Rabbit Antibodies Research Articles

Association of the combined parameters including the frequency of primary cilia, PD-L1, Smoothened protein, membranous β-catenin and cytoplasmic β-catenin expression with the outcome of patients with clear cell renal cell carcinoma.

The objective of this study was to investigate the association and combined prognostic significance of the PD-L1, Smoothened protein and β-catenin expressions in patients with clear cell renal cell carcinoma (ccRCC). The PD-L1, Smoothened protein and β-catenin expression were evaluated in 104 ccRCC patients. All studied tumor samples were acquired from nephrectomy specimens of primary tumors and not from biopsies or metastases. An indirect immunohistochemistry using polyclonal rabbit anti-Smoothened antibody, monoclonal mouse anti-human β-catenin-1 antibody, immunohistochemical assay PD-L1 28-8 pharmDx using monoclonal rabbit anti-PD-L1 antibody and anti-VHL (C- terminal) rabbit antibody was used. Immunohistochemistry was scored semiquantitavely. Median overall survival (OS) was significantly better in patients with lower PD-L1 expression (≤5%), Smoothened protein (SMO) expression (<5%) or cytoplasmic β-catenin expression (≤75%) than in patients with higher expressions of these biomarkers (P<0.001, P=0.047, and P<0.001, respectively). Membranous β-catenin showed an opposite effect with its lower expression (≤75%) being associated with longer OS (P=0.020). There was significant association between PD-1 and PD-L1 expression (P=0.007) and significant association of tumor grade (WHO 2016) with membranous β-catenin (P<0.001), cytoplasmic β-catenin (P=0.005), pVHL (P=0.042), PD-L1 (P=0.049) and PD-1 (P=0.028) expression. The present study provides the first data on the potential association and combined prognostic significance of frequency of primary cilia, PD-L1, Smoothened protein and β-catenin expression with the outcome in clear cell renal cell carcinoma.

Comparison of Shigella GMMA and glycoconjugate four-component formulations in animals.

Shigellosis is leading bacterial cause of diarrhea with high prevalence in children younger than 5years in low- and middle-income countries, and increasing number of reports of Shigella cases associated to anti-microbial resistance. No vaccines against Shigella are still licensed, but different candidates based on the O-antigen portion of lipopolysaccharides are in clinic. Generalized Modules for Membrane Antigens (GMMA) have been proposed as an alternative delivery system for the O-antigen, and a 4-component vaccine candidate (altSonflex1-2-3), containing GMMA from S. sonnei and S. flexneri 1b, 2a and 3a is being tested in a phase 1/2 clinical trial, with the aim to elicit broad protection against the most prevalent Shigella serotypes. Here, the 4-component GMMA vaccine candidate has been compared to a more traditional glycoconjugate formulation for the ability to induce functional antibodies in mice and rabbits. In mice, in the absence of Alhydrogel, GMMA induce higher IgG antibodies than glycoconjugates and stronger bactericidal titers against all Shigella serotypes. In the presence of Alhydrogel, GMMA induce O-antigen specific IgG levels similar to traditional glycoconjugates, but with a broader range of IgG subclasses, resulting in stronger bactericidal activity. In rabbits, GMMA elicit higher functional antibodies than glycoconjugates against S. sonnei, and similar responses to S. flexneri 1b, 2a and 3a, independently from the presence of Alhydrogel. Different O-antigen based vaccines against Shigella are now in clinical stage and it will be of particular interest to understand how the preclinical findings in the different animal models translate in humans.

New Anti-RSV Nucleoprotein Monoclonal Antibody Pairs Discovered Using Rabbit Phage Display Technology.

Human respiratory syncytial virus (hRSV) is one of the major contagious viruses and causes complicated respiratory issues, especially in young children. The sensitive and fast detection of hRSV is critical for taking the most effective actions. In the present study, rabbit antibodies against the hRSV nucleoprotein (NP) were developed using phage display technology. A female rabbit was immunized with an hRSV strain A2 recombinant NP. A Fab library was built and sorted during two successive panning rounds for strain B and the A2 NP (recombinant preparations), respectively. The choice of candidates was performed using ELISA on the two NP strains. The obtained library was 3 × 106 cfu/mL, with an insertion rate of >95%. The two panning rounds permitted an enrichment factor of 100. ELISA screening allowed us to obtain 28 NP-specific Fab candidates. Among them, 10 retained candidates were reformatted into rabbit full IgG; thereafter, pairing tests on the recombinant strains and native lysate samples were performed. After the pairing tests on the recombinant strains, 53 pairs were identified. Eleven pairs were identified as being able to detect RSVs from native lysates. This work presents new high-potential monoclonal antibodies mAbs (mAbs), which would benefit from lateral flow testing data with patient materials.

Expression of G protein-coupled receptor GPR19 in normal and neoplastic human tissues

Little is known about the expression of the orphan G protein-coupled receptor GPR19 at the protein level. Therefore, we developed a rabbit antibody, targeting human GPR19. After verification of the antibody specificity using GPR19-expressing cell lines and a GPR19-specific siRNA, the antibody was used for immunohistochemical staining of a variety of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. In normal tissues, GPR19 expression was detected in a distinct cell population within the cortex, in single cells of the pancreatic islets, in intestinal ganglia, gastric chief cells, and in endocrine cells of the bronchial tract, the gastrointestinal tract, and the prostate. Among the 30 different tumour entities investigated, strong GPR19 expression was found in adenocarcinomas, typical and atypical carcinoids of the lung, and small cell lung cancer. To a lesser extent, the receptor was also present in large cell neuroendocrine carcinomas of the lung, medullary thyroid carcinomas, parathyroid adenomas, pheochromocytomas, and a subpopulation of pancreatic neuroendocrine neoplasms. In lung tumours, a negative correlation with the expression of the proliferation marker Ki-67 and a positive interrelationship with patient survival was observed. Overall, our results indicate that in adenocarcinomas and neuroendocrine tumours of the lung GPR19 may serve as a suitable diagnostic or therapeutic target.

Role of CD68+ and CD206+ cells in the progression of toxic liver fibrosis in rats

The aim of the work was to evaluate the role of stellate macrophages in a large number of points of toxic liver fibrosis in rats. Liver fibrosis and cirrhosis in male Wistar rats were induced with thioacetamide at a dose of 200 mg/kg animal weight for 17 weeks. Histological preparations of the liver were stained with hematoxylin and eosin according to the Mallory method. Immunohistochemical examination was performed on paraffin sections using monoclonal mouse antibodies CD68 and polyclonal rabbit antibodies CD206. The fibrosis degree was determined according to the Ishak semi-quantitative scale. Toxic liver fibrosis before the start of its transformation into cirrhosis (9 weeks) was accompanied by an increase in the number of CD68+ cells compared with the control. At all subsequent experiment stages, no differences were found in comparison to the control. In the liver of control rats, CD206+ cells were practically absent. Throughout the experiment, their number remained above the control point – 3 weeks. With the progression of liver cirrhosis, a decrease in the number of CD206+ cells was noted, but it did not reach a level of 3 weeks. Morphologically, two different groups of CD68+ cells were identified. One group of cells had a pterygoid shape and they were located mainly in the liver sinusoids. The second group of CD68+ cells had a round shape and different localization. They were detected around the vessels of portal zones, surrounded brown pigment accumulations in connective tissue septa, were observed near single lying groups or groups of giant hepatocytes and liver cells containing brown pigment in the cytoplasm, and were also noted in the foci of necrosis of hepatocytes. Cells, expressing the CD206 marker, are round in shape and are elongated and located in the liver sinusoids. Presumably, round-shaped CD68+ cells perform a phagocytic function, and pterygoid-shaped CD68+ cells transdifferentiate into CD206+ cells that have anti-inflammatory properties.

Development and diagnostic evaluation of indirect enzyme linked immunosorbent assay for Epidermophyton floccosum infection in humans

BackgroundEpidermophyton floccosum is an anthrophophilic dermatophyte widely distributed in the tropics and subtropics. It can invade keratinized tissues of humans and cause superficial mycoses called dermatophytosis (ringworm). ObjectiveThe main objective of this study was to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) and to evaluate its performance for the immunological diagnosis of E. floccosum infection in humans. MethodsAn in-house indirect ELISA was developed using partially purified E. floccosum antigens, pre immunized rabbit serum as negative control, immunized rabbit polyclonal antibodies as positive control, enzyme labeled goat anti rabbit antibodies and goat anti human antibodies. A total of 50 serum samples from E. floccosum infected patients as confirmed by direct microscopy and culture and 30 samples from humans without history of dermatophyte infection that served as controls were used to evaluate the performance of an in-house indirect ELISA developed in this study. Analytical and diagnostic performance characteristics were determined to evaluate its diagnostic value. ResultsThe diagnostic sensitivity, specificity, positive and negative predictive values of E. floccosum indirect ELISA were 90.00 %, 83.33 %, 90.83 %, 83.83 % respectively. The performance of indirect ELISA assay was compared with gold standard diagnostic tests such as KOH hydrolysis test and fungal culture. The correlation coefficients of E. floccosum indirect ELISA with KOH hydrolysis and fungal culture method were 0.612** and 0.826** (P < 0.01) respectively indicating significant correlation between these tests. ConclusionThis revealed the great potentiality of E. floccosum indirect ELISA in early, specific and precise detection of E. floccosum infection in humans.

Preclinical evaluation of a SARS-CoV-2 variant B.1.351-based candidate DNA vaccine

The SARS-CoV-2 pandemic revealed the critical shortfalls of global vaccine availability for emergent pathogens and the need for exploring additional vaccine platforms with rapid update potential in response to new variants. Thus, it remains essential, for the present evolving SARS-CoV-2/Covid-19 and future pandemics, to continuously develop and characterize new and different vaccine platforms. Here, we describe an expression-optimized DNA vaccine candidate based on the SARS-CoV-2 spike protein of the Beta variant (B.1.351), pNTC-Spike.351, and, in animal models, compare its immunogenicity with a similar DNA vaccine encoding the ancestral index strain spike protein, pNTC-Spike. Both DNA vaccines induced neutralizing antibodies and a Th1 biased immune response. In contrast to the index-specific vaccine, the Beta-specific DNA vaccine induced antibodies in mice and rabbits that, even at low levels, efficiently neutralize the otherwise antibody resistant Beta variant. It similarly neutralized unrelated variants bearing the neutralization resistant E484K spike mutation. Intensive priming using two vaccinations with pNTC-Spike and a single booster immunization with the pNTC-Spike.351 induced a more robust neutralizing antibody response with comparable magnitude against different variants of concern. Thus, DNA vaccine technology with heterologous spike protein prime-boost should be explored further using the Beta derived pNTC-Spike.351 to broaden neutralizing antibody responses against emerging variants of concern.

Biopharmaceutical properties of polyvalent bacteriophage capsules

Scientific relevance. Rational phage therapy is a highly effective way to combat bacterial infections, especially in conditions of steadily increasing antibiotic resistance. Most bacteriophage preparations are formulated as oral and topical solutions. However, oral administration of liquid phage preparations results in significant inactivation in the stomach. To shield active ingredients from the acidic environment, Sextaphag® Pyobacteriophage, polyvalent, has been formulated into capsules.Aim. This study evaluated the polyvalent bacteriophage preparation in capsules in terms of its potency and biopharmaceutical properties.Materials and methods. The study compared the polyvalent bacteriophage preparation formulated as capsules with the polyvalent bacteriophage preparation formulated as a solution. The potency was evaluated by the Appelmans method, and phage particles were quantified by the Gratia method. To evaluate the acid-neutralising capacity, the authors placed test samples of the bacteriophage preparation in 0.1 M hydrochloric acid and analysed their potency by the Appelmans method. Chinchilla rabbits were used to analyse anti-phage immune responses, and their antibody levels were measured using an enzyme immunoassay test system developed by the authors. The pharmacokinetic parameters were studied in outbred white mice after oral dosing.Results. The polyvalent bacteriophage preparation exhibited high lytic activity towards Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aegidinosa, Staphylococcus aureus, and Streptococcus pneumoniae, which accounted for a dilution factor of 10-6. Following oral administration of the polyvalent bacteriophage preparation in capsules to mice, the level of absorption was 3.1–3.7 times higher than that observed with the solution. Repeated oral administration of therapeutic doses did not induce anti-phage antibodies in rabbits. The stability study showed that the polyvalent bacteriophage preparation retained high lytic activity for 18 months.Conclusions. According to the study results, the polyvalent bacteriophage preparation in capsules exerts significant antibacterial activity against the studied microorganisms, has a high level of absorption, retains its lytic activity for a long time, and does not induce anti-phage antibodies after oral dosing, which confirms its safety and efficacy.

Low Frequency of Aflatoxin Induced TP53 Gene Codon 249 Mutation in Hepatocellular Carcinoma from Egyptian Patients Living in the Nile Delta Region.

Study the frequency of codon 7 (c.747 G>T, p. R249S) mutation associated with Aflatoxin B1 (AFB1) exposure in Egyptian patients with hepatocellular carcinoma (HCC). We utilized restriction fragment polymorphism and direct sequencing to assess codon 7 mutations in 104 hepatocellular carcinomas. The expression of TP53 protein in the tumors were assessed in 44 tumors by a monoclonal rabbit antibody. We identified a single 1/104 (1%) with c.747 G>T, p. R249S variant. 28/44 (63.6%) tumors showed no or occasional (less than < 5%) nuclear staining; 9/44 (20.4%) showed mild to moderate (5-49%) and 7/44 (15.9%) showed strong ≥ 50% staining. We observed much lower frequency of TP53 gene than previously published results suggesting geographical alterations in AFB1 exposure in Egypt.

Neutralizing antibodies against EBV gp42 show potent in vivo protection and define novel epitopes

ABSTRACT Epstein–Barr virus (EBV) is the first reported human oncogenic virus and infects more than 95% of the human population worldwide. EBV latent infection in B lymphocytes is essential for viral persistence. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into B cells. The C-type lectin domain (CTLD) of gp42 plays a key role in receptor binding and is the major target of neutralizing antibodies. Here, we isolated two rabbit antibodies, 1A7 and 6G7, targeting gp42 CTLD with potent neutralizing activity against B cell infection. Antibody 6G7 efficiently protects humanized mice from lethal EBV challenge and EBV-induced lymphoma. Neutralizing epitopes targeted by antibodies 1A7 and 6G7 are distinct and novel. Antibody 6G7 blocks gp42 binding to B cell surface and both 1A7 and 6G7 inhibit membrane fusion with B cells. Furthermore, 1A7- and 6G7-like antibodies in immunized sera are major contributors to B cell neutralization. This study demonstrates that anti-gp42 neutralizing antibodies are effective in inhibiting EBV infection and sheds light on the design of gp42-based vaccines and therapeutics.

Translational physiologically-based pharmacokinetic model for ocular disposition of monoclonal antibodies.

We have previously published a PBPK model comprising the ocular compartment to characterize the disposition of monoclonal antibodies (mAbs) in rabbits. While rabbits are commonly used preclinical species in ocular research, non-human primates (NHPs) have the most phylogenetic resemblance to humans including the presence of macula in the eyes as well as higher sequence homology. However, their use in ocular research is limited due to the strict ethical guidelines. Similarly, in humans the ocular samples cannot be collected except for the tapping of aqueous humor (AH). Therefore, we have translated this rabbit model to monkeys and human species using literature-reported datasets. Parameters describing the tissue volumes, physiological flows, and FcRn-binding were obtained from the literature, or estimated by fitting the model to the data. In the monkey model, the values for the rate of lysosomal degradation for antibodies (Kdeg), intraocular reflection coefficients (σaq, σret, σcho), bidirectional rate of fluid circulation between the vitreous chamber and the aqueous chamber (QVA), and permeability-surface area product of lens (PSlens) were estimated; and were found to be 31.5h-1, 0.7629, 0.6982, 0.9999, 1.64 × 10-5 L/h, and 4.62 × 10-7 L/h, respectively. The monkey model could capture the data in plasma, aqueous humor, vitreous humor and retina reasonably well with the predictions being within twofold of the observed values. For the human model, only the value of Kdeg was estimated to fit the model to the plasma pharmacokinetics (PK) of mAbs and was found to be 24.4h-1 (4.14%). The human model could also capture the ocular PK data reasonably well with the predictions being within two- to threefold of observed values for the plasma, aqueous and vitreous humor. Thus, the proposed framework can be used to characterize and predict the PK of mAbs in the eye of monkey and human species following systemic and intravitreal administration. The model can also facilitate the development of new antibody-based therapeutics for the treatment of ocular diseases as well as predict ocular toxicities of such molecules following systemic administration.

THE MORPHOLOGICAL AND FUNCTIONAL ASSESSMENT OF FAP+ AND α-SMA+-CELLS IN DIFFERENT TIMES OF RAT TOXIC LIVER FIBROSIS

Qualitative study of the source of the fibro-genic cell population in relation to the etiology and stage of fibrosis, as well as an understanding of the molecular mechanisms that regulate changes in the phenotype of hepatic fibroblasts, are of paramount importance in the development of pharmacological drugs. The purpose of the study was a morphological and functional assessment of activated portal fibroblasts (FAP+) and fat-accumulating cells (α-SMA+) of the liver at various stages of toxic liver fibrosis in rats. Liver fibrosis and cirrhosis in male Wistar rats were induced with thioacetamide solution for 17 weeks. Morphological examination of the liver was carried out on paraffin sections stained with hematoxylin and eosin using the Mallory method; immunohistochemical examination was carried out using polyclonal rabbit antibodies to the portal fibroblast antigen FAP and using monoclonal mouse antibodies to the α-SMA+ cell antigen. Before the onset of liver fibrosis stage F3/F4, from weeks 3 to 7, the number of FAP+ and α-SMA+ cells increased alternately. During the stages of transformation of fibrosis into cirrhosis from 7 to 11 weeks, their number increased slightly. At the stage of incomplete (F5) and before the onset of significant cirrhosis (F6) from weeks 11 to 15, the number of FAP+ and α-SMA+ cells were inconsistent and there was an alternating increase and decrease in their number. α-SMA+ cells before the start of the process of transformation of fibrosis into cirrhosis (F4/F5) were observed in sinusoids and foci of necrosis. Then they were detected both in sinusoids and in connective tissue trabeculae. FAP+ cells at the stage of portal fibrosis (F1) were localized near the interlobular vessels and interlobular bile ducts of the portal zones, and from the F2/F3 period they were detected in connective tissue trabeculae and sinusoids. In quantitative terms, α-SMA+ cells predominated at all stages of fibrosis. Based on the results obtained, it can be assumed that FAP+ cells make a major contribution to the development of the portal and initial stages of bridging fibrosis. They should be considered as one of the myofibroblast populations in thioacetamide-induced liver fibrogenesis.

Development of a specific and sensitive sandwich enzyme-linked immunosorbent assay for the quantification of dasatinib

This study sought to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of dasatinib, a tyrosine kinase inhibitor. Anti-dasatinib antibodies were obtained from mice or rabbits by using two partial structures of dasatinib as haptens: 2-amino-N-(2-chloro-6-methylphenyl)-thiazole-5-carboxamide and 2-{4-(2-hydroxyethyl)-1-piperazinyl}-isonicotinic acid. The best combination of two antibodies for sandwich ELISA of dasatinib was determined using four anti-dasatinib antibodies derived from mice and rabbits. Using two dasatinib-specific rabbit antibodies, we successfully developed an ultra-specific and highly sensitive sandwich ELISA that is hardly affected by the main metabolite of dasatinib. The sandwich ELISA showed a linear detection range from 320 pg/mL to 1000 ng/mL. Serum dasatinib concentrations lower than 320 pg/mL were reproducibly measurable using the sandwich ELISA. The ELISA was specific to dasatinib and there were no cross-reactivities with the major metabolites 4′-hydroxy dasatinib and dasatinib carboxylic acid. The developed sandwich ELISA will be a valuable tool for pharmacokinetic studies of dasatinib. Furthermore, this study revealed that rabbit antibodies can sandwich drug molecules of a smaller size than mouse antibodies in sandwich ELISA.

#4744 MODELIZATION OF THE GUT-RENAL AXIS OF IGA NEPHROPATHY BY DEVELOPING (A1KI+/+ PIGR−/−) GENOTYPE IN MICE

Abstract Background and Aims IgA nephropathy is the most frequent primary glomerulonephritis worldwide, responsible for end stage renal disease within 20 years after diagnosis. The origin of the nephritogenic IgA1 in plasma remains poorly understood, but elements accumulate to suspect a mucosal origin after abnormal immune regulation. Herein, we propose a mouse model of IgA nephropathy, based on the modification of mucosal IgA synthesis and transport which drives an excess of polymeric forms of IgA1 in plasma. This model is based on (i) the presence of an IgA1 repertoire produced by mucosal plasma cells (Α1KI+/+ mice) and (ii) the absence of mucosal transcytosis of IgA allowed by the knockout of PIGR (PIGR−/−). This study aimed at describing the phenotype of such a mouse model which is supposed to circulate increased polymeric IgA1 derouted from a mucosal origin, which is a key feature of the human IgAN. The present study is aimed at describing the phenotype of this model (Α1KI+/+ PIGR−/−) through a mucosa-plasma-kidney axis, in comparison to wild type controls (WT), and Α1KI+/+ PIGR+/+. Method Tissue fragments were used for immunofluorescence. Antibodies anti-IgA humain (Igah) FITC, antibodies anti-IgA mice (IgAm) HRP, antibodies goat anti-IgG alexa fluor and antibodies goat anti mice C3 FITC are used. To determine the concentration of murine IgA in the mice studied, ELISA tests were performed with anti- IgAm antibodies not conjugated or conjugated with HRP. The determination of IgAh in mice, anti-IgAh rabbit antibodies and antibody IgAh AP were used. Electrophoresis was performed in non-reducing conditions on polyacrylamide gel, transferred to a blot and revealed using anti-mouse IgA or anti- human IgA. Results At mucosal level, the observation of ileal slices of (Α1KI+/+ PIGR−/−) mice, revealed that both IgA1h and IgAm were strongly localized in the subepithelial chorion of villi and lamina propria, much more than in WT and the Α1KI+/+ PIGR−/− conditions (figure 1). In serum, human IgA concentration was higher in sera issued from (Α1KI+/+ PIGR−/−) mice than serum from A1KI+/+PIGR+/+ and WT mice (figure 2A). The difference was even more striking for murine IgA with a 2log10 gap (figure 2B). The western blot revealed a marked accumulation of circulating polymeric forms of human IgA1 and murine IgA, mostly dimeric and trimeric (figure 2C). In kidney, immunofluorescence shows the presence of human IgA1, C3 and IgG deposit in renal glomeruli in (Α1KI+/+ PIGR−/−) mice (figure 3), with less IgA and C3 in A1KI+/+PIGR+/+ and no deposit in glomeruli of WT mice. It is noticeable that IgG can codeposit with human IgG in both Α1KI+/+ conditions. Conclusion This double genetically altered mouse model of IgA nephropathy (Α1KI+/+ PIGR−/−) display the mucosal-kidney axis of the human disease, by derouting polymeric IgA1 from a mucosal origin. Those IgA1 accumulate at a subepithelial stage, increase in serum with marked proportion of polymeric forms and deposit in kidney as pIgA1-IgG-C3 immune complexes. So this new murine model of IgA nephropathy could be helpful to investigate some potential therapeutic targets and increases the conviction of the role of mucosa to drive the pathogeny of the disease.

Cross-Reactivity of HBe Antigen-Specific Polyclonal Antibody with HBc Antigen.

Hepatitis B virus (HBV) infection is a major health problem worldwide and causes almost one million deaths annually. The HBV core gene codes for two related antigens, known as core antigen (HBcAg) and e-antigen (HBeAg), sharing 149 residues but having different amino- and carboxy-terminals. HBeAg is a soluble variant of HBcAg and a clinical marker for determining the disease severity and patients' screening. Currently available HBeAg assays have a shortcoming of showing cross-reactivity with HBcAg. In this study, for the first time, we evaluated whether HBcAg-adsorbed anti-HBe polyclonal antibodies could specifically recognize HBeAg or still show cross-reactivity with HBcAg. Recombinant HBeAg was cloned in pCold1 vector and successfully expressed in Escherichia coli and after purification by Ni-NTA resin was used to generate polyclonal anti-HBe antibodies in rabbit. Purified HBeAg was further characterized by assessing its reactivity with anti-HBe in the sera of chronically infected patients and HBeAg-immunized rabbit. Sera from patients with chronic HBV infection, containing anti-HBe, specifically reacted with recombinant HBeAg, implying antigenic similarity between the prokaryotic and native HBeAg in the serum of HBV-infected patients. In addition, the designed enzyme-linked immunosorbent assay (ELISA) with rabbit anti-HBe polyclonal antibodies could detect recombinant HBeAg with high sensitivity, while high cross-reactivity with HBcAg was observed. It is noteworthy that HBcAg-adsorbed anti-HBe polyclonal antibodies still showed high cross-reactivity with HBcAg, implying that due to the presence of highly similar epitopes in both antigens, HBcAg-adsorbed polyclonal antibodies cannot differentiate between the two antigens.

Preferential rabbit antibody responses to C-termini of NOTCH3 peptide immunogens

Antibodies raised in peptide-immunized rabbits have been used in biological research for decades. Although there has been wide implementation of this approach, specific proteins are occasionally difficult to target for multiple reasons. One consideration that was noted in mice is that humoral responses may preferentially target the carboxyl terminus of the peptide sequence which is not present in the intact protein. To shed light on the frequency of preferential rabbit antibody responses to C-termini of peptide immunogens, we present our experience with generation of rabbit antibodies to human NOTCH3. A total of 23 antibodies were raised against 10 peptide sequences of human NOTCH3. Over 70% (16 of 23) of these polyclonal antibodies were determined to be C-terminal preferring: NOTCH3 peptide-reactive antibodies largely targeted the terminating free carboxyl group of the immunizing peptide. The antibodies that preferred C-terminal epitopes reacted weakly or not at all with recombinant target sequences with extension the C-terminus that eliminated the free carboxyl group of the immunogen structure; furthermore, each of these antisera revealed no antibody reactivity to proteins truncated before the C-terminus of the immunogen. In immunocytochemical applications of these anti-peptide antibodies, we similarly found reactivity to recombinant targets that best binding to cells expressing the free C-terminus of the immunizing sequence. In aggregate, our experience demonstrates a strong propensity for rabbits to mount antibody responses to C-terminal epitopes of NOTCH3-derived peptides which is predicted to limit their use against the native protein. We discuss some potential approaches to overcome this bias that could improve the efficiency of generation of antibodies in this commonly utilized experimental paradigm.

Efficacy and muscle safety assessment of fukutin-related protein gene therapy

Limb-girdle muscular dystrophy type R9 (LGMDR9) is a muscle-wasting disease that begins in the hip and shoulder regions of the body. This disease is caused by mutations in fukutin-related protein (FKRP), a glycosyltransferase critical for maintaining muscle cell integrity. Here we investigated potential gene therapies for LGMDR9 containing an FKRP expression construct with untranslated region (UTR) modifications. Initial studies treated an aged dystrophic mouse model (FKRPP448L) with adeno-associated virus vector serotype 6 (AAV6). Grip strength improved in a dose- and time-dependent manner, injected mice exhibited fewer central nuclei and serum creatine kinase levels were 3- and 5-fold lower compared to those in non-injected FKRPP448L mice. Treatment also partially stabilized the respiratory pattern during exercise and improved treadmill running, partially protecting muscle from exercise-induced damage. Western blotting of C2C12 myotubes using a novel rabbit antibody confirmed heightened translation with the UTR modifications. We further explored the question of FKRP toxicity in wild-type mice using high doses of two additional muscle-tropic capsids: AAV9 and AAVMYO1. No toxic effects were detected with either therapeutic agent. These data further support the feasibility of gene therapy to treat LGMDR9.

Poly(allylamine) coated layer-by-layer assembly decorated 2D carbon backbone for highly sensitive and selective detection of Tau-441 using surface plasmon resonance biosensor

The determination of clinically significant amounts of tau protein in bodily fluids is a major problem in Alzheimer's disease (AD) diagnosis. As a result, the present work aims to develop a simple, label-free, fast, highly sensitive, and selective 2D carbon backbone graphene oxide (GO) patterned surface plasmon resonance (SPR) mediated affinity biosensor for Tau-441 monitoring. Initially, non-plasmonic nanosized GO was made using a modified Hummers' method, whereas green synthesized gold nanoparticles (AuNPs) were subjected to a layer-by-layer (LbL) design employing anionic and cationic polyelectrolytes. Several spectroscopical evaluations were carried out to ensure the synthesis of GO, AuNPs, and LbL assembly. Following that, the Anti-Tau rabbit antibody was immobilized on the designed LbL assembly using carbodiimide chemistry, and various studies such as sensitivity, selectivity, stability, repeatability, spiked sample analysis, etc., were conducted using the constructed affinity GO@LbL-AuNPs-Anti-Tau SPR biosensor. As an output, it shows a broad concentration range and a very low detection limit of 150 ng/mL to 5 fg/mL and 13.25 fg/mL, respectively. The remarkable sensitivity of this SPR biosensor represents the merits of a combination of plasmonic AuNPs and a non-plasmonic GO. It also exhibits great selectivity for Tau-441 in the presence of interfering molecules, which may be because of the immobilization of the Anti-Tau rabbit on the surface of the LbL assembly. Furthermore, it ensured high stability and repeatability, while spiked sample analysis and AD-induced animal samples analysis confirmed the practicability of GO@LbL-AuNPs-Anti-Tau SPR biosensor for Tau-441 detection. In conclusion, fabricated sensitive, selective, stable, label-free, quick, simple, and minimally invasive GO@LbL-AuNPs-Anti-Tau SPR biosensor will provide an alternative for AD diagnosis in the future.

A mammalian cell display platform based on scFab transposition.

In vitro display technologies have been successfully utilized for the discovery and evolution of monoclonal antibodies (mAbs) for diagnostic and therapeutic applications, with phage display and yeast display being the most commonly used platforms due to their simplicity and high efficiency. As their prokaryotic or lower eukaryotic host organisms typically have no or different post-translational modifications, several mammalian cell-based display and screening technologies for isolation and optimization of mAbs have emerged and are being developed. We report here a novel and useful mammalian cell display platform based on the PiggyBac transposon system to display mAbs in a single-chain Fab (scFab) format on the surface of HEK293F cells. Immune rabbit antibody libraries encompassing ~7×107 independent clones were generated in an all-in-one transposon vector, stably delivered into HEK293F cells and displayed as an scFab with rabbit variable and human constant domains. After one round of magnetic activated cell sorting and two rounds of fluorescence activated cell sorting, mAbs with high affinity in the subnanomolar range and cross-reactivity to the corresponding human and mouse antigens were identified, demonstrating the power of this platform for antibody discovery. We developed a highly efficient mammalian cell display platform based on the PiggyBac transposon system for antibody discovery, which could be further utilized for humanization as well as affinity and specificity maturation.

Detection of bacteria causing mastitis in cows with immunoelectric tool.

This study has investigated designing, producing, and evaluating a newly developed immunoelectric device for the identification of the Staphylococcus aureus and Escherichia coli in experimentally or clinically polluted milk samples. The design of the immunoelectric tool parts was carried out and produced by using a 3D printer. The animals were immunized against S. aureus and E. coli; purification of the specific IgG from hyperimmune serum was carried out by ion exchange and affinity chromatography methods. Coupling of the specific rabbit antibodies to the polystyrene filters was performed by DTPA linker. The purified rat antibodies were coupled to Fe nanoparticles and used as detector elements. The experimentally polluted milk and PBS samples were prepared. After optimization, the minimum traceable number of the bacteria was determined using immunoelectric tool. Also, the cow's milk samples with clinical mastitis were tested for bacterial detection by bacterial culture and immunoelectric methods. Coupling of the antibody to the filters and capturing of the target bacteria to the filters were successfully confirmed using enzyme immune assay and electron microscopy. The detection limit of the developed immunoelectric tool was equal to 15 cells/mL in PBS or milk samples for 30 min. Also, in agreement with bacterial cell culture, the clinically infected milk samples by S. aureus and E. coli were successfully detected using the immunoelectric device. The developed immunoelectric device could be utilized for rapid and cost-benefit detection of the bacterial organism.