Rabbit Serum Research Articles

Examination of the cross-reactivity between vaccinia virus Tiantan strain and monkeypox virus

AimTo investigate the cross-reactivity between the sera collected from Vaccinia Virus Tiantan Strain vaccinated rabbits and viral antigens of monkeypox virus. MethodsVaccinia viruses were prepared on chicken embryo fibroblasts (CEF) and Vero cells respectively named as CEF-VTT NVSI-1 and Vero-VTT NVSI-1. Rabbits were inoculated with a total of three doses of adjuvanted 1.3 × 108 PFU CEF-VTT NVSI-1 each dose or adjuvanted 3.9 × 107 PFU Vero-VTT NVSI-1 (Freunds complete adjuvant) via the subcutaneous route. We then performed the enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI) for determination of the binding activity and affinity of immune sera to five crucial surface antigens on monkeypox virus including A35, B6R, H3 and to corresponding homologous antigens A33R, B5 and L1R of vaccinia virus. For comparison, plaque reduction neutralizing tests were used to evaluate the neutralization of immune sera against vaccinia virus. ResultsBoth CEF-VTT NVSI-1 and Vero-VTT NVSI-1 vaccinations following planned schedule could induce neutralizing antibody titers greater than 1:2048 in rabbit sera. Binding antibodies targeting monkeypox viral antigens were confirmed by both indirect ELISA and BLI methods. Indirect ELISA for rabbit sera revealed 1:51200 binding antibody titers to A35/B6R/H3 monkeypox virus antigens while BLI tests yielded affinities at 2 × 10−6 to 8 × 10−7 between the sera and the three antigens. Similarly, such sera showed binding strength to vaccinia virus antigens A33R/B5/L1R consistent with that to three preceding monkeypox virus antigens. These results demonstrated the cross-reactivity between the sera of vaccinia virus vaccinated animals and monkeypox virus antigens. Traditional ELISA test and BLI method displayed a high consistency in antigen screening and they were further proved to correlate to the results of plaque reduction neutralizing test, which indicates that BLI could be utilized as an indirect alternative for assessment of neutralizing activity of samples in response to live virus. ConclusionsSera of vaccinia virus-vaccinated rabbits exhibited cross-reactivity with viral antigens of monkeypox virus. Potential in improving the accuracy of antigen discovery while reducing the lengthy work needed for the screening as BLI method possesses, it contributes greatly to the rapid preliminary evaluation of immune response generated by vaccines.

Construction of Antibody Library and Production of Polyclonal Antibodies Specific to Human Protein C

Background: Thromboembolism can occur at any age, from infancy to adulthood. The genetic risk factors for thromboembolism in the Thai population are protein C and protein S mutations. The diagnosis depends on the activity level of proteins in the plasma. Management of acute thrombotic events in severe protein C deficiency typically requires replacement with protein C in the form of fresh frozen plasma or protein C concentrate. At present, the measurement of protein C and protein S activities level is limited due to the cost and availability of the tests. Therefore, it can take a lengthy time to determine protein C levels, resulting in a delay in treatment, especially in severe cases. Objective: To create an antibody library on filamentous bacteriophage for generating antigen specific monoclonal antibodies and to produce polyclonal antibodies specific to protein C. Results: Recombinant protein C was constructed and produced from human embryonic kidney 293 cells. Protein C was used as an antigen to immunize mice and rabbits. The antibody titers after final boosting immunization reached up to 50,000 in mice and 10,000 in rabbits. The phage antibody library with a capacity of approximately 3×10⁵ CFU was obtained from mice lymphocytes. Polyclonal antibodies were purified from rabbit sera using affinity chromatography and achieved a yield of 2 mg/mL rabbit sera. The purified polyclonal antibodies were able to detect protein C by immunoblotting and ELISA. Conclusion: The present study demonstrated that polyclonal antibodies could be produced from immunized rabbits and the antibody library obtained from immunized mice is beneficial for further isolation of monoclonal antibodies against protein C. Keywords: Protein C; Recombinant protein; Antibody library; Monoclonal antibody; Polyclonal antibody

Fabrication of metal–organic framework based electrochemical Leishmania immunosensor

Leishmaniasis is a disease with a high impact on public health in many countries. In this study, an electrochemical immunosensor for Leishmania major surface protease (Gp63) antibody detection was developed using copper metal organic framework (Cu-(NH2-BDC)-MOF) modified gold screen printed electrode (AuSPE). For this purpose, after modification step of AuSPE with Cu-(NH2-BDC)-MOF, recombinant L.donovani antigen was immobilized on to electrode surface via crosslinker where change in electrochemical signal was monitored with electrochemical impedance spectroscopy technique. After conducting comprehensive optimization studies on experimental parameters of developed Leishmania immunosensor, analytical characteristics were searched. As a result, it was observed that anti-gp63 solution diluted up to the ratio of 1:1500 could be detected with the developed sensor. Meanwhile, relative standard deviation value was found as 4.67% for anti-gp63 diluted in the ratio of 1:80 (n = 3). Accordingly, real sample analysis was also performed by using Leishmania parasite crude antigen and rabbit serum which were positive and negative control samples, respectively. According to the results obtained, it can be concluded that a practical immunosensor that can allow the early diagnosis of leishmaniasis was developed.

RP-UFLC based Bioanalytical Method Development, Optimization, and Validation for the Estimation of Isradipine in Rabbit Serum

Introduction: The objective of this study is to provide a rapid, sensitive, consistent, and costeffective method for quantifying isradipine using ultra-fast liquid chromatography. Methods: Quality by Design principles will form the basis of this approach, grounded on response surface analysis. Shimadzu liquid chromatographic system equipped with a photodiode array detector and LC solution software was used to conduct the RP-UFLC method development and validation. An ODS C18 (250 x 4.6 mm; 5 μm) UFLC column was used to complete the analysis. The RSM methodology utilized a central composite design to perform the optimization studies. Results: The mobile phase ratio and flow rate were considered crucial method parameters, as well as the peak area, retention time, and USP plate were considered critical analytical attributes. The optimal conditions for chromatographic separation were followed using 80% acetonitrile and water (20% v/v) as mobile phase, a 1 mL/min flow rate, an injection volume of 20 μL, 40°C of column oven temperature, and maximum absorption at λmax 254 nm using graphical optimization technique. When examining concentrations between 5 and 150 ng/mL, linearity was observed with an R2 of 0.999. The method created was validated by employing stability testing per the recommendations provided by ICH Q2 (R1). The analysis of blood serum was modified so that it could be used to examine the pharmacokinetic parameters. Conclusion: As a result, high accuracy, precision, sensitivity, linearity, and robustness were established for predicting the amount of isradipine present in its freeze-dried nano-formulations.

Coronavirus spike protein-specific antibodies indicate frequent infections and reinfections in infancy and among BNT162b2-vaccinated healthcare workers

The prevalence of seasonal human coronavirus (HCoV) infections in early childhood and adults has not been well analyzed in longitudinal serological studies. Here we analyzed the changes in HCoV (229E, HKU1, NL63, OC43, MERS, and SARS-CoV-2) spike-specific antibody levels in follow-up serum specimens of 140 children at the age of 1, 2, and 3 years, and of 113 healthcare workers vaccinated for Covid-19 with BNT162b2-vaccine. IgG antibody levels against six recombinant HCoV spike subunit 1 (S1) proteins were measured by enzyme immunoassay. We show that by the age of three years the cumulative seropositivity for seasonal HCoVs increased to 38–81% depending on virus type. BNT162b2 vaccinations increased anti-SARS-CoV-2 S1 antibodies, but no increase in seasonal coronavirus antibodies associated with vaccinations. In healthcare workers (HCWs), during a 1-year follow-up, diagnostic antibody rises were seen in 5, 4 and 14% of the cases against 229E, NL63 and OC43 viruses, respectively, correlating well with the circulating HCoVs. In 6% of the HCWs, a diagnostic antibody rise was seen against S1 of HKU1, however, these rises coincided with anti-OC43 S1 antibody rises. Rabbit and guinea pig immune sera against HCoV S1 proteins indicated immunological cross-reactivity within alpha-CoV (229E and NL63) and beta-CoV (HKU1 and OC43) genera.

Porcine circovirus type 3: immunohistochemical detection in lesions of naturally affected piglets.

This study aimed to evaluate the relationship between porcine circovirus type 3 (PCV3) viral load and histopathological findings in perinatal piglet tissues and to develop an immunohistochemical method for detecting the virus in lesions. The quantitative polymerase chain reaction (qPCR) cycle threshold (Ct) when amplifying PCV3 DNA and the area of perivascular inflammatory infiltrates in different organs [central nervous system (CNS), lung, heart, liver, spleen, and lymph nodes] were compared. To develop an immunohistochemistry technique, rabbit sera were produced against PCV3-capsid protein peptides selected using bioinformatic analyses. The assay was initially implemented using a tissue sample previously tested using qPCR and in situ hybridization to optimize the procedure and reagent dilutions. To evaluate immunohistochemistry performance, tissue samples from another 17 cases were analyzed using standardized parameters. The most common microscopic lesion was multisystemic periarteritis, with associated vasculitis, as the mesenteric vascular plexus is one of the most affected organs. Other tissues, such as the heart, lung, CNS, and skeletal muscle, were also affected. Comparison of the Ct values for different tissues showed no significant difference, except in lymphoid organs (spleen and lymph nodes), which had significantly higher viral loads than the CNS tissues. There was no correlation between Ct values and perivascular inflammatory infiltrates. PCV3 immunohistochemistry revealed granular immunolabeling, mainly in the cytoplasm of cells in the vascular mesenteric plexus, heart, lung, kidney, and spleen.

Effectiveness of Polyclonal Antibody Immunoconjugate Treatment with Propamidine Isethionate for Amoebic Keratitis in Golden Hamsters.

Acanthamoeba griffini is known to cause amoebic keratitis (AK); its main causes are inadequate hygiene when contact lenses are handled and/or its prolonged use at night, as well as the use of contact lenses during underwater activities. The most used treatment for AK is the combination of propamidine isethionate combined with polyhexamethylene biguanide, which disrupts the cytoplasmic membrane, and damages cellular components and respiratory enzymes. We proposed an immunoconjugate treatment obtained from Acanthamoeba immunized rabbit serum combined with propamidine isethionate; the corneas of hamsters inoculated with A. griffini (MYP2004) were treated with the combined, at 1, 2, and 3 weeks. Propamidine isethionate is frequently used for AK treatment, in vivo study we are found IL-1β and IL-10 expression and caspase 3 activity is significantly increased with respect to the group that was inoculated with the amoeba without receiving any treatment, suggesting that it may be an effect of the toxicity of this drug on the corneal tissue. Application of the immunoconjugate showed enhanced amoebicidal and anti-inflammatory activities, with comparison to propamidine isethionate only. The aim of this study is to evaluate the effect of the immunoconjugate of propamidine isethionate and polyclonal antibodies as a treatment of AK in golden hamsters (Mesocricetus auratus).

Rapid purification of rabbit immunoglobulins using a single-step, negative-selection chromatography

Custom polyclonal antibodies raised in rabbits are routinely used in immunoblotting and other protein analysis techniques. Custom rabbit polyclonal antisera are generally purified using immunoaffinity or Protein A-affinity chromatography; however, these methods require harsh elution conditions that can compromise the antigen binding efficacy. We evaluated the utility of Melon™ Gel chromatography for purification of IgG from crude rabbit serum. We show that Melon Gel-purified rabbit IgGs are active and perform well in immunoblotting. In summary, the Melon Gel method is a rapid, one-step, negative-selection approach that can be employed in either preparative or small-scale format to purify IgG from crude rabbit serum without the need for denaturing eluent.

Development of sandwich ELISA and lateral flow assay for the detection of Bungarus multicinctus venom

Snakebite envenoming adversely affects human health and life worldwide. Presently, no suitable diagnostic tools for snakebite envenoming are available in China. Therefore, we sought to develop reliable diagnostic tests for snakebite management. We conducted affinity purification experiments to prepare species-specific antivenom antibody (SSAb). In brief, affinity chromatography with an antibody purification column (Protein A) was conducted to purify immunoglobulin G from Bungarus multicinctus (BM) venom hyperimmunized rabbit serum. The cross-reactive antibodies were removed from commercial BM antivenin by immune adsorption on the affinity chromatography columns of the other three venoms, Bungarus Fasciatus (FS), Naja atra (NA), and O. hannah (OH), generating SSAb. The results of western blot analysis and enzyme-linked immunosorbent assay (ELISA) showed the high specificity of the prepared SSAb. The obtained antibodies were then applied to ELISA and lateral flow assay (LFA) to detect BM venom. The resulting ELISA and LFA could specifically and rapidly detect BM venom in various samples with the limits of quantification as 0.1 and 1 ng/ml, respectively. This method could effectively detect snake venom in experimentally envenomed rats (simulating human envenomation), which could distinguish positive and negative samples within 10-15 min. This method also showed promise in serving as a highly useful tool for a rapid clinical distinguishing of BM bites and rational use of antivenom in emergency centers. The study also revealed cross-reactivity between BM and heterogenous venoms, suggesting that they shared common epitopes, which is of great significance for developing detection methods for venoms of the snakes belonging to the same family.

Encephalitozoon cuniculi Infection of Domestic Rabbits (Oryctolagus cuniculus) in Slovenia between 2017 and 2021.

Encephalitozoon cuniculi is a microsporidial parasite that primarily infects domestic rabbits (Oryctolagus cuniculus). It is the causative agent of encephalitozoonosis, a disease with an internationally recognized seroprevalence among rabbits. This study determines the presence, clinical manifestation, and serological status of encephalitozoonosis in pet rabbits in Slovenia using various diagnostic procedures. From 2017 to 2021, 224 pet rabbit sera were collected and tested for encephalitozoonosis with the indirect immunofluorescence assay. Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against E. cuniculi were confirmed in 160 (65.6%) cases. Most seropositive rabbits suffered from neurological clinical signs or signs of gastrointestinal disorders such as recurrent hypomotilities, chronic weight loss, cachexia, or anorexia, and fewer showed clinical signs related to the urinary system or phacoclastic uveitis. A quarter of the positively tested rabbits presented without clinical signs. Hematological and biochemical blood analysis confirmed that seropositive animals had elevated globulin and deviated albumin levels in comparison to the normal reference values of non-infected animals. Furthermore, rabbits with neurological clinical signs showed statistically significant higher levels of globulins and total protein. Sixty-eight whole-body radiographs and thirty-two abdominal ultrasound reports were analyzed, looking for changes in the shape or size of the urinary bladder, presence of urinary sludge or uroliths, and any abnormalities related to the kidneys (shape, size, or nephrolites). The results suggest that neurological defects in the urinary bladder caused by E. cuniculi lead to a distended urinary bladder and consequently dysuria, incontinence, urine scalding, and sludgy urine.

Calcium-binding motif-mediated binding of redundant calcium offers a chimeolysin enhanced bactericidal activity and extended host range under physiological conditions.

Calcium-binding motifs are shared by multiple bacteriophage lysins; however, the influence of calcium on the enzymatic activity and host range of these enzymes is still not understood. To address this, ClyF, a chimeric lysin with a putative calcium-binding motif, was used as a model for in vitro and in vivo investigations. The concentration of calcium bound to ClyF was determined by atomic absorption spectrometry. The influence of calcium on the structure, activity and host range of ClyF was assessed by circular dichroism and time-kill assays. The bactericidal activity of ClyF was evaluated in various sera and a mouse model of Streptococcus agalactiae bacteraemia. ClyF has a highly negatively charged surface around the calcium-binding motif that can bind extra calcium, thereby increasing the avidity of ClyF for the negatively charged bacterial cell wall. In line with this, ClyF exhibited significantly enhanced staphylolytic and streptolytic activity in various sera containing physiological calcium, including human serum, heat-inactivated human serum, mouse serum and rabbit serum. In a mouse model of S. agalactiae bacteraemia, intraperitoneal administration of a single dose of 25 μg/mouse ClyF fully protected the mice from lethal infection. The present data collectively showed that physiological calcium improves the bactericidal activity and host range of ClyF, making it a promising candidate for the treatment of infections caused by multiple staphylococci and streptococci.

Using dot-immunoassay in decoding the outbreak of pseudotuberculosis in the Tomsk region

Background. Pseudotuberculosis remains a serious healthcare problem, which determines the expediency of developing the express methods for its early diagnosis. To detect the pathogen, we designed test system for dot-immunoassay (DIA) based on antibodies labeled with silver nanoparticles (SNPs) isolated from hyperimmune rabbit serum obtained against killed cells of Yersinia pseudotuberculosis of O:1b serovariant.The aim. To assess the possibility of using dot-immunoassay for express identification of Y. pseudotuberculosis cultures isolated from clinical material and environmental objects at the initial stage of bacteriological study during laboratory diagnosis of the disease.Methods. We used the materials from the outbreak of pseudotuberculosis in the Krylovskaya Boarding School of the Bakcharsky district of the Tomsk region in 2021. Specific antibodies from hyperimmune rabbit sera obtained against Y. pseudotuberculosis 3704 particulate antigen of O:1b serotype were labeled with SNPs and used in DIA on nitrocellulose membranes with visualization of reaction results with a solution of a physical developer. The presence of the causative agent of pseudotuberculosis in the test material was inferred by the formation of gray spots of different intensity (from 4+ to 1+).Results. All Y. pseudotuberculosis strains isolated using bacteriological method on the second day of the study from clinical material obtained from sick people and environmental objects were detected in DIA at concentrations ≥ 3.1 × 104 microbial cells per milliliter (m.c./ml).Conclusion. The designed test system for dot-immunoassay using SNPs as a marker of specific antibodies for the detection of Y. pseudotuberculosis in cultures isolated from swabs from vegetables and clinical material from patients, including those with mixed infection, allows us to detect a specific corpuscular antigen with a high sensitivity (≥ 3.1 × 104 m.c./ml), providing express identification of isolated cultures at the initial stage of bacteriological study.

Enhanced in vivo anti-tumor efficacy of whole tumor lysate in combination with whole tumor cell-specific polyclonal antibody.

Despite the widespread utilization of cancer vaccines with specified antigens, the use of whole tumor cell lysates in tumor immunotherapy would be a very promising approach that can overcome several significant obstacles in vaccine production. Whole tumor cells provide a broad source of tumor-associated antigens and can activate cytotoxic T lymphocytes and CD4+ T helper cells concurrently. On the other hand, as an effective immunotherapy strategy, recent investigations have shown that the multi-targeting of tumor cells with polyclonal antibodies, which are also more effective than monoclonal antibodies at mediating effector functions for target elimination, might minimize the escape variants. We prepared polyclonal antibodies by immunizing rabbits with the highly invasive 4T1 breast cancer cell line. In vitro investigation indicated that the immunized rabbit serum inhibited cell proliferation and induced apoptosis in target tumor cells. Moreover, in vivo analysis showed enhanced anti-tumor efficacy of whole tumor cell lysate in combination with tumor cell-immunized serum. This combination therapy proved beneficial in significant inhibition of the tumor growth and the established tumor was entirely eradicated in treated mice. Serial intravenous injections of tumor cell immunized rabbit serum significantly inhibited tumor cell proliferation and induced apoptosis in vitro and in vivo in combination with whole tumor lysate. This platform could be a promising method for developing clinical-grade vaccines and open up the possibility of addressing the effectiveness and safety of cancer vaccines.

Highly dispersed Cu and Ni nano cluster sensor for ultrasensitive electrochemical detection of antiviral drug lamivudine.

The accurate and rapid detection for the nucleoside reverse transcriptase inhibitor lamivudine (LAM, 3TC) in cellular systems is always a challenge in the clinic application. Here, a sensitive Cu and Ni nano cluster sensor for LAM is generated underhydrothermal conditions.TheCu and Ni atoms are highly dispersed and aggregated in the nanosized opening pore windows of the synthesized LTA zeolite, through the diatomic synergistic contribution of Cu and Ni and the enrichment of zeolitic channel pores. Using differential pulse voltammetry (DPV), the detection limit (LOD) of LAM at the potential (- 0.15V) can reach 0.001pM and the linear range is 0.002pM-0.002μM. Since the nano cluster is separated and restricted by the nanosized windows of the zeolite framework, the sensor provideshigh stability, good recovery (92.5-109%) and RSD (0.8-3.2%) in the analysis oftap water, RPMI 1640 medium, and rabbit serum. The Cu/Ni/LTA zeolite-modified glassy carbon electrode (Cu/Ni/LTA/GCE) exhibits excellent catalytic performance for LAM with high selectivity over potentiallyinterfering agents. A sensitive Cu and Ni nano cluster sensor for LAM is generated in the hydrothermal condition that the Cu and Ni atoms are highly dispersed and aggregated in the nanosized opening pore windows of the as-synthesized LTA zeolite. Through the diatomic synergistic contribution of Cu and Ni and the enrichment of zeolitic channel pores, the observed limit of detection (LOD) can reach 0.001 pM under differential pulse voltammetry (DPV) method with a wide linear relationship to 0.002 μM.

Experimental and Acomparative Study for the effect of GlucosAmine hydrochloride as Antioxidant in Sera of Adult Male Rabbits

This study involved giving the administration of Glucosamine hydrochloride to (Adult Male Rabbits) through the feeding tube .Was used in this experiment (21) Rabbit were distributed randomly into three groups . Group is control to each containing
 This study involved giving drug glucoseamine hydrochloride Secretary of the animals used in the experiment (rabbits adult males ) through the feeding tube as used in this experiment 21 animals were distributed randomly into 3 groups of 7 rabbits per group . Treated the first two sets glucoseamine hydrochloride doses of 70 and 9 mg / dl / day group first and second , respectively, for a period of 30 days, while the third group used Xatrh treated with saline Physiological was then assess the property on each of the antioxidants and the image of the fat and some liver enzymes in the blood , as well as liver tissue .
 The results showed that the significantly increased of the GPX morally P ≤ 0.05)) in the group G1 compared with C, either GSH significantly increased when the G2 compared to control , also showed MDA increased significantly for both groups compared with C, while showed peroxy nitrit have decreased significantly, with no significantly change in uric acid.
 The level of TG was significantly lower with C , while the cholesterol the same results when the G1 compared with C, either LDL-C and sessile density has increased significantly compared with C, HDL-C levels significantly elevated sera of G1 only, but in the G1 also , decreased the level of VLDL compared with C .
 The liver enzymes AST enzyme has shown a significant increase compared with C.
 with no significantly change in both ALT, ALP, total protein, albumin and Globulin any significant effect compared with the C group, also showed the results of the study tela affected liver tissue of the two groups , as results showed infiltration of the cells of the lymph cells Kuppffer cell around the central vein congested G1, with necrosis in a vein in the G2.

In vitro culture of the zoonotic nematode Anisakis pegreffii (Nematoda, Anisakidae)

BackgroundAnisakiasis is a foodborne disease caused by the third-stage larvae (L3) of two species belonging to the genus Anisakis: Anisakis pegreffii and Anisakis simplex sensu stricto. Both species have been the subject of different -omics studies undertaken in the past decade, but a reliable in vitro culture protocol that would enable a more versatile approach to functional studies has never been devised. In nature, A. pegreffii shows a polyxenous life-cycle. It reproduces in toothed whales (final host) and disseminates embryonated eggs via cetacean faeces in the water column. In the environment, a first- (L1) and second-stage larva (L2) develops inside the egg, and subsequently hatched L2 is ingested by a planktonic crustacean or small fish (intermediate host). In the crustacean pseudocoelom, the larva moults to the third stage (L3) and grows until the host is eaten by a fish or cephalopod (paratenic host). Infective L3 migrates into the visceral cavity of its paratenic host and remains in the state of paratenesis until a final host preys on the former. Once in the final host’s gastric chambers, L3 attaches to mucosa, moults in the fourth stage (L4) and closes its life-cycle by becoming reproductively mature.MethodsTesting two commercially available media (RPMI 1640, Schneider’s Drosophila) in combination with each of the six different heat-inactivated sera, namely foetal bovine, rabbit, chicken, donkey, porcine and human serum, we have obtained the first reliable, fast and simple in vitro cultivation protocol for A. pegreffii.ResultsSchneider’s Drosophila insect media supplemented with 10% chicken serum allowed high reproducibility and survival of adult A. pegreffii. The maturity was reached already at the beginning of the third week in culture. From collected eggs, hatched L2 were maintained in culture for 2 weeks. The protocol also enabled the description of undocumented morphological and ultrastructural features of the parasite developmental stages.ConclusionsClosing of the A. pegreffii life-cycle from L3 to reproducing adults is an important step from many research perspectives (e.g., vaccine and drug–target research, transgenesis, pathogenesis), but further effort is necessary to optimise the efficient moulting of L2 to infective L3.Graphical

Resolving Molecular Heterogeneity with Single-Molecule Centrifugation.

For many classes of biomolecules, population-level heterogeneity is an essential aspect of biological function─from antibodies produced by the immune system to post-translationally modified proteins that regulate cellular processes. However, heterogeneity is difficult to fully characterize for multiple reasons: (i) single-molecule approaches are needed to avoid information lost by ensemble-level averaging, (ii) sufficient statistics must be gathered on both a per-molecule and per-population level, and (iii) a suitable analysis framework is required to make sense of a potentially limited number of intrinsically noisy measurements. Here, we introduce an approach that overcomes these difficulties by combining three techniques: a DNA nanoswitch construct to repeatedly interrogate the same molecule, a benchtop centrifuge force microscope (CFM) to obtain thousands of statistics in a highly parallel manner, and a Bayesian nonparametric (BNP) inference method to resolve separate subpopulations with distinct kinetics. We apply this approach to characterize commercially available antibodies and find that polyclonal antibody from rabbit serum is well-modeled by a mixture of three subpopulations. Our results show how combining a spatially and temporally multiplexed nanoswitch-CFM assay with BNP analysis can help resolve complex biomolecular interactions in heterogeneous samples.

Stereoselective Synthesis and Immunological Evaluation of Common O‐antigen of P. mirabilisOE and P. vulgarisTG103

Comprehensive SummaryProteus species especially Proteus mirabilis and Proteus vulgaris are zoonotic pathogens which can cause public health disease. Owing to their antibiotic‐resistance, developing vaccines against these pathogens is urgently required. Herein, we describe the first synthesis of the common O‐antigen of Proteus mirabilis OE and Proteus vulgaris TG 103. The repeating unit incorporates two challenging 1,2‐cis‐glycosidic bonds: the 1,2‐cis‐2‐acetamido‐2‐deoxy‐glucosidic bonds were successfully constructed by use of conformation‐restrained 2‐nitroglucal donor under bifunctional organothiourea catalysis; while the 1,2‐cis‐2‐acetamido‐2‐deoxy‐galactosidic bonds were formed by use of di‐tert‐butylsilylidene protected 2‐azidogalactose donors. The synthetic fragments were screened by glycan microarray with immunized rabbit sera against purified LPS from Proteus mirabilis O54. The results revealed that the synthetic common O‐antigens both hexasaccharide 2 and nonasaccharide 3 can strongly bind with the IgG antibodies (Abs) in the sera, which is highly valuable for further immunological investigation in synthetic carbohydrate‐based vaccine development.

Seroprevalence of Toxoplasma gondii in domestic cats, dogs and rabbits from Poland

The seroprevalence of Toxoplasma gondii in domestic cats, dogs and rabbits was evaluated. Samples from cats and dogs were collected from five veterinary practices from various parts of Poland - Poznan (wielkopolskie voivodeship), Przemysl (podkarpackie voivodeship), Kluczbork (opolskie voivodeship), Lublin (lubelskie voivodeship) and Deblin (lubelskie voivodeship). Moreover, the samples from rabbits were collected in Poznan. In total, serum samples from 193 cats, 204 dogs and 71 rabbits were randomly selected and tested for specific antibodies against T. gondii using a commercial ELISA test. Pathogen seroprevalence among cats and dogs was calculated at a 95% confidence interval (CI) for each sex and age category (up to 12 months, 1–3 years, 4–7 years and over 8 years) and compared with a chi-squared test. The highest seroprevalence of T. gondii was noted in cats − 49.74% (96/193; 95% CI: 42.76–56.73). In dogs, it reached 28.92% (59/204; 95% CI: 23.13–35.49). Only 1 rabbit (3-year-old male) was seropositive, and the seroprevalence in rabbits was 1.41% (1/71; 95% CI: 0.25–7.56). A statistically significant correlation between seropositivity and age (p < 0.05) was observed in cats and dogs. No statistically significant difference in seroprevalence concerning gender or location was found in cats and dogs. Our findings indicate that cat and dog serum samples had a high frequency of anti-T. gondii antibodies, while rabbit serum samples had low frequency and that these species are exposed to T. gondii in Poland and develop humoral response due to infection.

Recombinant GMA56 and ROP17 of Eimeria magna conferred protection against infection by homologous species.

One of the most common rabbits coccidia species, Eimeria magna is mainly parasitic in the ileal and jejunal epithelial cells. E. magna infection can affect the growth performance of rabbits or cause other secondary diseases. Traditional methods of anticoccidial treatment typically result in drug resistance and drug residue. Therefore, vaccination is a promising alternative. Gametocyte antigen 56 (GAM56) and rhoptry kinase family proteins (ROPs) are involved in oocyst wall formation and parasite invasion, respectively. A virulence factor, ROP17 contains a serine/threonine kinase catalytic domain. In this study, recombinant E. magna GAM56 (rEmGAM56) and ROP17 (rEmROP17) proteins were obtained from a prokaryotic expression system and their reactogenicity was investigated with immunoblotting. To assess the potential of rEmGAM56 and rEmROP17 as coccidiosis vaccines, New Zealand White rabbits were subcutaneously immunized with 100 μg rEmGAM56 (rGC group) or rEmROP17 (rRC group) twice at 2-week intervals followed by homologous oocyst challenge. The rabbit serum was collected weekly to detect the specific antibody levels. The cytokine levels of pre-challenge serum were measured by enzyme-linked immunosorbent assay and the rabbits were observed and recorded post-challenge for the onset of clinical symptoms. The weight gain, oocyst output, and feed conversion ratio were calculated at the end of the experiment. The results showed that both rEmGAM56 and rEmROP17 had good reactogenicity. The rEmGAM56- or rEmROP17-immunized rabbits had milder clinical symptoms and feed conversion ratios of 3.27:1 and 3.37:1, respectively. The rEmGAM56-immunized rabbits had 81.35% body weight gain and 63.85% oocyst output reduction; the rEmROP17-immunized rabbits had 79.03% body weight gain and 80.10% oocyst output reduction. The ACI of rGC and rRC groups were 162.35 and 171.03, respectively. The specific antibody levels increased rapidly after immunization. Significantly increased interleukin (IL)-2, interferon (IFN)-γ, and IL-17 levels were evident in the rGC and rRC groups (p < 0.05). The rEmGAM56 and rEmROP17 elicited humoral and cellular responses, which protected against E. magna infection in rabbits. Thus, rEmGAM56 and rEmROP17 are potential vaccine candidates against E. magna, and rEmROP17 performed better than rEmGAM56.