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PurposeThe CIGMA study investigated a novel human polyclonal antibody preparation (trimodulin) containing ~ 23% immunoglobulin (Ig) M, ~ 21% IgA, and ~ 56% IgG as add-on therapy for patients with severe community-acquired pneumonia (sCAP).MethodsIn this double-blind, phase II study (NCT01420744), 160 patients with sCAP requiring invasive mechanical ventilation were randomized (1:1) to trimodulin (42 mg IgM/kg/day) or placebo for five consecutive days. Primary endpoint was ventilator-free days (VFDs). Secondary endpoints included 28-day all-cause and pneumonia-related mortality. Safety and tolerability were monitored. Exploratory post hoc analyses were performed in subsets stratified by baseline C-reactive protein (CRP; ≥ 70 mg/L) and/or IgM (≤ 0.8 g/L).ResultsOverall, there was no statistically significant difference in VFDs between trimodulin (mean 11.0, median 11 [n = 81]) and placebo (mean 9.6; median 8 [n = 79]; p = 0.173). Twenty-eight-day all-cause mortality was 22.2% vs. 27.8%, respectively (p = 0.465). Time to discharge from intensive care unit and mean duration of hospitalization were comparable between groups. Adverse-event incidences were comparable. Post hoc subset analyses, which included the majority of patients (58–78%), showed significant reductions in all-cause mortality (trimodulin vs. placebo) in patients with high CRP, low IgM, and high CRP/low IgM at baseline.ConclusionsNo significant differences were found in VFDs and mortality between trimodulin and placebo groups. Post hoc analyses supported improved outcome regarding mortality with trimodulin in subsets of patients with elevated CRP, reduced IgM, or both. These findings warrant further investigation.Trial registration: NCT01420744.

The unique cell wall of  Mycobacterium tuberculosis  (Mtb) creates a barrier to hydrophilic drugs, which is crucial for its survival and pathogenicity. However, the immune reactivity elicited by its components remains incompletely understood. We aimed to assess the antibody responses induced by  Mtb H37Rv  cell wall components and to develop and characterize antigen-specific polyclonal antibodies (pAbs). Rabbits were immunized with these components. Immune serum reactivity was tested against various Mtb antigens. Specific polyclonal antibodies (pAbs) were purified by affinity chromatography. The results showed that immune serum reacted with lipoarabinomannan (LAM), ESAT-6 secretion system-1 (Esx-1) secreted protein B (EspB), and Mtb8, but showed no reactivity with other tested Mtb antigens. LAM-, EspB-, or Mtb8-specific pAbs were subsequently affinity-purified. The affinity-purified LAM pAb, EspB pAb, and Mtb8 pAb each demonstrated high specificity and sensitivity, showing no cross-reactivity with non-target antigens. They recognized antigens in culture supernatants and cells from diverse mycobacterial strains, including both slow-growing mycobacteria (SGM) and rapid-growing mycobacteria (RGM). In a sandwich ELISA using LAM pAb as the capture antibody and biotinylated LAM-specific monoclonal Abs (BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio, or BJRbL76-Bio) as detection antibodies, the assay detected SGM but did not react with RGM species. EspB pAb recognized EspB in both cell lysate and culture supernatant fractions, where full-length and mature EspB are predominantly found, respectively. Mtb8 pAb reacted with monomeric and polymeric forms of Mtb8. In conclusion, we successfully generated novel pAbs against LAM, EspB, and Mtb8, providing promising research tools for investigating these critical molecules. Key points Rabbit antibodies against Mtb H37Rv cell wall components target LAM, EspB, and Mtb8 Novel LAM-, EspB-, and Mtb8-specific pAbs were generated and characterized Broad mycobacterial reactivity and specific target detection confirm pAb utility

Dengue, a vector-borne disease caused by the dengue virus (DENV), is spread by Aedes mosquitoes like, Aedes aegypti and Aedes albopictus. In the absence of antiviral drugs or vaccines, dengue prevention is centered on mosquito vector control. Laboratory confirmation of DENV infection is essential to offer the appropriate clinical care. The non-structural protein-1 (NS1) antigen of the virus, in its secretory form, is being employed as a diagnostic marker to identify active dengue infection in humans and mosquito vectors. Commercial dengue NS1 ELISA kits use monoclonal antibodies to detect the NS1 antigen. However, polyclonal antibodies offer several advantages over monoclonal antibodies in terms of shorter production time, lower cost and high stability. Therefore, in the present study, we have raised Rabbit Polyclonal Antibodies against the Dengue NS1 antigen (RPAD) and evaluated their sensitivity and specificity in detecting dengue virus. The RPAD were tested against commercial recombinant dengue virus-2 NS1 antigen (rDNS1Ag) by standardized ELISA and Western Blot assays. Further, the sensitivity of RPAD was checked against the NS1 antigens of different serotypes of DENV. The specificity of RPAD towards the NS1 antigens of other flaviviruses was also tested. The RPAD demonstrated consistent immunoreactivity toward recombinant NS1 antigens of all four dengue virus serotypes in ELISA and Western blot assays, with minimal cross-reactivity to NS1 antigens of other flaviviruses. These findings indicate the potential utility of RPAD as a laboratory reagent for NS1 antigen detection, warranting further validation using clinical samples and field-collected mosquito vectors.

Tau-based blood biomarkers are increasingly recognised as important for the diagnosis of Alzheimer’s disease (AD). More than 60 proteolytic cleavage sites of tau have been identified, and current assays may miss critical information from some of the smaller protein fragments. By capturing a broader range of tau species, a polyclonal approach may offer greater interrogation of this complex “tauosome” and deliver valuable insights into the onset or progression of AD. A sheep was hyper-immunised with 2N4R tau113-251 peptide, encompassing the proline-rich region. An affinity-purified proline region polyclonal antibody (P.pAb) was derived from sheep serum, after four rounds of immunisation. Following characterisation of P.pAb, utility as a plasma biomarker/diagnostic agent for AD was assessed using a single molecular array (Simoa) assay in a selected cohort consisting of clinically diagnosed AD patients and age-matched cognitively unimpaired (CU) individuals. Two assays were considered for this assessment including pairing the P.pAb with itself (P.pAb-P.pAb) to capture and detect multiple tau fragments in plasma, and pairing pTau217 capture mAb with P.pAb (pTau217-P.pAb). The P.pAb showed high affinity towards full-length tau and 113–251 peptide immunogen and bound smaller 13-amino acid (aa) fragments throughout the proline rich region. The selected patient cohort was initially assessed by commercial neurofilament light (NfL) and pTau217 assays, the results of which were consistent with AD-related neurodegeneration in the AD sample and not in the CU group. The P.pAb-P.pAb and the pTau217-P.pAb assays were each able to distinguish between CU and AD groups; values were greater in AD (1.4-fold, p  < 0.0001 and 2.8-fold, p  < 0.001, respectively). By contrast, a commercial total-tau (T-tau) assay did not distinguish between the two groups. We demonstrate the feasibility of an immunodiagnostic approach based on the detection of tau species containing the proline-rich region. The development of an affinity-purified proline region-specific pAb, capable of detecting multiple tau species in plasma, provides the foundation for a novel approach with potential applications in AD diagnosis and monitoring of disease progression.

A strategy to identify high-quality commercially available antibodies for research reveals extensive use of non-specific antibodies and offers solutions for future large-scale testing.A strategy to identify high-quality commercially available antibodies for research reveals extensive use of non-specific antibodies and offers solutions for future large-scale testing.

Specific antibodies, which can be used in various experiments, are critical tools for unraveling genes’ function, but many commercial antibodies are not tested for these properties. GPATCH1 is a novel G-patch family protein. Genome-wide association studies (GWAS) revealed it as a gene associated with human osteoporosis, and yeast-based research suggested it may be a splicing factor; however, its molecular mechanism remains a mystery. We report here that currently available commercial GPATCH1 antibodies have poor specificity and are not recommended for immunoprecipitation. We elucidated the apparent molecular weight of GPATCH1 to evaluate the antibodies’ specificity. Based on this, a specific polyclonal antibody against GPATCH1 that can be used for Western blotting, immunoprecipitation and immunofluorescence was prepared. With the antibodies, we found that GPATCH1 may be a tissue-specific splicing factor. Our study lays the groundwork for further investigations into the molecular mechanisms by which GPATCH1 affects bone metabolism in the future.

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 TiO 2 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. Graphical abstract

Goatpoxvirus (GTPV), sheeppoxvius (SPPV), and the Lumpy skin disease virus (LSDV) is a Capripoxvirus belonging to the family poxviridae . They can cause significant economic losses in countries where this disease are endemic. However, effective and convenient diagnostic tools against sera antibody are not readily available until now. Toward this goal, a polyclonal antibody competitive enzyme-linked immunosorbent assay (c-ELISA) of detecting serogroup-specific antibody is established based on major LSDV antigen A33. Serum samples ( n  = 605) were collected to optimize the c-ELISA from different areas. The cut-off value for the c-ELISA was estimate using percent inhibition (PI) values. The diagnostic performance of test including sensitivity (sn) and specificity (sp) were obtained by receiver operator characteristic (ROC) analysis. Among these analysis, > 57.61% PI value was accepted as cut-off of the c-ELISA, the diagnostic sn an diagnostic sp were reached to 96.4% and 98.5%, at > 95% confidence interval. These results show that the developed competitive ELISA is sensitive, specific, and reliable, which make it appropriate for serological investigation.

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.

BackgroundCocoonase 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.ResultsIn 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.ConclusionThe 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.