detection of both the viruses. We evaluated seventy-five field samples both for ASPV and ApMV of which 25 samples for ASPV and 21 samples for ApMV were tested positive by DAS-ELISA, corroborated by RT-PCR analyses. An Immunocapture (IC)-PCR was standardized for ASPV detection using the developed antibody. This is also the first report for development of any indigenous isolate based immunodiagnostics for detection of both Apple mosaic virus and Apple stem pitting virus.

Vibrio parahaemolyticus is one of the major pathogens responsible for vibriosis and zoonotic infections in teleosts, marine invertebrates, and also humans through consumption of contaminated or unprocessed seafood. Emergence of resistance against current accessible antibiotics and spread to the food chain and environment necessitate the development of safe and effective subunit vaccine against this bacterium. Many bacteria including V. parahaemolyticus produce extracellular curli fibrils, heteropolymeric filaments of major and minor subunit, which have been implicated in adhesion, biofilm formation, and virulence. Adhesins are the primary contact points with the host which help in establishing infection and colonization. CsgA, an adhesin, is the major structural component of the curli fiber that forms homopolymers of several hundred units. Due to their exposure on the cell surface, the curli fibers are recognized by the host's immune system, would generate high immune response, and therefore can serve as effective vaccine candidate. In the present study, we describe characterization of the csgA gene, and preparation of recombinant soluble CsgA of V. parahaemolyticus (rVpCsgA), and evaluation of its vaccine potential. Immunization of BALB/c mice with the rVpCsgA mounted a strong immune response. Cellular immune assays such as antibody isotyping, in vitro splenocyte proliferation analysis, and cytokine profiling revealed mixed T-helper cell immune response. The anti-rVpCsgA antiserum was agglutination positive and specifically cross-reacted with the curli CsgA present on the outer membrane of V. parahaemolyticus cells, thus demonstrating its neutralization potential. One hundred percent survival of the immunized mice upon challenge with the lethal dosage of the bacterium established that the rVpCsgA could serve as an effective vaccine against the bacterium. KEY POINTS: • Recombinant histidine-tagged CsgA of V. parahaemolyticus, rVpCsgA, was purified. • The rVpCsgA immunization produced mixed immune response and agglutinating antibodies. • Immunization with the rVpCsgA protected mice against V. parahaemolyticus challenge.

The aim of this study was to isolate Babesia canis soluble antigens and to investigate the effect of their conjugates with gold nanoparticles on the immunogenicity in laboratory animals. A procedure was developed for isolating and purifying B. canis antigens. The isolated culture antigen of B. canis 495 was coupled to gold nanoparticles, and the conjugate was used to immunize laboratory mice. Western blotting showed that the resultant antiserum specifically recognized the proteins of the B. canis strains isolated from naturally infected dogs. The antibody titer, the respiratory activity of peritoneal macrophages, the proliferative activity of splenocytes, and the production of cytokines were maximal when the animals were immunized with the antigen–nanoparticle conjugate emulsified in complete Freund’s adjuvant. Without adjuvant, the babesial antigen was weakly immunogenic. Therefore, the use of gold nanoparticles as an antigen carrier induced a broad immune response involving both cellular and humoral responses. The antibodies raised by the proposed procedure are potentially effective at immunodetection of Babesia canis infections in dogs.

Two known Clostridiodes (Clostridium) difficile surface antigens, a lipoteichoic acid (LTA) and a polysaccharide (PS-II) were isolated and purified in order to prepare glycoconjugate vaccines to the carrier protein human serum albumin utilising a reductive amination strategy. Mice and rabbits were immunized with a prime and two boost strategy and the resulting sera were examined for their ability to recognise the purified homologous antigens and subsequently killed whole cells of C. difficile strains and other Clostridia species. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, with generally similar titers from animals that received the LTA or the PS-II conjugates. Sera raised to the LTA conjugates were able to recognise other Clostridia species C. butyricum, C. bifermentans and C. subterminale whereas sera raised to the PS-II conjugates were not. These LTA and PS-II sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has confirmed that the LTA and PS-II polysaccharides are both highly conserved surface polymers of C. difficile that are easily accessible to the immune system and as such may have potential as vaccine antigens or as targets for therapeutics to combat C. difficile infection.

Aggregation of hyperphosphorylated TDP-43 is the hallmark pathological feature of the most common molecular form of frontotemporal lobar degeneration (FTLD–TDP) and in the vast majority of cases with amyotrophic lateral sclerosis (ALS–TDP). However, most of the specific phosphorylation sites remain to be determined, and their relevance regarding pathogenicity and clinical and pathological phenotypic diversity in FTLD–TDP and ALS–TDP remains to be identified. Here, we generated a novel antibody raised against TDP-43 phosphorylated at serine 375 (pTDP-43S375) located in the low-complexity domain, and used it to investigate the presence of S375 phosphorylation in a series (n = 44) of FTLD–TDP and ALS–TDP cases. Immunoblot analysis demonstrated phosphorylation of S375 to be a consistent feature of pathological TDP-43 species, including full-length and C-terminal fragments, in all FTLD–TDP subtypes examined (A–C) and in ALS–TDP. Of particular interest, however, detailed immunohistochemical analysis showed striking differences in the immunoreactivity profile of inclusions with the pTDP-43S375 antiserum among pathological subtypes. TDP-43 pathology of ALS–TDP, FTLD–TDP type B (including cases with the C9orf72 mutation), and FTLD–TDP type C all showed strong pTDP-43S375 immunoreactivity that was similar in amount and morphology to that seen with an antibody against TDP-43 phosphorylated at S409/410 used as the gold standard. In stark contrast, TDP-43 pathology in sporadic and genetic forms of FTLD–TDP type A (including cases with GRN and C9orf72 mutations) was found to be almost completely negative by pTDP-43S375 immunohistochemistry. These data suggest a subtype-specific, conformation-dependent binding of pTDP-43S375 antiserum to TDP-43 aggregates, consistent with the idea of distinct structural TDP-43 conformers (i.e., TDP-43 strains) as the molecular basis for the phenotypic diversity in TDP-43 proteinopathies.

Thladiantha dubia mosaic virus (ThDMV) from the wild Manchurian tuber gourd (Thladiantha dubia Bunge, Cucurbitaceae), is a novel tentative specie of the genus Potyvirus within the family Potyviridae. It may pose a potential risk to both cultivated crops and plants in the natural ecosystem. In the present study, we developed an ELISA procedure for detection of ThDMV. Coat protein (CP) of ThDMV was successfully expressed in Escherichia coli, and used to immunize rabbit for antiserum after being purified by using Ni-NTA affinity column. Results of indirect ELISA showed that the optimum dilutions of the coating antigen and antiserum were 1:160 (1 μg per well), and 1:2000 respectively. Western blot indicated that antibodies in the antiserum could specifically recognize and bind CP of ThDMV. An ELISA method was developed with the antiserum to detect ThDMV in plant extracts. Samples collected from potato fields in Northeast China and Northwest China were analyzed by ELISA and RT-PCR and both gave consistent results. The ELISA assay developed here has potential to be used as a sensitive, reliable and cost-effective tool for ThDMV detection in epidemiological studies.

Tephrosia, an important medicinal plant, and a potential livestock feed, was found to be affected by a leaf yellowing disease at the Ziway field site of the International Livestock Research Institute. A total of fifty samples from 300 plants were collected from twenty Tephrosia species in three consecutive planting seasons; 2015, 2016 and 2017. The samples were screened for viral infection by dot-blot assay with antiserum targetting eight viruses. RT-PCR of dot-blot positive samples using virus specific primers gave an amplification product of the expected size (867 bp) only for cucumber mosaic virus (CMV) in Tephrosia senna. The amplified products were sequenced; coat protein sequence (657 bp) extracted, and submitted to NCBI database (Tep-Et; KY041651). Sequence alignment and phylogenetic analyses indicated that the isolate, Tep-Et, shared maximum identity [88.8–97.5% nucleotide (nt) and 89.4–96.3% amino acid (aa)] with CMV belonging to members of subgroup-I. To our knowledge, this is the first report of molecular characterisation of a CMV isolate infecting a new host, T. senna in Ethiopia.

The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.

DOCK10, a guanine-nucleotide exchange factor (GEF) for Rho GTPases, represents the example of a gene that gives rise to alternative first exon mRNA isoforms, named DOCK10.1 and DOCK10.2. Expression of human DOCK10.2 protein in cell lines, and its induction by interleukin-4 (IL-4) in normal B lymphocytes and chronic lymphocytic leukemia (CLL) cells, were previously demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.2. Here, expression of human DOCK10.1 protein was demonstrated using an antiserum raised against a peptide encoded by sequences from exon 1.1. Specificity of the DOCK10.1 and DOCK10.2 antisera for their respective isoforms was demonstrated using transfected human 293 T cells. Their specificity for endogenous DOCK10 was strongly suggested by the high significance of the correlations between the levels of their expected signals at the molecular size of 250 kDa and the levels of DOCK10.1 and DOCK10.2 mRNAs, respectively, in human hematopoietic cell lines. Specificity of the DOCK10.1 antiserum for DOCK10 was also demostrated in mouse using the DOCK10 knockout model. The DOCK10.1 protein was induced by IL-4 in CLL cells, which demonstrates that the mechanism by which IL-4 regulates DOCK10 is not isoform-specific. Last, to get insights into differential regulation of the DOCK10 isoforms, their protein levels in cell lines were compared with their gene expression profiles retrieved from the Cancer Cell Line Encyclopedia (CCLE), leading to the identification of BCL3 and KLF12 as potential transcriptional regulators of DOCK10.1 and DOCK10.2, respectively.

Apple stem grooving virus (ASGV) belonging to the genus Capillovirus under family Betaflexiviridae is one of the widely distributed latent viruses mainly on pome fruit trees. It infects apple causing considerable economic losses that is a threat to the apple industry. To study the occurrence and incidence of ASGV disease, sensitive antisera based diagnostic tool has been developed, which would be helpful in large scale indexing, certification and quarantine programmes. Coat protein gene of ASGV was cloned into pET-32a(+) and pHIS-Parallel expression vectors. Expression characteristics of the proteins expressed from both the systems were compared and taken for raising the antisera. Sensitivity and specificity of the antiserum against virus infection was compared by Double Antibody Sandwich (DAS-ELISA). It was observed that the antisera raised from the protein expressed from pHIS-Parallel expression system (smaller ~ 3 kDa His tag) was more sensitive as compared to the antisera raised from the protein expressed from pET-32a(+) expression system (~ 18 kDa His tag) and the commercially available antisera. The antisera could also be successfully used for western blotting of infected samples and in DAS-ELISA. Thus, this study presents the first report on production of polyclonal antiserum against recombinant coat protein of ASGV from India, for reliable detection of the virus.

The origin of SARS-CoV-2 variants of concern remains unclear. Here, we test whether intra-host virus evolution during persistent infections could be a contributing factor by characterizing the long-term SARS-CoV-2 infection dynamics in an immunosuppressed kidney transplant recipient. Applying RT-qPCR and next-generation sequencing (NGS) of sequential respiratory specimens, we identify several mutations in the viral genome late in infection. We demonstrate that a late viral isolate exhibiting genome mutations similar to those found in variants of concern first identified in UK, South Africa, and Brazil, can escape neutralization by COVID-19 antisera. Moreover, infection of susceptible mice with this patient’s escape variant elicits protective immunity against re-infection with either the parental virus and the escape variant, as well as high neutralization titers against the alpha and beta SARS-CoV-2 variants, B.1.1.7 and B.1.351, demonstrating a considerable immune control against such variants of concern. Upon lowering immunosuppressive treatment, the patient generated spike-specific neutralizing antibodies and resolved the infection. Our results suggest that immunocompromised patients could be a source for the emergence of potentially harmful SARS-CoV-2 variants., Here, in a longitudinal case study, Weigang et al. demonstrate that evolution of SARS-CoV-2 within a persistently infected immunosuppressed patient can result in the emergence of novel variants with reduced sensitivity to antibody neutralization.

Background The natural antibody responses to B-cell epitopes from dengue structural proteins were assessed using immune sera from people having well-defined past dengue infections with one of the four serotypes. Method Based on an immune-computational analysis previously conducted, nineteen epitopes from the envelope (E) and eight epitopes from pre-membrane (prM), which were more than 50% conserved across all the four DENV serotypes, were selected. Peptides to represent these B-cell epitopes were obtained from commercially available arrays, and were subjected to enzyme linked immunosorbent assay with sera obtained from dengue seropositive healthy volunteers (DENV1 n = 12: DENV2 n = 12: DENV3 n = 12 and DENV4 n = 12), and 10 dengue seronegative healthy volunteers from Sri Lanka. The cut-off value for the positive antibody response was set by taking the mean response of a peptide to the negative sera plus three standard deviations. The peptides (N = 7) showing the broad immune responses were used to generate antibodies in three mice (Balb/c) batches. The mice antisera were then subjected to microneutralization assays against all the four DENV serotypes. An EC50 viral neutralization ≥ 40 times the serum dilution was considered as neutralizing. Results Five of the E-peptide and two prM peptides were recognised by most individuls exposed to infections with each of the four serotypes, showing a serotype cross-reactive broad antibody response. The mice immune sera against the peptides representing the five E protein epitopes neutralized all the four DENV serotypes. Two of these five epitopes are from the Domain II, whereas one of them includes the whole bc-loop region. Conclusion The antibody responses of highly conserved epitopes across the serotypes, were broadly responsive with sera of all four DENV serotypes collected from individuals infected with only one DENV serotype. Weakly conserved epitopes showed rather specific antibody responses dominated by one or few serotypes.

Background Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. Methods To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. Results Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 μg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. Conclusions Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage. Graphical abstract

Background Humoral immunity plays an important role in the prevention of canine distemper. Anti-CD virus (CDV) antibody has strong antiviral activity and is widely used in the treatment of CD. However, with the increase of CD cases, the availability of therapeutic CD antibody fell short of the clinical needs. Results The high-titer antiserum with the high-titer neutralizing activity against CDV was obtained from the donkeys (Dezhou Donkey) immunized with the inactivated CDV vaccine. The donkey anti-CDV IgG was purified from the donkey serum, which was identified to significantly inhibit the CDV replication in the cultured Vero cells and effectively reduce the clinical symptoms and increase the survival rates (75%) of CDV-infected dogs (Shih-tzu Dog), similar to that treated with the dog-derived anti-CDV IgG. These results indicate that donkey-derived IgG is a potential substitute for dog-derived IgG to treat the CD in clinic. Conclusions Administration of donkey-derived anti-CDV IgG can ameliorate clinical symptoms and inhibit virus replication, thereby increasing the survival of CDV-infected dogs. This study opens up a new source of therapeutic antibody for CD treatment.

Background Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. Results We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. Conclusions The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.

Interleukin-4 is a signature cytokine of T-helper type 2 (Th2) cells that play a major role in shaping immune responses. Its role in highly relevant animal model of tuberculosis (TB) like guinea pig has not been studied till date. In the current study, the guinea pig IL-4 gene was cloned and expressed using a prokaryotic expression vector (pET30 a(+)). This approach yielded a recombinant protein of 19 kDa as confirmed by mass spectrometry analysis and named as recombinant guinea pig (rgp)IL-4 protein. The authenticity of the expression of rgpIL-4 protein was further verified through polyclonal anti-IL4 antiserum raised in rabbits that showed specific and strong binding with the recombinant protein. The biological activity of the rgpIL-4 was ascertained in RAW264.7 cells where LPS-treated nitric oxide (NO) production was found to be suppressed in the presence of this protein. The three-dimensional structure of guinea pig IL-4 was predicted by utilizing the template structure of human interleukin-4, which shared a sequence homology of 58%. The homology modeling result showed clear resemblance of guinea pig IL-4 structure with the human IL-4. Taken together, our study indicates that the newly expressed, biologically active rgpIL-4 protein could provide deeper understanding of the immune responses in guinea pig to different infectious diseases like TB and non-infectious ones.

The effect of the response surface methodology was applied to the production of Caulobacter crescentus (strain NA1000) β-xylosidases using corn cob. The components of the medium that presented the greatest influence on the enzyme production were chosen for optimization including the concentration of the corn cob residue and temperature variation. Optimal concentrations were determined by a central composite rotational design and a combination of 3.5% (w/v) corn cob concentration and 27 °C temperature was found to be optimal. When C. crescentus was cultivated using the optimized conditions, a maximum activity of 393.36 U/mL of β-xylosidases was achieved in 24-h cultures with a yield of 95% in real test conditions compared to the predicted one. In parallel, there was an increase of 3.6 times in the production of intracellular xylanases when compared to cultures without statistical application. In the C. crescentus genome, 5 genes that encode β-xylosidases are present. In order to evaluate which of them would be induced in the optimized conditions, the quantitative expression (qPCR) of the xynB1–xynB5 genes was evaluated in the presence of 1 or 3.5% corn cob (w/v) and surprisingly, all showed a constitutive expression in relation to the control. Assays of Western Blot performed with a polyclonal antiserum against C. crescentus β-xylosidase II in the optimized condition also did not show mass variation of the referred protein. These data strongly suggest that post-transcriptional controls are operating in the induced condition to increase the activity of C. crescentus β-xylosidases I-V, but β-xylosidase II. To our knowledge, this is the first time that these data are reported in literature for a bacterial system.

Banana streak viruses (BSVs) are considered a major constraint to banana production worldwide. The integrated BSV sequence in the banana genome makes the nucleo-based detection unreliable and the serological-based methods require high quality polyclonal antiserum. In this study, reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for a specific amplification of episomal banana streak Mysore virus (BSMYV) in different banana cultivars. A total of 50 symptomatic and asymptomatic leaf samples of different banana cultivars were evaluated and BSMYV was efficiently detected by RT-RPA using a crude leaf sap template. Similar results were obtained with conventional reverse transcription-polymerase chain reaction (RT-PCR) using purified RNA as template. RT-RPA was significantly faster than RT-PCR with results obtained in less than one hour. The rapid and reliable diagnosis of episomal BSMYV will be most useful for virus indexing in plants including the banana tissue culture certification programme.

An immunoassay was previously developed as a technique to improve methods for detection and analysis of fly artifacts found at crime scenes. The dot blot assay utilized a polyclonal antiserum (anti-md3) based on a unique digestive cathepsin D found in cyclorrhaphous Diptera. In this study, artifacts produced by adults of Calliphora vicina, Cynomya cadaverina, Sarcophaga bullata, and Protophormia terraenovae were examined using the immunoassay to determine if insect-derived stains could be distinguished from a range of human body fluid stains. A lift technique was developed which permitted transfer of fly artifacts from test materials to filter paper for dot blot analyses. All species readily deposited artifacts on all test household materials regardless of diet consumed. Despite differences in texture and porosity of the household materials, artifacts of all species transferred to the filter paper. With all fly species, anti-md3 serum bound to artifacts produced after feeding on semen, blood, feces, urine, and saliva. By contrast, anti-md3 serum did not react with any of the human fluids tested, nor with any of the lifts from household materials not exposed to flies. There was no evidence of false positives with any of the fly species tested, regardless of diet consumed. There was also no indication of false negatives with any of the dot blot assays. These observations suggest that immunoassays using anti-md3 serum performed on a simple lift of suspected fly artifacts can be used effectively as a confirmatory assay to distinguish fly regurgitate and fecal stains from human body fluids.

Diphacinone (DPN) is an extensively used anticoagulant rodenticide that is also considered a hazardous chemical, which poses a threat to nontarget species. DPN poisoning cases in humans or other species frequently occur, while rapid and sensitive detection methods are rarely reported. Thus, it is meaningful to develop an immunoassay for DPN detection with high sensitivity and specificity. In this study, a hapten was synthesized and then conjugated with carrier proteins to prepare the immunogens with different conjugation ratios for the preparation of antibody. After evaluation of the antisera using an indirect competitive enzyme-linked immunosorbent assay (icELISA) and statistical analysis, we found that the immunogen prepared using the N,N-dicyclohexylcarbodiimide (DCC) method with a conjugation ratio of 28.5 could elicit mice to generate antibodies with high performance. Using hybridoma technology, we obtained the specific monoclonal antibody (mAb) 4G5 with a half maximal inhibitory concentration (IC50) of 0.82 ng/mL in buffer solution. We initially explored the recognition mechanism of DPN/CLDPN and mAb from both conformational and electronic aspects. Then, mAb 4G5 was applied to develop icELISA for biological samples. The limits of detection (LODs) of icELISA were 0.28 μg/L, 0.32 μg/L, and 0.55 μg/kg for swine plasma, urine, and liver samples, respectively, and the recoveries ranged from 72.3 to 103.3% with a coefficient of variation (CV) of less than 12.3% in spiked samples. In summary, we developed a sensitive, specific, and accurate icELISA for the detection of DPN in biological samples, which showed potential in food safety analysis and clinical diagnosis.

Equine influenza virus is an important pathogen for the horse industry because of its economic impact, and vaccination is a key control measure. Our previous work suggested that a mutation at position 144 in the hemagglutinin of Florida sublineage clade 2 viruses reduces the cross-neutralizing activity of antiserum against a former vaccine strain. To confirm this suggestion, here, we generated viruses by reverse genetics. Antibody titers against the mutated viruses were one-tenth to one-sixteenth of those against the former vaccine strain. Our findings confirm that this single amino acid substitution reduces the cross-reactivity of antiserum against this former Japanese vaccine.

The present studies prove that conjugation of meningococcal lipooligosaccharides through their non-reducing terminus conserves their inner epitopes resulting in conjugates potent to induce a protective immune response. Four different oligosaccharides were obtained by specific degradations of the same L7 lipooligosaccharide (L7-LOS), and each was linked to tetanus toxoid by direct reductive amination. Two were truncated oligosaccharides with incomplete inner epitopes and were obtained by mild acid hydrolysis of lipooligosaccharide. The terminal galactose of one oligosaccharide was additionally enzymatically oxidized. These oligosaccharides were conjugated through a newly exposed terminal Kdo in reducing end or through oxidized galactose localized at non-reducing end of the core, respectively. The third was a full-length oligosaccharide obtained by O-deacylation of the L7-LOS and subsequent enzymatic removal of phosphate substituents from its lipid A moiety. The fourth one was also a full-length O-deacylated lipooligosaccharide, but treated with galactose oxidase. This allowed direct conjugation to tetanus toxoid through terminal 2-N-acyl-2-deoxy-d-glucopyranose or through oxidized galactose, respectively. Comparison of the immune performance of four conjugates in mice revealed, that while each was able to induce significant level of L7-LOS-specific IgG antibody, the conjugates made with the full-length saccharides were able to induce antibodies with increased bactericidal activity against homologous meningococci. Only full-length oligosaccharides were good inhibitors of the binding of L7-LOS to the bactericidal antiserum. Moreover, induction of the significant level of the L7-LOS-specific antibody by full-length lipooligosaccharide conjugated from non-reducing end, provided also the direct evidence that internal core epitopes are fully responsible for the immunorecognition and immunoreactivity.

To have an opportunity for reliable detection of benyvirus Beet soil-borne mosaic virus (BSBMV) and its discrimination from Beet necrotic yellow vein virus (BNYVV), an attempt to generate a specific immunoassay was made. Using a purified recombinant polypeptide corresponding to the 55 C-terminal amino acid residues of viral coat protein as antigen, a specific polyclonal antiserum against BSBMV was raised. The obtained immunoglobulin G fraction reacted with high specificity in western blot analysis with both the recombinant polypeptide expressed in Escherichia coli and total protein extract from BSBMV-infected plants. In parallel, to improve the identification of benyviruses, a duplex RT-PCR method for simultaneous detection of BNYVV and BSBMV was developed. This method is based on the combination of multiple primer sets to different pathogens into single multiplexed amplification reaction, which share single standard amplification conditions.

Bacterial black node, a disease of barley and wheat, is caused by Pseudomonas syringae pv. syringae (synonym pv. japonica, hereafter Psj). Here a selective medium, serine-potassium tellurite-based Psj-selective agar (SPTPsjA) was developed to isolate and detect Psj. After 5–7 days on SPTPsjA at 25 °C, characteristic black Psj colonies formed with an efficiency equal to that on potato–peptone–glucose agar. Except for P. viridiflava, P. cichorii, and some strains of P. syringae group bacteria, SPTPsjA either inhibited growth of other phytopathogenic bacteria or enabled their discrimination. To facilitate diagnosis of Psj, we prepared three antisera against three strains; in a slide agglutination test, the antisera reacted to all tested strains of Psj and failed to react to P. viridiflava and P. cichorii although some strains of other P. syringae group bacteria were indistinguishable from Psj with SPTPsjA and antisera. When barley and wheat seeds from fields in which bacterial black node had occurred were placed on SPTPsjA in the dark at 25 °C for 7 days, black colonies grew on the medium around the seeds. Among these strains, only those responsive to the above antisera were pathogenic on barley. These results show that the combination of SPTPsjA and antisera can isolate Psj from seeds of barley and wheat.

Background Hemagglutinin is a major surface protein in influenza A virus (IAV), and HA2 is relative conserved among different IAVs. It will be meaningful to identify broad-spectrum epitopes based on the HA2 protein. Results Overlapping peptides of the HA2 protein of the H5N1 IAV A/Mallard/Huadong/S/2005 were synthesized and loaded on modified silica gel film to form a microarray, and antisera against different subtypes of IAVs were used to screen universal epitopes. The selected epitope was further confirmed by western blotting using anti-peptide immune serum and viruses rescued with amino acid substitution. The results showed that 485-FYHKCDNECME-495 of the H5 14th peptide in HA2 had broad-spectrum binding activity with antisera against H1, H3, H4, H5, H6, H7, H8, H9, and H10 subtype IAV. Substitution of amino acids (K or D) in rescued viruses resulted in decreased serum binding, indicating that they were critical residues for serum binding activity. In Immune Epitope Database, some epitopes containing 14–4 peptide were confirmed as MHC-II-restricted CD4 T cell epitope and had effects on releasing IL-2 or IFN. Conclusion The identified epitope should be a novel universal target for detection and vaccine design and its ability to generate immune protection needs further exploration.

Sterile inflammation (SI) is an essential process in response to snakebite and injury. The venom induced pathophysiological response to sterile inflammation results into many harmful and deleterious effects that ultimately leads to death. The available treatment for snakebite is antiserum which does not provide enough protection against venom-induced pathophysiological changes like haemorrhage, necrosis, nephrotoxicity and often develop hypersensitive reactions. In order to overcome these hindrances, scientists around the globe are searching for an alternative therapy to provide better treatment to the snake envenomation patients. In the present study TiO2 (Titanium dioxide)-NPs (Nanoparticles) has been assessed for antisnake venom activity and its potential to be used as an antidote. In this study, the synthesis of TiO2-NPs arrays has been demonstrated on p-type Silicon Si substrate (∼30 ohm-cm) and the surface topography has been detected by Field-emission scanning electron microscopy (FESEM). The TiO2-NPs successfully neutralized the Daboia russelii venom (DRV) and Naja kaouthia venom (NKV)-induced lethal activity. Viper venom induced haemorrhagic, coagulant and anticoagulant activities were effectively neutralized both in in-vitro and in vivo studies. The cobra and viper venoms-induced sterile inflammatory molecules (IL-6, HMGB1, HSP70, HSP90, S100B and vWF) were effectively neutralised by the TiO2-NPs in experimental animals.

Four new H9N2 avian influenza viruses (AIVs) were isolated from domestic birds in Guangdong between December 2015 and April 2016. Nucleotide sequence comparisons indicated that most of the internal genes of these four strains were highly similar to those of human H7N9 viruses. Amino acid substitutions and deletions found in the HA and NA proteins indicated that all four of these new isolates may have an enhanced ability to infect humans and other mammals. A cross-hemagglutinin-inhibition assay, conducted with two vaccine strains that are broadly used in China, suggested that antisera against vaccine candidates could not provide complete inhibition of the new isolates.

The major yolk protein in sea urchins is a transferrin-like protein. In this report, a new component was detected in gonad extracts of Strongylocentrotus intermedius and Mesocentrotus nudus, which cross-reacts with antiserum against egg yolk proteins. We tentatively named them egg yolk-related proteins siYRP and mnYRP. The siYRP was purified from testis of S. intermedius by ammonium sulfate fractionation, anion exchange chromatography, affinity chromatography and gel filtration. Purified siYRP was > 900 kDa in size. The siYRP purified on SDS-PAGE under reducing conditions gave seven bands, corresponding to 93, 213 and > 250 kDa. Purified mnYRP displayed similar structural characteristics as siYRP. Purified siYRP and mnYRP were identified by tandem mass spectrometry and renamed as siVitellogenin (Vtg)-like and mnVtg-like proteins, respectively. Both Vtg-like proteins were confirmed to be lipoglycoproteins by staining with Sudan black and periodic acid-Schiff. A specific antiserum against the siVtg-like protein was raised in rabbit. Antiserum against the siVtg-like protein immunostained siVtg-like and mnVtg-like proteins. Immunochemical methods using the antiserum revealed that siVtg-like and mnVtg-like proteins were present in the ovary, testis and unfertilized eggs of both species. These results indicated that Vtg-like proteins have important physiological functions for gonadal growth and gametogenesis in sea urchins.

Following acute infection of mucosal surfaces by bovine herpesvirus 1 (BoHV-1), sensory neurons are a primary site for lifelong latency. Stress, as mimicked by the synthetic corticosteroid dexamethasone, consistently induces reactivation from latency. Two viral regulatory proteins (VP16 and bICP0) are expressed within 1 h after calves latently infected with BoHV-1 are treated with dexamethasone. Since the immediate early transcription unit 1 (IEtu1) promoter regulates both BoHV-1 infected cell protein 0 (bICP0) and bICP4 expressions, we hypothesized that the bICP4 protein is also expressed during early stages of reactivation from latency. In this study, we tested whether bICP4 and bICP22, the only other BoHV-1 protein known to be encoded by an immediate early gene, were expressed during reactivation from latency by generating peptide-specific antiserum to each protein. bICP4 and bICP22 protein expression were detected in trigeminal ganglionic (TG) neurons during early phases of dexamethasone-induced reactivation from latency, operationally defined as the escape from latency. Conversely, bICP4 and bICP22 were not readily detected in TG neurons of latently infected calves. In summary, it seems clear that all proteins encoded by known BoHV-1 IE genes (bICP4, bICP22, and bICP0) were expressed during early stages of dexamethasone-induced reactivation from latency.

Enzymatic removal of blood groups antigens A and B is an efficient method for production of universal red blood cells. In this research, an α-N-acetylgalactosaminidase (NAGA) enzyme was expressed in Pichia pastoris for digestion of the A blood antigen. DNA sequence of the gene NAGA, originally expressed in Elizabethkingia meningosepticum (NAGA-EM), was ordered for optimization and synthesis. It was then expressed in P. pastoris (KM71H and GS115 strains). Expression of the recombinant NAGA was evaluated by dot blot, SDS-PAGE, and Western blotting. The activity of the enzyme was measured using a synthetic substrate in addition to the conversion of group A red blood cells to the O cells. Expression of NAGA-EM with an apparent molecular mass of 55 kDa was verified by dot blot, SDS-PAGE and Western blot analysis. The maximum enzyme activity in the supernatant of KM71H was higher than that in the GS115 (250 vs. 200 U/ml). Treated group A RBCs did not react with the anti-A antiserum or with the sera from individuals with blood groups B and O. The results of this study indicated that NAGA-EM is an efficient enzyme for production of universal O blood cells.

Phytophthora infestans is a global challenging pathogen among the biotic stresses which affect healthy and sustainable potato production. Desirable and successful management strategies depend on understanding of its biology, disease cycle and more importantly rapid and accurate detection of the pathogen during early stage of infection. Here, we report production of recombinant antibodies against whole cellulose binding domain (CBD-1) protein of P. infestans and a synthetic signal peptide of CBD1 and their effectiveness in detection of P. infestans both in infected potato leaf and tuber. The recombinant whole CBD1 protein and synthetic signal peptide of CBD1 protein was used to immunize the rabbits and blood serum was collected, purified and measured by SDS–PAGE. Among the two antibodies, synthetic peptide based antibody showed high specificity for mycelia, sporangia and zoospores. It also showed strong signal intensity for P. infestans infected leaf and tuber tissues. The data indicated that the produced synthetic peptide antiserum was efficient and accurate in discrimination of negative and positive samples in ELISA. Reaction of antiserum with early stage of infected leaf and tuber tissue confirmed the expression of the CBD protein, which in turn confirmed the presence of the pathogen in the plant tissues. Therefore it will enable to detect the pathogen in the potato seed before planting as well as in the standing crop in the field thus, would be helpful to ensure production of healthy seed potatoes and for effective schedule for management practices.

Plants are promising host systems for recombinant protein production. However, progress in the commercialization of plant-made proteins (PMPs) has been slow. Only one PMP drug is commercially available. In this study, we explored the possibility of using plants to produce E2 of classical swine fever virus (CSFV) and the use of this plant-produced E2 as a vaccine. We designed high-level expression vectors for transgenic plants by considering the transcription, translation, and storage of E2 in the cell. We incorporated a cellulosebinding domain sequence into the expression vector as an affinity tag for cost-effective, one-step purification. Using this vector, we generated multiple lines of transgenic Arabidopsis thaliana plants expressing a fusion protein of E2 from CSFV at high levels (0.7% of total soluble proteins). ER-targeted E2 fusion protein was successfully purified via a one-step purification process using amorphous cellulose resin. Arabidopsis-produced E2 was recognized by an antibody that detects CSFV antigen. Finally, antisera from mice immunized with E2 fusion protein reacted strongly to the antigens in a CSFV antibody detection kit. Therefore, we propose that plant-produced E2 fusion proteins could be further developed for use as a green vaccine against CSFV in animals.

To heterologously produce the Shigella dysenteriae serotype 1 O-polysaccharide (O-PS, O-antigen) in Escherichia coli by transferring the minimum number of genes instead of the entire O-PS gene cluster. The three glycosyltransferase genes (rfbR, rfbQ and rfp) responsible for the formation of the O-repeat unit were introduced into E. coli K-12 W3110 to synthesize S. dysenteriae 1 O-PS. The specific O-antigen ladder type with different chain lengths of O-repeat units was observed in the recombinant E. coli strain by SDS-PAGE silver staining and western blotting using S. dysenteriae 1 lipopolysaccharide antiserum. Analysis by mass spectrometry and ion chromatography suggested generation of the specific S. dysenteriae 1 O-repeat unit structure with an extra glucose residue attached. Recombinant E. coli expressing specific glycosyltransferase genes can generate the O-PS of S. dysenteriae 1 and might be able to synthesize heterologous O-antigens of various pathogenic bacteria for vaccine preparation.

The Nuclear Mitotic Apparatus protein (NuMA) is a nuclear protein that plays critical role in the mitosis as an organizer of the mitotic spindles and is a prevalent protein in malignant urothelial cells which makes it an appropriate marker for bladder cancer early detection. In this study, NuMA protein sequence was obtained from Genbank based on literature studies. Immunodominant epitopes which are located between residues 1 and 300 were reverse translated and optimized for expression in E. coli cells. Final sequence was then synthesized, cloned into pGS21a vector and expressed in BL21 cells. The expressed protein was examined by SDS-PAGE and also Western blotting to confirm expression by using anti-His antibody-HRP. The protein was then purified using nickel chromatography column and 18 mg of purified truncated NuMA was obtained from 1 l of bacterial culture. ELISA was performed to evaluate the reactivity of commercial anti-NuMA antibody towards truncated protein. Finally, the antigen was injected and polyclonal antibody was produced in rabbits. The immunoglobulins of antiserum were precipitated by ammonium sulfate and purified by DEAE–Cellulose chromatography column. To evaluate antibody accuracy, it was tested on HEp-2 cells with permeable nucleus membranes fixed on slides. The antibody detected NuMA protein in the nuclei of HEp-2 cells, which means it can have diagnostic value in the bladder cancer patients. The truncated NuMA is a suitable protein as a single multiepitope antigen that further may be used for studies to develop assays for early diagnosis of bladder cancer.

A previously cloned coat protein (CP) gene of Grapevine leafroll-associated virus 3 (GLRaV-3) from cultivar Cabernet Souvignon was over-expressed in Escherichia coli strain BL21 expression system as ~ 43 kDa fusion protein containing polyhistidine tag (6His) at its N terminal. The protein was purified from insoluble fraction and reacted positively in western blotting with commercial anti GLRaV-3 polyclonal antiserum (Bioreba, Switzerland) and hence, used as immunogen for the production of polyclonal antisera in New Zealand white rabbits. Polyclonal antiserum specific to GLRaV-3 detected the virus by double antibody sandwich enzyme linked immunosorbent assay using commercial alkaline phosphatase (ALP) conjugated globulin fraction (Bioreba, Switzerland) in GLRaV-3 positive grapevine samples. The immunoreactivity of the antiserum was confirmed through western blotting. The purified antiserum was conjugated with ALP. The primary antiserum along with ALP conjugate successfully detected the GLRaV-3 from the infected sample at 1:8000 and 1:10,000 dilutions, respectively. To the best of our knowledge, it is the first global study wherein the CP of GLRaV-3 was cloned in pET28a(+) expression vector having many advantages over the earlier used expression vectors. The cloned CP gene was expressed, purified and subjected to the production of immunoreagents. The developed immunoreagents will be useful for certification programmes as well as for large scale virus screening to produce GLRaV-3 free grapevines. The indigenously developed immunereagents will provide a cost-effective way of managing grapevine leafroll disease in Indian sub-continent.

Conglutinin, a high molecular weight protein detected in bovine serum, belongs to the family of innate immunity markers called collectins. It plays a key role as antimicrobial agent against all kinds of micro-organisms by binding to sugar moieties present on them. In the present study, partial recombinant conglutinin consisting neck and carbohydrate recognition domain of buffalo and nilgai was expressed in prokaryotic system and found to be of 27 kDa through SDS-PAGE and western blotting using antiserum raised against buffalo conglutinin. In the presence of calcium, protein showed high binding activity towards mannan coated to microtitre plate but in contrast, binding activity towards lipopolysaccharide was calcium independent, which needs to be explored further. The partial protein was unable to inhibit haemagglutination by Newcastle disease virus; this could be attributed to lack of complete protein, as complete protein forms tetramer which can cross link the virus. The protein was also found to reduce bovine herpes virus-1 titre propagated in MDBK cells. This paves path to check the effect of complete conglutinin protein on different enveloped viruses, which will provide insight into protein’s importance in innate immunity. Since the concentration of serum conglutinin is found to be hereditary dependent, this could be also helpful in better parentage selection.

The format of an immunochromatographic multiassay is first proposed with native antisera and a universal conjugate of antispecies antibodies with gold nanoparticles. This format allows (1) the exclusion of purification and conjugation stages for specific antibodies and (2) significant reduction of the concentration of specific antibodies in the system. The independent use of specific antibodies and a conjugated marker provided a low detection limit and high signal intensity. The proposed format was implemented for the simultaneous detection of two herbicides. The instrumental limits for the detection of atrazine and chlorsulfuron were 0.1 and 0.7 ng/mL, respectively, and the analysis time was 20 min. The suitability of the test system for monitoring these herbicides in nontreated apple and blackcurrant juices is shown. The assay technique is simple, sensitive, and easily transferrable to any other antigen. Graphical abstract The proposed format of the immunochromatographic multiassay is based on the use of native antisera and a universal conjugate of antispecies antibodies with gold nanoparticles. In this way purification and conjugation stages for specific antibodies are excluded, and the concentrations of specific antibodies and the conjugated marker can be varied independently to obtain a low detection limit.

To develop a safe and effective oral vaccine against Helicobacter pylori using its HpaA protein expressed in Lactococcus lactis. The gene encoding HpaA was obtained by PCR and ligated to pNZ8110-lysM following digestion with NaeI + SphI. The recombinant plasmid was transferred into E. coli for multiplication, and then into L. lactis. The recombinant L. lactis was induced to express HpaA, resulting in two products of 29 and 25 kDa, both of which yielded positive immunoreaction with mouse antisera against H. pylori, as confirmed by immunoblot assays. The 29 kDa product constituted 12% of the cell lysates. Oral inoculation with the engineered L. lactis evoked significantly elevated serum IgG level in mice (P

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3’-N-P-M-F-HN-L-5’), and showed low sequence identity to other APMVs (

During infection host histo-blood group antigens (HBGAs) act as attachment factors that interact with rabbit hemorrhagic disease virus (RHDV) and participate in the infectious process. In the present study, baculovirus expressing recombinant RHDV capsid protein (VP60r) as a vaccine immunogen was used to test its antigenicity and immunogenicity via immunization experiments. Each group of rabbits immunized with VP60r was found to be fully protected against RHDV challenge. The duration of immunity of the vaccine following the inoculation of a single dose was determined to be at least 240 days. RHDV-specific humoral responses in antisera from inoculated rabbits were analyzed using VP60r virus-like particle (VLP)-based ELISA. Anti-VP60-specific antibody was produced by 7 days post-primary immunization. Following this stage, the levels of this antibody increased steadily, peaking at 90 days and maintaining a high level until 240 days. We developed a synthetic carbohydrate assay to detect blockage in attachment of RHDV VLPs to HBGAs by the rabbit antisera. On day 7 post-immunization, serum samples were demonstrated to block the binding of H type 2 to RHDV VLPs, with a blocking rate of almost 60%, a value that then increased steadily over time. From day 60 to day 240 post-immunization, serum samples completely blocked the binding of H type 2 to RHDV VLPs, with a blocking rate of almost 100%. This indicated that VP60-induced antibodies neutralize the interaction of RHDV with HBGAs.

Maintenance of virus-free citrus in nurseries and orchards is essential to control the spread of aphid-transmitted Citrus tristeza virus (CTV). To this end, a lateral flow immunoassay (LFIA) was developed to detect CTV that was user-friendly and field-deployable. Polyclonal antisera was generated in rabbit against purified virions from Nicotiana benthamiana inoculated with an infectious recombinant cDNA clone of CTV (rCTV). Affinity purified rCTV IgG (1 mg/ml) and Mouse IgG (0.5 mg/ml) were conjugated with gold nanoparticles and coated onto a glass fiber membrane conjugate pad. Affinity purified rCTV IgG and anti-mouse IgG were used as test line and control line, respectively on the test strip. The LFIA detected CTV within 10 min and was sensitive up to a 1:80 dilution of crude plant sap extract. The LFIA was validated using CTV strains T30, T36, VT and RB from greenhouse and field sources and results were 100% in agreement with Enzyme-Linked Immunosorbent Assay and Reverse Transcription quantitative PCR. The immunostrip readily detected CTV after six months in storage in a sealed bag with silica gel at room temperature indicating good stability. The LFIA test strips offered a cost-effective tool to detect CTV in the field or nursery by non-skilled personnel without laboratory equipment.

To evaluate residues of olaquindox (OLA) and its marker compound methyl-3-quinoxaline-2-carboxylic acid (MQCA) in aquatic products in the field, five types of hapten and corresponding antigens with exposure of different antigenic determinants were designed and synthesized. The optimal immunogen-coating combinations were obtained by antiserum detection and heterologous coating screening, successfully achieving a highly specific monoclonal antibody (mAb) against MQCA. Based on the highly specific mAb 1A11 showing no cross-reactivity with other analogs, an indirect competitive ELISA and an immunochromatographic strip for MQCA detection in fish were developed using the MQCA derivative MQCA-(NH2)4 coupled to KLH as the immunogen and MQCA-NH2 coupled to OVA as the heterologous coating antigen. The IC50 value of the indirect competitive enzyme-linked immunosorbent assay (icELISA) was 3.17 ng/mL, and the linear range was 0.58–17.29 ng/mL for MQCA under optimized concentrations of coating antigen and antibody of 0.03 and 0.3 μg/mL, respectively. The recoveries of MQCA by the developed icELISA in fish samples ranged from 81.6 to 90.7%. The cutoff value of the strip was 5 ng/mL, and the limit of detection was 0.25 ng/mL under optimal working conditions of 4 μL 0.1 M K2CO3 and 50 μL 0.2 mg/mL per 1 mL colloidal gold solution. The developed icELISA and immunochromatographic strip will be very useful tools for the primary screening of MQCA in fish.

Cucumber mosaic virus (CMV) causes severe agricultural losses in Korea and has a very large host range. To produce virus-free plants, it is necessary to develop efficient, sensitive virus detection methods. In this study, we collected leaf samples from Lilium showing characteristic symptoms of CMV infection from Gang-won Province, Korea in 2011 and amplified the coat protein (CP) gene from CMV from the samples by RT-PCR. Three CMV isolates were found to belong to subgroup IA based on the nucleotide sequence of the CP gene. We cloned the complete CP gene into the pET21d(+) expression vector to generate recombinant coat proteins. After expressing His-tagged coat proteins in Escherichia coli strain BL21 (DE3) by IPTG induction, we purified the proteins using Ni-NTA agarose beads and used the purified CMV recombinant CP for antiserum production. The resulting polyclonal antisera specifically recognized CMV from infected lily samples, as determined by indirect enzyme-linked immunosorbent assay (ID-ELISA) and Western blotting assays. In addition, we developed an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay using the polyclonal antiserum to enable sensitive, reliable, cost-effective detection of CMV. This study demonstrates that three CMV isolates belong to subgroup IA, and that CMV-specific PAbs can be used to detect CMV in epidemiological studies.