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Background: An antigen is a small foreign substance, such as a microorganism structural protein, that may trigger an immune response once inside the body. Antigens are preferentially used rather than completely attenuated microorganisms to develop safe vaccines. Unfortunately, not all antigens are able to induce an immune response. Thus, new adjuvants to enhance the antigen’s ability to stimulate immunity must be developed. Objectives: Therefore, this work aimed to evaluate the molecular-structure adjuvant activity of tannic acid (TA) coupled to a protein antigen in Balb/c mice. Method: Bovine serum albumin (BSA) was used as an antigen. The coupling of BSA and TA was mediated by carbodiimide crosslinking, and verified by SDS-PAGE. Forty-two Balb/c mice were divided into seven groups, including two controls without antigen, an antigen control, an adjuvant control, and two treatment groups. An additional group was used for macrophages isolation. A 30-day scheme was used to immunize the mice. The analysis of humoral immunity included immunoglobulin quantification, isotyping and antigen-antibody precipitation. The analysis of cell-mediated immunity included the quantification of nitric oxide from peritoneal macrophages and splenocytes’ proliferation assay after treatment stimulation. Results: No differences were found in the antibodies’ concentration or isotypes induced with the conjugate or the pure BSA. However, an immunogenicity improvement (p < 0.05) was observed through the specific anti-BSA antibody titers in mice immunized with the conjugate. Besides, macrophage activation (p < 0.05) was detected when stimulated with the treatments containing TA. Conclusion: Tannic acid exhibited macrophages’ activation properties. Moreover, when TA was incorporated into the structure of a protein antigen, such as BSA, an antibody specificity enhancement was observed. This was a consequence of antigen processing by activated antigen-presenting cells. These results showed the use of tannic acid as a novel candidate for vaccine molecular-structure adjuvant.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for ‘on-demand’ degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The overexpression of PDPN contributes to the malignant progression of tumors. Therefore, the development of anti-PDPN monoclonal antibodies (mAbs) to animals is essential to evaluate the pathogenesis and cellular functions. Using peptide immunization, we previously developed an anti-elephant PDPN (elePDPN) mAb, PMab-295, which is useful for flow cytometry, Western blotting, and immunohistochemistry. In this study, we determined the critical epitope of PMab-295 by enzyme-linked immunosorbent assay (ELISA). We performed ELISA with the alanine-substituted peptides of elePDPN extracellular domain (amino acids 38-51), and found that PMab-295 did not recognize the alanine-substituted peptides of M41A, P44A, and E47A. Furthermore, these peptides could not inhibit the recognition of PMab-295 to elePDPN-expressing cells by flow cytometry and immunohistochemistry. The results indicate that the binding epitope of PMab-295 includes Met41, Pro44, and Glu47 of elePDPN.

Antibodies against HLA antigens are ubiquitous in the sera of transplant patients. Analysis of anti-HLA antibodies specificity has gone through a long history of development using assays like agglutination and lymphocytotoxicity, which utilize lymphocytes, and flow cytometry, which utilize multiplex beads coupled with single antigens. Hundreds of HLA antigens are identified to date, and the realization that antibody reactivity against the antigens is multispecific presented difficulties in accurately defining antibody specificity. Although Cross Reacting Groups (CREG), describing cross reactivity among HLA antigens, were helpful with determining specificity, they proved to be inadequate for the highly sensitized patients. Amino acid sequencing and three-dimensional modeling of the HLA molecules significantly advanced our understating of the HLA antigens and their epitopes. Although sensitive assays for antibody testing advanced analysis, they unmasked additional specificities undetectable by traditional methods, and the presence of naturally occurring anti-HLA antibodies in sera further complicated analysis and underscored the need to understand antibody reactivity and their epitopes. Hundreds of HLA class-I and class-II epitopes were defined by the Tekasaki and Duquesnoy groups and their usefulness in organ transplants were further advanced by a great number of transplant centers. Alloantibody specificities, CREGs, and nondonor specific antigens (NDSA) are now explained by public epitopes.

The sequence diversity of HIV-1 is the biggest hurdle for the design of a prophylactic vaccine. Mosaic (Mos) antigens consisting of synthetically shuffled epitopes from various HIV-1 strains are currently tested in the clinical vaccine trial Mosaico (NCT03964415). Besides adenovirus vectors encoding variants of Mos.Gag-Pol and soluble Mos.Env proteins, the Mosaico vaccine entails vectors mediating gene transfer and expression of the membrane-anchored Env-variant Mos2S.Env. We thus examined whether the expression of mosaic Gag mediates the formation of virus-like particles (VLPs). Mos1.Gag- and Mos2.Gag-VLP-formation was readily detected using Western blot- and electron microscopic-analysis. Upon co-expression of both mosaic Gag variants with Mos2S.Env, incorporation of Env into Gag-formed VLPs was observed. The display of the respective neutralization-sensitive target epitopes on Mos2S.Env-decorated VLPs was demonstrated employing a panel of broadly neutralizing antibodies (bNAbs) in a VLP-capture assay. This opens new perspectives for future HIV vaccine designs.

CXC chemokine receptor 4 (CXCR4), a member of the G-protein-coupled receptor family, plays an important role in host immune responses. Within the teleost lineage, there are two paralogs of CXCR4; however, the role of CXCR4 in teleost B cells is poorly understood. In this study, we determined the cDNA sequences of the two CXCR4 paralogs from the Japanese sea bass (Lateolabrax japonica; LjCXCR4a and LjCXCR4b). Sequence and phylogenetic tree analyses revealed that LjCXCR4a and LjCXCR4b are most closely related to CXCR4a and CXCR4b, respectively, in the large yellow croaker (Larimichthys crocea). CXCR4 transcripts were mainly expressed in the gills, and their expression in different tissues was altered upon infection with Vibrio harveyi. LjCXCR4a and LjCXCR4b protein levels were upregulated in infected B cells. Knockdown of LjCXCR4a and LjCXCR4b in B cells by RNA interference, the phagocytic activity of B cells was not affected. Furthermore, knockdown of LjCXCR4a, not of LjCXCR4b, was observed to inhibit LjIgM expression in lipopolysaccharide-stimulated B cells. In addition, knockdown of LjCXCR4a, not of LjCXCR4b, was found to reduce reactive oxygen species levels in B cells. Our results indicate that LjCXCR4a and LjCXCR4b modulate the immune response of Japanese sea bass B cells against bacterial infection, albeit via different pathways.

Monoclonal antibodies (mAbs) that specifically target podoplanin (PDPN), a marker for type I alveolar cells, are required for immunohistochemical analyses. Anti-PDPN mAbs are available for many species, including human, mouse, rat, rabbit, dog, cat, bovine, pig, Tasmanian devil, alpaca, tiger, whale, goat, horse, bear, sheep, and California sea lion PDPNs. However, no anti-Steller sea lion PDPN (stePDPN) antibody has been developed. Immunohistochemical analysis showed that an anti-California sea lion PDPN mAb (PMab-269) reacted with type I alveolar cells from the Steller sea lion lung, renal glomeruli and Bowman's capsules from kidney, and lymphatic endothelial cells from the colon, indicating that PMab-269 is useful for detecting stePDPN.

Therapeutic antibodies have become one of the most widely used classes of biotherapeutics due to their unique antigen specificity and their ability to be engineered against diverse disease targets. There is significant interest in utilizing truncated antibody fragments as therapeutics, as their small size affords favorable properties such as increased tumor penetration as well as the ability to utilize lower-cost prokaryotic production methods. Their small size and simple architecture, however, also lead to rapid blood clearance, limiting the efficacy of these potentially powerful therapeutics. A common approach to circumvent these limitations is to enable engagement with the half-life extending neonatal Fc receptor (FcRn). This is usually achieved via fusion with a large Fc domain, which negates the benefits of the antibody fragment's small size. In this work, we show that modifying antibody fragments with short FcRn-binding peptide domains that mimic native IgG engagement with FcRn enables binding and FcRn-mediated recycling and transmembrane transcytosis in cell-based assays. Further, we show that rational, single amino acid mutations to the peptide sequence have a significant impact on the receptor-mediated function and investigate the underlying structural basis for this effect using computational modeling. Finally, we report the identification of a short peptide from human serum albumin that enables FcRn-mediated function when grafted onto a single-chain variable fragment (scFv) scaffold, establishing an approach for the rational selection of short-peptide domains from full-length proteins that could enable the transfer of non-native functions to small recombinant proteins without significantly impacting their size or structure.

As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed ‘MILKSHAKE,’ is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue’s modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

Cancer treatment has gradually developed from toxic chemotherapy to targeted therapy with fewer side effects. Approximately 30% of breast cancer patients overexpress human epidermal growth factor receptor 2 (HER‐2). Previous studies have successfully produced single‐chain antibodies (scFv) targeting HER‐2+ breast cancer; however, scFv have poor stability, easy aggregation and a shorter half‐life, which have no significant effect on targeting therapy. Moreover, scFv has been considered as a drug delivery platform that can kill target cells by effector molecules. However, the functional killing domains of immunotoxins are mainly derived from plant or bacterial toxins, which have a large molecular weight, low tissue permeability and severe side effects. To address these concerns, we designed several apoptotic immune molecules to replace exogenous toxins using endogenous apoptosis‐related protein DNA fragmentation factor 40 (DFF40) and tandem‐repeat Cytochrome c base on caspase‐3 responsive peptide (DEVD). Our results suggest that DFF40 or Cytc fusion scFv specifically targets HER‐2 overexpressing breast cancer cells (SK‐BR‐3 and BT‐474) rather than HER‐2 negative cells (MDA‐MB‐231 and MCF‐7). Following cellular internalization, apoptosis‐related proteins inhibited tumour activity by initiating endogenous apoptosis pathways, which significantly reduced immunogenicity and toxic side effects. Therefore, we suggest that immunoapoptotic molecules may become potential drugs for targeted immunotherapy of breast cancer.

The American Zika virus (ZIKV) epidemic has highlighted the need to gain a better understanding of this emerging virus. The goal of this study was to describe the clinical symptoms, laboratory findings, and risk factors for symptomatic ZIKV infection in an area with ongoing transmission of other arboviral infections. We recruited patients at least 2 years of age seeking care at public health centers in León, Nicaragua, between January 2016 and August 2017, for fever, maculopapular rash, and/or nonsuppurative conjunctivitis with a duration of less than 1 week. A laboratory diagnosis of ZIKV was established using a combination of molecular and serological tests. Clinical and laboratory findings and potential risk factors were compared between participants with and without acute ZIKV infection. Fifty-eight (26%) of the 225 participants included in the analysis were found to have acute ZIKV infection. Pregnancy and reports of previous arboviral infection were associated with a higher risk of ZIKV infection. Rash, conjunctivitis, sore throat, and lower absolute neutrophil counts were associated with acute ZIKV infection. The clinical characteristics and risk factors identified were consistent with those identified by previous studies; however, we found sore throat to be a feature of ZIKV infection. We also found that neutrophil counts were lower in ZIKV-infected subjects. These clinical symptoms and laboratory data may help clinicians suspect ZIKV infection during future outbreaks.

Specific antibody interactions with short peptides have made epitope tagging systems a vital tool employed in virtually all fields of biological research. Here, we present a novel epitope tagging system comprised of a monoclonal antibody named GD-26, which recognises the TD peptide (GTGATPADD) derived from Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant. The crystal structure of the antigen-binding fragment (Fab) of GD-26 complexed with the TD peptide was determined to a resolution of 1.45 A. The TD peptide was found to adopt a 310 helix conformation within the binding cleft, providing a characteristic peptide structure for recognition by GD-26 Fab. Based on the structure information, polar and non-polar forces collectively contribute to the strong binding. Attempts to engineer the TD peptide show that the proline residue is crucial for the formation of the 310 helix in order to fit into the binding cleft. Isothermal calorimetry (ITC) reported a dissociation constant KD of 12 ± 2.8 nM, indicating a strong interaction between the TD peptide and GD-26 Fab. High specificity of GD-26 IgG to the TD peptide was demonstrated by Western blotting, enzyme-linked immunosorbent assays (ELISA) and immunofluorescence as only TD-tagged proteins were detected, suggesting the effectiveness of the GD-26/TD peptide tagging system. In addition to already-existing epitope tags such as the FLAG tag and the ALFA tag adopting either extended or α-helix conformations, the unique 310 helix conformation of the TD peptide together with the corresponding monoclonal antibody GD-26 offers a novel tagging option for research.

Classical swine fever virus (CSFV) is a member of the genus Pestivirus, which causes serious economic losses. The re-emergence of the disease in Japan in 2018 has increased awareness of CSFV. In this study, Balb/c mice were immunized with plant-derived E2 protein, and four monoclonal antibodies (mAbs) 4B11, 7B3, 11A5 and 6F3 were generated. Two of these mAbs, 4B11 and 7B3, effectively blocked CSFV infection of PK-15 cells. Both mAbs recognized a novel linear epitope, 256CLIGNTTVKVHASDER271. The neutralizing ability of anti-CSFV serum decreased 63%, when pre-incubated with the linear peptide at 200 μg/mL. Structural analysis showed that this linear epitope is present at the border of Domain C and Domain D on the surface of the E2 protein. Alignment of amino acid sequences showed that the epitope was conserved in different subgroups of CSFV but not in other members of the Pestivirus genus. Consistently with the analysis above, this epitope distinguished antibodies against CSFV from those against bovine viral diarrhea virus (BVDV). Our study provides an ideal candidate peptide for new vaccine design and differential diagnosis of CSFV. These findings will contribute to the control and eradication of classical swine fever.

Chimeric antigen receptors (CAR)-T cells are genetically engineered T-lymphocytes redirected with a predefined specificity to any target antigen, in a non-HLA restricted manner, therefore combining antibody-type specificity with effector T-cell function. This strategy was developed some thirty years ago, after extensive work established the key role of the immune system against cancer. The first-engineered T-cell with chimeric molecule was designed in 1993 by Israeli immunologist Zelig Eshhar. Since then, several modifications took place, including the addition of co-stimulatory domain, to further improve CAR-T cell anti-tumor potency. The first clinical application of CAR-T cell was done in Rotterdam in 2005 for metastatic renal cell carcinoma and simultaneously at the National Cancer Institute (NCI) for metastatic ovarian cancer. These pioneered studies failed to demonstrate a therapeutic benefit, but warning emerged concerning their safety of use. The real clinical success came with anti-CD19 CAR-T cells, used since 2009 by Steven Rosenberg at the NCI in a patient with refractory follicular lymphoma and in 2011 by Carl June and David Porter from the University of Pennsylvania in patients with chronic lymphocytic leukemia and B-cell acute lymphoblastic leukemia. From that time, large centers in North America have embarked in several early phase and pivotal trials that have demonstrated unprecedent response rate in heavily pretreated chemo refractory patient with B-cell malignancies. Theses clinical success have led to the approval of three anti-CD19 CAR-T cells products for the management of B-cell malignancies in the United States and in Europe as of December 2020.

Tumor-associated glycolipids such as NeuGc GM3 are auspicious molecular targets in antineoplastic therapies and vaccine strategies. 14F7 is a monoclonal IgG1 with high clinical potential in cancer immunotherapy as it displays extraordinary specificity for NeuGc GM3, while it does not recognize the very similar, ubiquitous NeuAc GM3. Here we present the 2.3 Å crystal structure of the 14F7 antigen-binding domain (14F7 scFv) in complex with the NeuGc GM3 trisaccharide. Modeling analysis and previous mutagenesis data suggest that 14F7 may also bind to an alternative NeuGc GM3 conformation, not observed in the crystal structure. The most intriguing finding, however, was that a water molecule centrally placed in the complementarity-determining region directly mediates the specificity of 14F7 to NeuGc GM3. This has profound impact on the complexity of engineering in the binding site and provides an excellent example of the importance in understanding the water structure in antibody–antigen interactions.

Rabbit hemorrhagic disease virus (RHDV) typically causes a fatal disease in rabbits. In Australia, RHDV was imported to control the feral rabbit population, while it poses a severe threat to native rabbits in other countries. RHDV variants are genetically diverse and serological studies using antibodies isolated from infected rabbits or raised against RHDV virus-like particles (VLPs) have found RHDV variants antigenically distinct. In this study, we determined the X-ray crystal structure of an RHDV GI.2 (N11 strain) protruding (P) domain in complex with a diagnostic monoclonal antibody (2D9) Fab. We showed that 2D9 interacted with conserved and variable residues on top of the P domain with nanomolar affinity. To better illustrate 2D9 specificity, we determined the X-ray crystal structure of an RHDV GI.1b (Ast89 strain) that was a 2D9 non-binder. Structural analysis indicated that amino acid substitutions on the GI.1b P domain likely restricted 2D9 binding. Interestingly, a model of the GI.2 P domain-Fab complex superimposed onto a cryo-EM structure of an RHDV VLP revealed that 2D9 Fab molecules clashed with neighboring Fabs and indicated that there was a reduced antibody binding occupancy. Moreover, the RHDV GI.2 histo-blood group antigen (HBGA) co-factor binding site appeared obstructed when 2D9 was modeled on the VLP and suggested that 2D9 might also function by blocking HBGA attachment. Overall, this new data provides the first structural basis of RHDV antibody specificity and explains how amino acid variation at the binding site likely restricts 2D9 cross-reactivity. IMPORTANCE Isolated RHDV antibodies have been used for decades to distinguish between antigenic variants, monitor temporal capsid evolution, and examine neutralizing capacities. In this study, we provided the structural basis for an RHDV GI.2 specific diagnostic antibody (2D9) binding and reveal that a small number of amino acid substitutions at the binding site could differentiate between RHDV GI.2 and GI.1b. This novel structural information provides a framework for understanding how RHDV displays a specific antigenic epitope and engages an antibody at the atomic level. Importantly, part of the 2D9 binding region was earlier reported to contain a neutralizing epitope and our structural modeling as well as recent human norovirus antibody-mediated neutralization studies, suggest that the 2D9 antibody has the potential to block HBGA attachment. These new findings should aid in characterizing antigenic variants and advance the development of novel monoclonal antibodies for diagnostics and therapeutics.

Antibiotics in aquaculture prevent bacterial infection of fish, but their misuse is a public health risk and contributes to the unintentional creation of multiresistant pathogens. Regulatory agencies cannot do the rigorous, expensive testing required to keep up with the volume of seafood shipments. Current rapid test kits for these drugs enable the increase in testing needed for adequate monitoring of food supply chains, but they lack a high degree of accuracy. To combat this, we set out to discover and engineer single-domain antibodies (VHHs) that bind to small molecule antibiotics, and that can be used in rapid test kits. The small size, solubility, and stability of VHHs are useful properties that can improve the reliability and shelf-life of test kits for these adulterants. Here, we report a novel anti-chloramphenicol VHH (Chl-VHH) with a disassociation constant of 57 nM. This was achieved by immunizing a llama against a chloramphenicol-keyhole limpet hemocyanin (KLH) conjugate and screening for high affinity binders through phage display. The crystal structure of the bound-VHH to chloramphenicol was key to identifying a mutation in the binding pocket that resulted in a 16-fold improvement in binding affinity. In addition, the structure provides new insights into VHH-hapten interactions that can guide future engineering of VHHs against additional targets.

Objective Mice were immunized with purified virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) to prepare polyclonal antibody and identify specificity. Methods BALB/c mice were injected with duck tembusu virus to generate viperin in mouse brain by intracranial injection. Viperin gene, cloned from mouse brain tissue by reverse transcription PCR, was inserted into pGEX-6p-1 prokaryotic expression vector and transformed into E. coli Rosetta. The recombinant viperin protein was induced by isopropyl thiogalactoside (IPTG) and its solubility was analyzed. The protein was purified by potassium chloride (KCl) staining and gel cutting method. Polyclonal antibody was prepared by immunizing mice with purified recombinant viperin protein subcutaneously through abdomen, and the titer of polyclonal antibody was determined by indirect ELISA. Western blot analysis and indirect fluorescence assay (IFA) were used to detect the transient expression of viperin protein in BHK-21 cells to identify the specificity and sensitivity of the prepared polyclonal antibody against viperin protein. Results The mouse viperin gene was successfully cloned and the viperin protein was expressed. The titer of the prepared anti-viperin polyclonal antibody reached 1:25 600. The mouse anti-viperin polyclonal antibody could specifically recognize the transient expression of viperin protein in BHK-21 cells. Conclusion Mouse polyclonal antibody against viperin protein with high specificity and sensitivity was successfully prepared.

Four amino acids are involved in epitope formation of human neutrophil antigens (HNA)-1 alleles, located at positions 36, 65, 78, and 82. HNA-1a and HNA-1b alloantibody epitopes were recently characterized. The HNA-1b allele also carries the HNA-1d epitope p.78Ap.82N. The current study aimed to identify compound antibody specificities in HNA-1b alloantisera, especially the presence of anti-HNA-1d.For investigation of binding epitopes for HNA-1b alloantibodies, cells stably expressing different HNA-1 alleles were generated and tested against previously well-characterized HNA-1b antisera (n = 11) in an antigen capture assay. Sera with p.82N specificity or p.36S and p.82N specificity were additionally analyzed using adsorption and elution methods.Three amino acids, p.36S, p.78A, and p.82N, are involved in epitope formation of HNA-1b. The following specificities were identified in 11 HNA-1b alloantisera: p.36S (6/11), p.82N (9/11), and p.78Ap.82N (8/11), of which p.36S was identified as a sole entity in 2/11, whereas 9/11 antisera contained a polyspecific mixture of anti-p.36S, p.82N (1/11), and anti-p.78Ap.82N in combination with anti p.82N (5/11) or compound specificities of anti-p.36S, p.82N, and p.78Ap82N (3/11). In seven of eight antisera with p.82N specificity, anti-p.78Ap.82N was detected.Analysis of HNA-1b antisera indicates compound specificities for HNA-1b alloantibodies with a high variation between HNA-1b immunized individuals. Amino acids p.36S, p.82N, and p.78Ap.82N are necessary for HNA-1b epitope formation. The HNA-1d epitope is recognized by 73% (8/11) of HNA-1b immunized individuals.

Antibody tests for SARS-CoV-2, the virus that causes COVID-19, are widely available. This living review summarizes the evidence on the prevalence, levels, and durability of detectable antibodies after SARS-CoV-2 infection and whether antibodies to SARS-CoV-2 confer protective immunity. The review will be updated as more evidence becomes available., Background: The clinical significance of the antibody response after SARS-CoV-2 infection remains unclear. Purpose: To synthesize evidence on the prevalence, levels, and durability of detectable antibodies after SARS-CoV-2 infection and whether antibodies to SARS-CoV-2 confer natural immunity. Data Sources: MEDLINE (Ovid), Embase, CINAHL, Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, World Health Organization global literature database, and Covid19reviews.org from 1 January through 15 December 2020, limited to peer-reviewed publications available in English. Study Selection: Primary studies characterizing the prevalence, levels, and duration of antibodies in adults with SARS-CoV-2 infection confirmed by reverse transcriptase polymerase chain reaction (RT-PCR); reinfection incidence; and unintended consequences of antibody testing. Data Extraction: Two investigators sequentially extracted study data and rated quality. Data Synthesis: Moderate-strength evidence suggests that most adults develop detectable levels of IgM and IgG antibodies after infection with SARS-CoV-2 and that IgG levels peak approximately 25 days after symptom onset and may remain detectable for at least 120 days. Moderate-strength evidence suggests that IgM levels peak at approximately 20 days and then decline. Low-strength evidence suggests that most adults generate neutralizing antibodies, which may persist for several months like IgG. Low-strength evidence also suggests that older age, greater disease severity, and presence of symptoms may be associated with higher antibody levels. Some adults do not develop antibodies after SARS-CoV-2 infection for reasons that are unclear. Limitation: Most studies were small and had methodological limitations; studies used immunoassays of variable accuracy. Conclusion: Most adults with SARS-CoV-2 infection confirmed by RT-PCR develop antibodies. Levels of IgM peak early in the disease course and then decline, whereas IgG peaks later and may remain detectable for at least 120 days. Primary Funding Source: Agency for Healthcare Research and Quality. (PROSPERO: CRD42020207098)

Monoclonal antibodies are extremely valuable functional biomaterials that are widely used not only in life science research but also in antibody drugs and test drugs. There is also a strong need to develop high-quality neutralizing antibodies as soon as possible in order to stop the rapid spread of new infectious diseases such as the SARS-CoV-2 virus. This study has developed a membrane-type immunoglobulin-directed hybridoma screening (MIHS) method for obtaining high-quality monoclonal antibodies with high efficiency and high speed. In addition to these advantages, this paper demonstrates that the MIHS method can selectively obtain monoclonal antibodies that specifically recognize the functional structure of proteins. The MIHS method is a useful technology that greatly contributes to the research community because it can be easily introduced in any laboratory that uses a flow cytometer.

The Kidd-null phenotype, Jk(a-b-), is rare, and a patient with this phenotype may develop anti-Jk3, a red blood cell (RBC) antibody reactive with a domain common to both Jka and Jkb. Like other antibodies to high-prevalence antigens, the presence of this antibody poses challenges in the immunohematologic evaluation of these patients. Thoughtful laboratory testing is necessary to resolve the antibody specificity and to reveal other underlying antibodies. Moreover, the rarity of the Kidd-null phenotype makes finding blood donors difficult for those who need transfusion and have developed anti-Jk3. This review describes methods used in identifying anti-Jk3 in four pregnant patients. Blood bank records were retrospectively reviewed to illustrate the common approach in anti-Jk3 identification. In all cases, pertinent blood bank history was gathered, and extended RBC phenotyping was performed, followed by adsorption studies and testing of selected RBCs. Underlying antibodies were found in two of the cases. This review also reiterates some common challenges encountered with Kidd antibody analysis and highlights the importance of patient ethnic ancestry and obtaining accurate patient transfusion history.The Kidd-null phenotype, Jk(a–b–), is rare, and a patient with this phenotype may develop anti-Jk3, a red blood cell (RBC) antibody reactive with a domain common to both Jka and Jkb. Like other antibodies to high-prevalence antigens, the presence of this antibody poses challenges in the immunohematologic evaluation of these patients. Thoughtful laboratory testing is necessary to resolve the antibody specificity and to reveal other underlying antibodies. Moreover, the rarity of the Kidd-null phenotype makes finding blood donors difficult for those who need transfusion and have developed anti-Jk3. This review describes methods used in identifying anti-Jk3 in four pregnant patients. Blood bank records were retrospectively reviewed to illustrate the common approach in anti-Jk3 identification. In all cases, pertinent blood bank history was gathered, and extended RBC phenotyping was performed, followed by adsorption studies and testing of selected RBCs. Underlying antibodies were found in two of the cases. This review also reiterates some common challenges encountered with Kidd antibody analysis and highlights the importance of patient ethnic ancestry and obtaining accurate patient transfusion history.

Podoplanin (PDPN) is an essential marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) that can specifically recognize PDPN in immunohistochemistry are important to analyze the development of tissues and the pathogenesis of diseases, including cancers. We have developed anti-PDPN mAbs against many animal species; however, mAbs that can recognize elephant-derived membrane proteins and distinguish the specific cell types in immunohistochemistry are limited. In this study, a novel anti-elephant PDPN (elePDPN) mAb, PMab-295 (IgG

Chinese hamster (

This retrospective study aimed to evaluate the relationship between plasma autoantibody species and rhTPO response in adult ITP patients who failed the first-line treatments. Plasma anti-glycoprotein (GP) IIb/IIIa and anti-GPIb/IX autoantibodies were detected in 47·2% and 40·6% of the 123 patients, respectively. Overall response rate to rhTPO treatment in patients without anti-GPIb/IX autoantibodies was significantly higher than patients with anti-GPIb/IX autoantibodies (82·2% vs. 60·0%, P = 0·006). By contrast, no statistical difference in response rate was observed between patients with or without anti-GPIIb/IIIa autoantibodies (74·1% vs. 72·3%, P = 0·819). Therefore, the presence of anti-GPIb/IX autoantibodies might serve as a predictive factor for poor response to rhTPO treatment in ITP.

Serological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect antibodies against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various antigen-specific antibody isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via polymerase chain reaction test. We developed IgG, IgM and IgA measurement assays for each antigen, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full length S protein, S trimer and nucleocapsid (N) domain, based on enzyme-linked immunosorbent assay. The assays of the S protein for all isotypes showed high specificity, while the assays for all isotypes against N protein showed lower specificity. The sensitivity of all antigen-specific antibody isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 days post-symptom onset. The best correlation with virus neutralizing activity was found for IgG against RBD (RBD-IgG), and levels of RBD-IgG in sera from four severe COVID-19 patients increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

Vaccination is crucial in combatting the severe acute respiratory syndrome coronavirus 2 pandemic. The rare complication of thrombocytopenia and thrombotic complications at unusual sites after ChAdOx1 nCov-19 vaccination is caused by platelet-activating antibodies directed against platelet factor 4 (PF4). We present a widely applicable whole-blood standard flow cytometric assay to identify the pathogenic antibodies associated with vaccine-induced immune-mediated thrombotic thrombocytopenia (VITT) after ChAdOx1 nCov-19 vaccination. This assay will enable rapid diagnosis by many laboratories. This trial was registered at www.clinicaltrials.gov as #NCT04370119.

OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.

The optimization of therapeutic antibodies is time-intensive and resource-demanding, largely because of the low-throughput screening of full-length antibodies (approximately 1 x 10(3) variants) expressed in mammalian cells, which typically results in few optimized leads. Here we show that optimized antibody variants can be identified by predicting antigen specificity via deep learning from a massively diverse space of antibody sequences. To produce data for training deep neural networks, we deep-sequenced libraries of the therapeutic antibody trastuzumab (about 1 x 10(4) variants), expressed in a mammalian cell line through site-directed mutagenesis via CRISPR-Cas9-mediated homology-directed repair, and screened the libraries for specificity to human epidermal growth factor receptor 2 (HER2). We then used the trained neural networks to screen a computational library of approximately 1 x 10(8) trastuzumab variants and predict the HER2-specific subset (approximately 1 x 10(6) variants), which can then be filtered for viscosity, clearance, solubility and immunogenicity to generate thousands of highly optimized lead candidates. Recombinant expression and experimental testing of 30 randomly selected variants from the unfiltered library showed that all 30 retained specificity for HER2. Deep learning may facilitate antibody engineering and optimization., Therapeutic antibodies can be optimized using deep-learning models trained on antibody-mutagenesis libraries to generate antibody variants and predict their antigen specificity.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.

Alpha-methylacyl-coenzyme A-racemase (AMACR), also known as p504s, is overexpressed in prostatic adenocarcinoma and is frequently used in combination with basal cell markers to aid in diagnosing difficult prostate adenocarcinoma cases. In this retrospective method comparison study, we examined the sensitivity and specificity of the ready-to-use anti-p504s (SP116) Rabbit Monoclonal Primary Antibody compared to the monoclonal rabbit anti-human AMACR clone 13H4 in prostatic adenocarcinoma samples. De-identified prostatic adenocarcinoma tissue samples were stained with either the SP116 or 13H4 antibody clone in combination with the VENTANA Basal Cell Cocktail (34βE12+p63) and scored as positive or negative for prostatic adenocarcinoma. The scoring pathologist was blinded to the known historical diagnosis of each sample. The scoring pathologist correctly diagnosed each sample regardless of which p504s clone was used. Both assays using either clone were 100% concordant in their sensitivity and specificity. This study demonstrates that the ready-to-use anti-p504s (SP116) Rabbit Monoclonal Primary Antibody is equivalent to clone 13H4 concentrate when used according to package insert instructions in combination with the VENTANA Basal Cell Cocktail (34βE12+p63) to aid pathologists in the diagnosis of prostatic adenocarcinoma.

Some Plasmodium falciparum repetitive interspersed families of polypeptides (RIFINs)—variant surface antigens that are expressed on infected erythrocytes1—bind to the inhibitory receptor LAIR1, and insertion of DNA that encodes LAIR1 into immunoglobulin genes generates RIFIN-specific antibodies2,3. Here we address the general relevance of this finding by searching for antibodies that incorporate LILRB1, another inhibitory receptor that binds to β2 microglobulin and RIFINs through their apical domains4,5. By screening plasma from a cohort of donors from Mali, we identified individuals with LILRB1-containing antibodies. B cell clones isolated from three donors showed large DNA insertions in the switch region that encodes non-apical LILRB1 extracellular domain 3 and 4 (D3D4) or D3 alone in the variable–constant (VH–CH1) elbow. Through mass spectrometry and binding assays, we identified a large set of RIFINs that bind to LILRB1 D3. Crystal and cryo-electron microscopy structures of a RIFIN in complex with either LILRB1 D3D4 or a D3D4-containing antibody Fab revealed a mode of RIFIN–LILRB1 D3 interaction that is similar to that of RIFIN–LAIR1. The Fab showed an unconventional triangular architecture with the inserted LILRB1 domains opening up the VH–CH1 elbow without affecting VH–VL or CH1–CL pairing. Collectively, these findings show that RIFINs bind to LILRB1 through D3 and illustrate, with a naturally selected example, the general principle of creating novel antibodies by inserting receptor domains into the VH–CH1 elbow. Plasmodium antigens called RIFINs bind to specific antibodies that incorporate the inhibitory receptor LILRB1 through its D3 domain, illustrating the principle of receptor-containing antibodies.

Objectives Our study purpose was to detect the distribution of anti-nuclear antibody (ANA) IgG subclasses in patients with systemic lupus erythematosus (SLE) and to evaluate their influence on the inflammatory process in SLE. Methods We determined the serum levels of ANA IgG subclasses from 70 SLE patients, 25 patients with other autoimmune diseases (OAD), and 25 healthy controls using ELISA. The serum level of total ANA IgG and the avidity of ANA IgG, dsDNA IgG, and dsDNA IgG subclasses were analysed by ELISA. Results The results indicated that levels of four ANA IgG subclasses (IgG1, IgG2, IgG3 and IgG4) and total IgG were significantly higher in SLE patients than in OAD patients and healthy controls ( p Conclusions First, we demonstrated that the ANA IgG subclasses were diagnostic tools in SLE patients. Furthermore, HA IgG ANAs might affect the distribution of ANA IgG3 and IgG4. Moreover, ANA IgG3 might play a particular role in the activity of SLE disease and therapy. Therefore, an altered ANA IgG subclass distribution might be a risk factor influencing the inflammatory process in SLE.

Most ovarian cancers are infiltrated by prognostically relevant activated T cells1–3, yet exhibit low response rates to immune checkpoint inhibitors4. Memory B cell and plasma cell infiltrates have previously been associated with better outcomes in ovarian cancer5,6, but the nature and functional relevance of these responses are controversial. Here, using 3 independent cohorts that in total comprise 534 patients with high-grade serous ovarian cancer, we show that robust, protective humoral responses are dominated by the production of polyclonal IgA, which binds to polymeric IgA receptors that are universally expressed on ovarian cancer cells. Notably, tumour B-cell-derived IgA redirects myeloid cells against extracellular oncogenic drivers, which causes tumour cell death. In addition, IgA transcytosis through malignant epithelial cells elicits transcriptional changes that antagonize the RAS pathway and sensitize tumour cells to cytolytic killing by T cells, which also contributes to hindering malignant progression. Thus, tumour-antigen-specific and -antigen-independent IgA responses antagonize the growth of ovarian cancer by governing coordinated tumour cell, T cell and B cell responses. These findings provide a platform for identifying targets that are spontaneously recognized by intratumoural B-cell-derived antibodies, and suggest that immunotherapies that augment B cell responses may be more effective than approaches that focus on T cells, particularly for malignancies that are resistant to checkpoint inhibitors. In patients with high-grade serous ovarian cancer, robust and protective humoral responses are dominated by B-cell-derived polyclonal IgA that binds to polymeric IgA receptors that are universally expressed on ovarian cancer cells.

B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic malignancy of B-type lymphoid precursor cells. Minimal/measurable residual disease (MRD) is an important prognostic factor for B-ALL relapse. Traditional flow cytometry detection mainly relies on CD19-based gating strategies. However, relapse of CD19-negative B-ALL frequently occurs in patients who receive cellular and targeted therapy. This review will summarize the technical aspects of standard MRD assessment in B-ALL by flow cytometry, and then discuss the challenges of MRD strategies to deal with the scenario of CD19 negative or dim B-ALL relapse.

Platelet transfusion refractoriness results in adverse outcomes and increased health care costs. Managing refractoriness resulting from HLA alloimmunization necessitates the use of HLA antigen–matched platelets but requires a large platelet donor pool and does not guarantee full matching. We report the first randomized, double-blind, noninferiority, crossover trial comparing HLA epitope–matched (HEM) platelets with HLA standard antigen–matched (HSM) platelet transfusions. Alloimmunized, platelet-refractory, thrombocytopenic patients with aplastic anemia, myelodysplastic syndrome, or acute myeloid leukemia were eligible. HEM platelets were selected using HLAMatchMaker epitope (specifically eplet) matching. Patients received up to 8 prophylactic HEM and HSM transfusions provided in random order. The primary outcome was 1-hour posttransfusion platelet count increment (PCI). Forty-nine patients were randomized at 14 UK hospitals. For intention to treat, numbers of evaluable transfusions were 107 and 112 for HEM and HSM methods, respectively. Unadjusted mean PCIs for HEM and HSM methods were 23.9 (standard deviation [SD], 15) and 23.5 (SD, 14.1), respectively (adjusted mean difference, −0.1; 95% confidence interval [CI], −2.9 to 2.8). Because the lower limit of the 95% CI was not greater than the predefined noninferiority limit, the HEM approach was declared noninferior to the HSM approach. There were no differences in secondary outcomes of platelet counts, transfusion requirements, and bleeding events. Adequate 1-hour PCI was more frequently observed, with a mean number of 3.2 epitope mismatches, compared with 5.5 epitope mismatches for inadequate 1-hour increments. For every additional epitope mismatch, the likelihood of an adequate PCI decreased by 15%. Epitope-matched platelets should be considered to support HLA alloimmunized patients. This trial was registered at www.isrctn.com as #ISRCTN23996532.

Factor V inhibitors are a rare cause of life-threatening bleeding. We present a case of an acquired factor V inhibitor likely caused by coronavirus disease 2019 infection. Bleeding was manifested by severe anemia requiring frequent red-cell transfusion, left psoas muscle hematoma, and left retroperitoneal cavity hematoma. Factor V activity was less than 1% and the factor V inhibitor titer was 31.6 Bethesda units. Severe acute respiratory syndrome coronavirus 2 RNA testing of the nasopharynx was positive 2 weeks before presentation and continued to be positive for 30 days. The patient failed treatment with intravenous immunoglobulin and dexamethasone. Three cycles of plasmapheresis with fresh frozen plasma replacement resulted in correction of the bleeding and laboratory coagulopathy. This is the first reported case of a factor V inhibitor in a coronavirus disease 2019 patient and suggests that plasmapheresis may be a successful treatment strategy.

Breast cancer susceptibility gene 1 (BRCA1) encodes a tumor suppressor that is frequently mutated in familial breast and ovarian cancer patients. BRCA1 functions in multiple important cellular processes including DNA damage repair, cell cycle checkpoint activation, protein ubiquitination, chromatin remodeling, transcriptional regulation, as well as R-loop formation and apoptosis. A large number of BRCA1 antibodies have been generated and become commercially available over the past three decades, however, many commercial antibodies are poorly characterized and, when widely used, led to unreliable data. In search of reliable and specific BRCA1 antibodies (Abs), particularly antibodies recognizing mouse BRCA1, we performed a rigorous validation of a number of commercially available anti-BRCA1 antibodies, using proper controls in a panel of validation applications, including Western blot (WB), immunoprecipitation (IP), immunoprecipitation-mass spectrometry (IP-MS), chromatin immunoprecipitation (ChIP) and immunofluorescence (IF). Furthermore, we assessed the specificity of these antibodies to detect mouse BRCA1 protein through the use of testis tissue and mouse embryonic fibroblasts (MEFs) from Brca1+/+ and Brca1Δ11/Δ11 mice. We find that Ab1, D-9, 07-434 (for recognizing human BRCA1) and 287.17, 440621, BR-64 (for recognizing mouse BRCA1) are specific with high quality performance in the indicated assays. We share these results here with the goal of helping the community combat the common challenges associated with anti-BRCA1 antibody specificity and reproducibility and, hopefully, better understanding BRCA1 functions at cellular and tissue levels.

We investigated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a group of convalescent, potential blood donors in Germany who had PCR-confirmed SARS-CoV-2 infection. Sixty days after onset of symptoms, 13/78 (17%) study participants had borderline or negative results to an ELISA detecting IgG against the S1 protein of SARS-CoV-2. We analyzed participants with PCR-confirmed infection who had strong antibody responses (ratio >3) as positive controls and participants without symptoms of SARS-CoV-2 infection and without household contact with infected patients as negative controls. Using interferon-γ ELISpot, we observed that 78% of PCR-positive volunteers with undetectable antibodies showed T-cell immunity against SARS-CoV-2. We observed a similar frequency (80%) of T-cell immunity in convalescent donors with strong antibody responses but did not detect immunity in negative controls. We concluded that, in convalescent patients with undetectable SARS-CoV-2 IgG, immunity may be mediated through T cells. OA platinum

International audience; Secretory intestinal IgA can protect from re-infection with rotavirus (RV), but very little is known about the mechanisms that induce IgA production during intestinal virus infections. Classical dendritic cells (cDCs) in the intestine can facilitate both T cell-dependent and -independent secretory IgA. Here, we show that BATF3-dependent cDC1, but not cDC2, are critical for the optimal induction of RV-specific IgA responses in the mesenteric lymph nodes. This depends on the selective expression of the TGF beta-activating integrin alpha v beta 8 by cDC1. In contrast, alpha v beta 8 on cDC1 is dispensible for steady state immune homeostasis. Given that cDC2 are crucial in driving IgA during steady state but are dispensable for RV-specific IgA responses, we propose that the capacity of DC subsets to induce intestinal IgA responses reflects the context, as opposed to an intrinsic property of individual DC subsets.

The ability to measure total and phosphorylated tau levels in clinical samples is transforming the detection of Alzheimer’s disease (AD) and other neurodegenerative diseases. In particular, recent reports indicate that accurate detection of low levels of phosphorylated tau (p-tau) in plasma provides a reliable biomarker of AD long before sensing memory loss. Therefore, the diagnosis and monitoring of neurodegenerative diseases progression using blood samples is becoming a reality. These major advances were achieved by using antibodies specific to p-tau as well as sophisticated high-sensitivity immunoassay platforms. This review focuses on these enabling advances in high-specificity antibody development, engineering, and novel signal detection methods. We will draw insights from structural studies on p-tau antibodies, engineering efforts to improve their binding properties, and efforts to validate their specificity. A comprehensive survey of high-sensitivity p-tau immunoassay platforms along with sensitivity limits will be provided. We conclude that although robust approaches for detecting certain p-tau species have been established, systematic efforts to validate antibodies for assay development is still needed for the recognition of biomarkers for AD and other neurodegenerative diseases.

Antibodies (Abs) are among the most important class of biologicals, showcasing a high therapeutic and diagnostic value. In the global therapeutic Ab market, fully-human monoclonal Abs (FH-mAbs) are flourishing thanks to their low immunogenicity and high specificity. The rapidly emerging field of single-cell technologies has paved the way to efficiently discover mAbs by facilitating a fast screening of the antigen (Ag)-specificity and functionality of Abs expressed by B cells. This review summarizes the principles and challenges of the four key concepts to discover mAbs using these technologies, being confinement of single cells using either droplet microfluidics or microstructure arrays, identification of the cells of interest, retrieval of those cells and single-cell sequence determination required for mAb production. This review reveals the enormous potential for mix-and-matching of the above-mentioned strategies, which is illustrated by the plethora of established, highly integrated devices. Lastly, an outlook is given on the many opportunities and challenges that still lie ahead to fully exploit miniaturized single-cell technologies for mAb discovery. ispartof: LAB ON A CHIP vol:21 issue:19 pages:3627-3654 ispartof: location:England status: published

Objective To evaluate the sensitivity and the stability of the monoclonal antibodies (Aa3c10, b10c1), against truncated Histidine-rich protein 2 (PfHRP2), developed using smart polymer, poly N-isopropylacrylamide, as adjuvant for malarial diagnostic applications in comparison with the available commercial antibodies. Methods Two hybridoma clones (Aa3c10, b10c1) were used for the production of ascites in BALB/c mice. Purification of monoclonal antibodies from the ascites was carried out using affinity columns. The thermal stability study of monoclonal antibodies was done by storing it at 37°C and 45°C for thirty days. The stored antibodies were analyzed using SDS-PAGE and flow-through device where the antigenantibody interaction was visualized by Protein A colloidal gold solution. Sensitivity was determined by endpoint dilution ELISA and the dissociation constant by competitive ELISA. Sensitive pair optimization was done by sandwich ELISA using biotinylated antibodies. Prototype preparation for lateral flow assay had a colloidal gold-based detection system. Results Thermal stability experiments showed that both mAbs (Aa3c10; b10c1) are stable up to thirty days at 45°C while the commercially available mAbs were stable up to fifteen days only. Compared to commercial antibodies, the mAb Aa3c10, showed the highest sensitivity in end-point titre. In sensitive pair optimization, it was observed that the mAb, b10c1, as a detector and the mAb, Aa3c10, as a capture antibody showed the highest absorbance to detect 50pg/ml PfHRP2 antigen. The prototype formulation of lateral flow assay using the mAbs (Aa3c10; b10c1) showed good reactivity with WHO panel and no false-positive results were observed with twenty clinically negative samples and five P. vivax positive samples. Conclusions The novel monoclonal antibodies (Aa3c10, b10c1) against truncated PfHRP2, could be a strong potential candidates that can be included in making RDTs with better sensitivity and stability.

Purification of antibodies has become a critical factor in antibody production. A high-purity specific antibody against antigens, especially small molecules, seems to be difficult to obtain, even with the help of a protein A affinity column, which is a conventional and broadly used ligand for the separation of antibody and non-antibody proteins. Therefore, it is urgent to develop a cheap, simple, efficient, and stable method to separate the specific antibody from other antibodies. In this study, to improve the sensitivity and accuracy of immunoassay results, enrofloxacin (ENR) was grafted onto polyethylenimine (PEI) by the abundant amino groups and then the whole ligand (ENR-PEI) was conjugated to CNBr-Sepharose 4B to prepare the affinity column for the purification of the specific antibody against ENR from polyclonal antibodies. Scanning electron microscopy and Fourier transform infrared spectroscopy verification showed that Sepharose 4B was successfully modified by ENR-PEI with excellent uniformity. The capacity of the prepared column could reach to 6.15 mg of specific antibody with high purity per milliliter resin due to the high coupling ratio (49.3:1) of ENR on PEI, and the IC50 value of the antibody after purification was 47.58 ng/mL with a lowest limit of detection (IC10) of 1.099 ng/mL-18 times lower than those of the antibody purified through the protein A column. All the results showed that this new kind of resin could be used as the potential ligand in the purification of the trace-specific antibody against antigens in complex mixtures with high efficiency and specificity.

Cytokeratin (CK) 18 is an intermediate filament protein that plays a major functional role in the integrity and mechanical stability of cells. Since both CK8 and CK18 are major components of simple epithelia, in the context of tumors, they are expressed in most carcinomas, and have been studied as diagnostic and prognostic markers in tumor pathology. CK18 is also cleaved by some caspases during apoptosis. Three-dimensional (3D)-cultured cancer cells are useful for cancer research as an intermediate model between in vitro cancer cell line cultures and in vivo tumors. In this study, we produced rat monoclonal antibodies (mAbs) through immunization of the lysate from 3D-cultured DLD-1 cells to elucidate a characteristic feature of a tumor, and our results showed that mAb 2H7 recognized human CK18. Furthermore, we indicated that mAb 2H7 was useful for immunoblotting, immunoprecipitation, and immunofluorescence staining. Therefore, it may be useful as a diagnostic tool for evaluating malignancy.

The antibody response magnitude and kinetics may impact clinical severity, serological diagnosis and long-term protection of COVID-19, which may play a role in why children experience lower morbidity. We therefore tested samples from 122 children in Hong Kong with symptomatic (n = 78) and asymptomatic (n = 44) SARS-CoV-2 infections up to 200 days post infection, relative to 71 infected adults (symptomatic n = 61, and asymptomatic n = 10), and negative controls (n = 48). We assessed serum IgG antibodies to a 14-wide antigen panel of structural and accessory proteins by Luciferase Immuno-Precipitation System (LIPS) assay and circulating cytokines. Infected children have lower levels of Spike, Membrane, ORF3a, ORF7a, ORF7b antibodies, comparable ORF8 and elevated E-specific antibodies than adults. Combination of two unique antibody targets, ORF3d and ORF8, can accurately discriminate SARS-CoV-2 infection in children. Principal component analysis reveals distinct pediatric serological signatures, and the highest contribution to variance from adults are antibody responses to non-structural proteins ORF3d, NSP1, ORF3a and ORF8. From a diverse panel of cytokines that can modulate immune priming and relative inflammation, IL-8, MCP-1 and IL-6 correlate with the magnitude of pediatric antibody specificity and severity. Antibodies to SARS-CoV-2 internal proteins may become an important sero surveillance tool of infection with the roll-out of vaccines in the pediatric population.