Determination Of Antibody Titers Research Articles

Determination of Chinese hamster ovary (CHO) cell densities and antibody titers from small volumes of cell culture supernatants using multivariate analysis and partial least squares regression of UV-Vis spectra

Antibody titer and viable cell density (VCD) are two important parameters that need to be closely monitored during the process of cell line development and manufacturing of therapeutic antibodies. Typically, determination of each parameter requires 10–100 μL of supernatant sample, which is not suitable for small scale cultivation. In this study, we demonstrated that as low as 2 μL of culture supernatants were sufficient for the analysis using UV-Vis spectrum assisted with partial least squares (PLS) model. The results indicated that the optimal PLS models could be used to predict antibody titer and VCD with the linear relationship between reference values and predicted values at R2 values ranging from 0.8 to > 0.9 in supernatant samples obtained from four different single clones and in polyclones that were cultured in various selection stringencies. Then, the percentage of cell viability and productivity were predicted from a set of samples of polyclones. The results indicated that while all predicted % cell viability were very similar to the actual value at RSEP value of 6.7 and R2 of 0.8908, the predicted productivity from 14 of 18 samples were closed to the reference measurements at RSEP value of 22.4 and R2 of 0.8522. These results indicated that UV-Vis combined with PLS has potential to be used for monitoring antibody titer, VCD, and % cell viability for both online and off-line therapeutic production process.Graphical abstractThe process of multivariate analysis and partial least squares regression of UV-Vis spectrum for the determination of CHO cell densities and antibody titers obtained from small volume of cell culture supernatant samples.

Evaluation of the immune response to COVID-19 vaccine mRNA BNT162b2 and correlation with previous COVID-19 infection.

BackgroundThe kinetics of immune response after vaccination with mRNA-BNT162b2 (Comirnaty®) and the correlation with previous COVID-19 infection are still unclear.MethodsThirty-six subjects receiving mRNA-BNT162b2 were prospectively studied [10 days after the first dose (Time 1), 7 days and 16 weeks after the second dose (Time 2 and Time 3)] to determine antibody titers against nucleocapside, trimeric spike protein (TSP) and receptor-binding-domain (RBD) of the spike protein. Ten subjects had a previous COVID-19 infection not requiring hospitalization (Group 1) and 26 did not (Group 2).ResultsAt Time 1 all subjects in Group 1 had IgG against TSP > 800 AU/mL compared to 11/26 (42.3%) in Group 2, whilst at Time 2 all subjects in both groups had > 800 AU/mL. The mean IgG against TSP titer at Time 3 was 711 AU/mL (95% CI 652–800) in Group 1 and 240 AU/mL (95% CI 112–375) in Group 2 (p < 0.0001). However, all subjects in both groups maintained antibody titers above the lower threshold limit at each time-point considered. These results were confirmed also using anti-RBD antibodiy tests. Antibodies against nucleocapside were reactive only in subjects in Group 1 and remained stable during the study period. No subject had a new onset of COVID-19 infection within 16 weeks of follow-up.ConclusionsSubjects with previous COVID-19 infection have a more rapid immune response to mRNA-BNT162b2 than others and maintained higher antibody titers during 16 weeks of follow-up. However, no new COVID-19 infection also in subjects with lower antibody titers.

Comparison of injectable trace minerals vs. adjuvant on measures of innate and humoral immune responses of beef heifers

• Injectable trace minerals (ITM), adjuvant (ADJ) and saline did not impact calf growth • Haptoglobin and ceruloplasmin were greatest for ADJ but similar between SAL and ITM • ITM enhanced calf antibody titers but not to the extent as ADJ injection Previous studies reported heightened innate and humoral immune responses among cattle administered injectable trace minerals (ITM) concurrent to immunogen challenge, thus suggesting a potential adjuvant-like activity of ITM. To pursue this concept further, a two-year study was conducted at the Mountain Research Station (North Carolina State University, Waynesville) to evaluate immunological responses of Angus heifers (n = 22, 235 ± 18 kg of BW and 225 ± 4 d of age in yr 1; n = 36, 237 ± 6 kg of BW and 247 ± 5 d of age in yr 2). On d 0 (30 days after weaning), heifers were administered two 5-mL i.m. injections of porcine red blood cells (PRBC; Lampire Biological Laboratories, Pipersville, PA) solution (25% PRBC and 75% sterile PBS; Amresco, Solon, OH), concurrent to either a single 2.5-mL s.c. injection of sterile saline (SAL; 0.9% NaCl), a squalene-based oil-in-water adjuvant (ADJ; AddaVax; InvivoGen, Inc. San Diego, CA) that specifically induces a humoral immune response, or injectable trace minerals (ITM; MultiMin 90; MultiMin USA, Inc. Fort Collins, CO; 60, 10, 15 and 5 mg/mL of Zn, Mn, Cu and Se). During the 28-d study, heifers were offered tall fescue hay ( Lolium arundinacea ) and concentrate at 1.2 and 1.0% of BW (DM basis), respectively, blended with 114 g daily of a complete mineral mix. Shrunk BW was recorded on d 0 and 28 following 12 h of feed and water withdrawal. Blood samples were collected on d 0, 2, 7, 15, and 21 for determination of antibody titers against PRBC and plasma concentrations of ceruloplasmin and haptoglobin. Liver biopsy samples were collected 1 mo prior to weaning and on d 15 after weaning for determination of trace mineral status. No differences were detected ( P ≥ 0.13) for BW change, ADG, G:F, or DMI. Liver Se concentrations on d 15 were greater ( P ≤ 0.01) for heifers receiving ITM compared to SAL and ADJ treatments. Plasma haptoglobin concentrations on d 2 were greatest for ADJ ( P < 0.0001) and did not differ between SAL and ITM heifers ( P ≥ 0.46). Plasma ceruloplasmin concentrations on d 2 were greatest for ADJ ( P ≤ 0.04) and did not differ between SAL and ITM heifers ( P = 0.39). On d 7, plasma concentrations of ceruloplasmin did not differ ( P = 0.37) between ADJ and ITM, were less for SAL vs. ADJ ( P = 0.005), and tended to differ ( P = 0.064) between ITM and SAL heifers. Serum PRBC titers were greatest for ADJ ( P ≤ 0.03) vs. SAL on d 7, 15, and 21 and ITM on d 7 ( P < 0.001). Serum PRBC titers did not differ between SAL and ITM on d 7 and 15 ( P ≥ 0.23), but were greater in ITM vs. SAL on d 21 ( P = 0.03). In summary, ITM prolonged the antibody titers against PRBC on d 21 compared to saline injection in mineral-adequate beef calves, but not as quickly or to the extent of ADJ injection.

A comparative study of Widal test with blood culture in the diagnosis of typhoid fever in febrile patients

Typhoid fever is a major health problem in developing countries and its diagnosis on clinical ground is difficult. Diagnosis in developing countries including Madhya Pradesh is mostly done by Widal test. However, the value of the test has been debated. Hence, evaluating the result of this test is necessary for correct interpretation of the result. A comparative study of Widal test with blood culture in the diagnosis of typhoid fever in febrile patients. Blood samples were collected from 62 febrile patients with symptoms clinically similar to typhoid fever and visiting red cross Hospitals at bhopal from mid May 2018 to August 2018. Blood culture was used to isolate S.typhi and S.paratyphi. Slide agglutination test and tube agglutination tests were used for the determination of antibody titer. Statistical analysis used: Data analysis by IBM SPSS Statistics 20 10 participants were females and 22 were males in blood culture and 20 were positive, 12 were negative. Total 62 cases of S. typhi were identified with the total prevalence of typhoid fever. The total number of patients who have indicative of recent infection by either of O and H antigens. Total 62 cases of S. Typhi or paratyphi were identify with the total prevalence of typhoid fever.Widal test is 30 patients were observed and blood culture were observed 32 patients.

Development of Zika NS1 ELISA methodology for seroprevalence detection in a cohort of Mexican patients in an endemic region

BackgroundZika (ZIKV) is a mosquito-borne virus that has caused multiple outbreaks in Americas. The early and accurate diagnosis of ZIKV is the key to minimize the health burden imposed on the infected patients. Many commercial ZIKV diagnosis kit are available based on ZIKV envelope antigen with varying sensitivity and specificity. ObjectiveThe aim of this study was to develop sensitive ELISA methodology based on recombinant ZIKV NS1 and NS1 β-ladder antigens for seroprevalence detection in a cohort of patients in an arbovirus endemic Mexican region in comparison to a commercial kit. Study designWe obtained serum samples from 60 patients presenting with febrile illness and from 10 healthy donors in Michoacán state in 2016–2017. These samples were screened for ZIKV by RT-PCR and used to develop NS1 based ELISA and compared to a commercial kit. ResultsOur results indicate that both ZIKV sNS1 and β-ladder-based ELISA can reliably detect anti-ZIKV NS1 antibodies in infected patients, relevant for the serodiagnosis of ZIKV. Determination of antibody titers showed that it offered higher sensitivity than a commercially available ZIKV ELISA. Over 90% seropositivity against ZIKV for the febrile patients was detected in Lázaro Cárdenas which is an arbovirus endemic region while lower seropositivity was observed in the healthy volunteers in Morelia (non-endemic area). ConclusionWe conclude that our simple and sensitive in-house NS1 based ELISA used in this study has excellent sensitivity, is easy to use and can provide fast results suitable for larger population-based seroprevalence studies in the future.

Uptake of Cost-Free Hepatitis B Vaccination among Healthcare Workers in Northern Tanzania

Background The World Health Organization and other global agencies recommend all healthcare workers (HCWs) to get vaccinated against hepatitis B virus before enrolment to clinical work. There are limited data in low-income countries on hepatitis B virus vaccination coverage among HCWs, which set back WHO Hepatitis B Elimination Strategy 2016-2021. The aim of this study was to determine the prevalence of hepatitis B virus vaccination uptake and associated factors among HCWs from a tertiary and teaching hospital in northern Tanzania. Methods A cross-sectional study was conducted among HCWs at Kilimanjaro Christian Medical Centre a referral and teaching hospital from August 2016 to June 2017. Questionnaire based interviews were used to obtain information about HCWs’ demographic characteristics, occupation risks and other invasive procedures such as intravenous, and intramuscular injections and knowledge of Hepatitis B Virus (HBV) infection. Multivariable logistic regression was used to determine factors associated with Engerex DNA-recombinant hepatitis B vaccine uptake. Results About two third 295 (67.4%) of 438 participants had received HBV vaccination. Among those who ever received HBV vaccination (n=295), 205 (70.5%) received 3 shots, 57 (19.5%) received 2 shots and 33 (10%) received 1 shot. HCWs working in clinical areas had 79% higher odds of vaccine uptake compared to those who were not (OR=1.79, 95%CI 1.11, 2.89). Ever having intravenous and intramuscular injections significantly increased the odds of vaccination uptake compared to those who never had (OR=2.21, 95%CI 1.33, 3.66) and (OR=2.42, 95%CI 1.36, 4.28) respectively. Also, HCWs with a fair and good knowledge on HBV infection had over three times higher odds of vaccine uptake compared to those with poor knowledge (OR=3.08, 95%CI 1.90, 5.01) and (OR=3.07, 95%CI 1.75, 5.40), respectively. After adjusting for other factors, those that remained to be significantly associated with HBV vaccine uptake were area of specialty and knowledge on HBV infection. Conclusion The current Engerex DNA-recombinant hepatitis B vaccine coverage among HCWs at KCMC, referral and teaching hospital in North-eastern Tanzania was only 67.4% despite of it been given free of charge. Factors that were associated with HBV vaccine uptake include area of specialty and knowledge of participants on HBV infection and vaccination. We encourage continuous medical education among HCWs about importance of hepatitis B vaccination. We also recommend hepatitis B vaccination as pre-employment requirement for HCWs as well as doing antibody titer determination post vaccination. Keywords: Cost-free hepatitis B vaccine, Prevention, Healthcare workers, vaccination coverage, Low income countries, Tanzania.

Quantitative Measurement of Anti-SARS-CoV-2 Antibodies: Analytical and Clinical Evaluation.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Molecular-based testing is used to diagnose COVID-19, and serologic testing of antibodies specific to SARS-CoV-2 is used to detect past infection. While most serologic assays are qualitative, a quantitative serologic assay was recently developed that measures antibodies against the S protein, the target of vaccines. Quantitative antibody determination may help determine antibody titer and facilitate longitudinal monitoring of the antibody response, including antibody response to vaccines. We evaluated the quantitative Roche Elecsys anti-SARS-CoV-2 S assay. Specimens from 167 PCR-positive patients and 103 control specimens were analyzed using the Elecsys anti-SARS-CoV-2 S assay on the cobas e411 (Roche Diagnostics). Analytical evaluation included assessing linearity, imprecision, and analytical sensitivity. Clinical evaluation included assessing clinical sensitivity, specificity, cross-reactivity, positive predictive value (PPV), negative predictive value (NPV), and serial sampling from the same patient. The Elecsys anti-SARS-CoV-2 S assay exhibited its highest sensitivity (84.0%) at 15 to 30 days post-PCR positivity and exhibited no cross-reactivity, a specificity and PPV of 100%, and an NPV between 98.3% and 99.8% at ≥14 days post-PCR positivity, depending on the seroprevalence estimate. Imprecision was <2% at 9.06 U/ml across 6 days, the negative quality control (QC) was consistently negative (<0.40 U/ml), the manufacturer's claimed limit of quantitation of 0.40 U/ml was verified, and linearity across the analytical measuring range was observed, except at the low end (<20 U/ml). Lastly, antibody response showed high interindividual variation in level and time of peak antibody titer and trends over time.

The Therapeutic Use of Plasma from Covid-19 Convalescent Patients

The outbreak of coronavirus disease (COVID-19) requires multidisciplinary teamwork to explore new methodological designs with the aim of approaching treatment strategies on different fronts. In principle, the rationale for the use of convalescent plasma is consistent from both an efficacy and safety point of view. Although there are still few completed randomized trials, the latest published results regarding efficacy are inconclusive. Immediate studies should target convalescent plasma preparations with a high titer of anti–SARS-CoV-2 antibodies and given early in the disease course. Regarding safety, its use with minimum risk factors or side effects is well documented. However, comparative research studies that involve the determination of antibody titers to support efficacy are required. Moreover, regulatory agencies and national blood systems should take a positive position via evaluation, supervisory, and control systems to maintain a blood-tissue store that meets the needs of the community and its hospitals.

Guideline: Vulvovaginal candidosis (AWMF 015/072, level S2k).

Approximately 70‐75% of women will have vulvovaginal candidosis (VVC) at least once in their lifetime. In premenopausal, pregnant, asymptomatic and healthy women and women with acute VVC, Candida albicans is the predominant species. The diagnosis of VVC should be based on clinical symptoms and microscopic detection of pseudohyphae. Symptoms alone do not allow reliable differentiation of the causes of vaginitis. In recurrent or complicated cases, diagnostics should involve fungal culture with species identification. Serological determination of antibody titres has no role in VVC. Before the induction of therapy, VVC should always be medically confirmed. Acute VVC can be treated with local imidazoles, polyenes or ciclopirox olamine, using vaginal tablets, ovules or creams. Triazoles can also be prescribed orally, together with antifungal creams, for the treatment of the vulva. Commonly available antimycotics are generally well tolerated, and the different regimens show similarly good results. Antiseptics are potentially effective but act against the physiological vaginal flora. Neither a woman with asymptomatic colonisation nor an asymptomatic sexual partner should be treated. Women with chronic recurrent Candida albicans vulvovaginitis should undergo dose‐reducing maintenance therapy with oral triazoles. Unnecessary antimycotic therapies should always be avoided, and non‐albicans vaginitis should be treated with alternative antifungal agents. In the last 6 weeks of pregnancy, women should receive antifungal treatment to reduce the risk of vertical transmission, oral thrush and diaper dermatitis of the newborn. Local treatment is preferred during pregnancy.

Health Status of Broiler Chickens Fed Diets with Equi-Protein Replacement of Soybean Meal with Groundunt Cake

This study was carried out to investigate the effect of equi-protein replacement of soybean meal with groundnut cake on immune status, haematology and serum biochemical parameters of broiler chickens. A total of One hundred and twenty (120) Cobb‘s day old chicks were randomly allocated to five (5) dietary treatments of three (3) replicates and eight (8) chicks per replicate in a completely randomized design. The Diet I was the control diet with 100% SBM as the major plant protein source while in other diets, SBM was replaced with 25, 50, 75 and 100% GNC in equi-protein basis and designated as Diets II, III, IV and V respectively. Feed and water were provided ad-libitum throughout the experimental period of 56 days. Newcastle disease (ND) vaccines were administered following a stipulated regime. At the end of the experiment two birds were randomly selected from each replicate and slaughtered. Blood and serum was collected for haematological analysis, serum bioassay and HA/HI tests to determine antibody titres against ND vaccines. All data collected were subjected to one-way analysis of variance using SPSS software package. Results showed that haematological parameters were significantly influenced by the dietary treatments in which birds fed 50% equi-protein replacement of SBM with GNC had better PCV, basophil, MCH and MCHC values compared to that of birds in the other dietary treatments. The antibody titres of experimental birds were not significantly different among treatment groups. The diet with 50% equi-protein replacement of SBM with GNC elicited the highest antibody titres of Log28 and Log210 after the 1st and 2nd ND vaccinations when compared to the other diets. The serum total protein, globulin, albumin, and glucose were significantly influenced by the dietary treatments. The present result indicate that 50% equi-protein replacement of SBM with GNC in diets is not detrimental to health status of broiler chickens.

Study of the teratogenicity of the vaccine strain of the Rubella virus «Orlov-V» (Matonaviridae: Rubivirus: Rubella virus) in experience on rhesus macaques.

Rubella virus has pronounced teratogenic properties that can cause generalized and persistent intrauterine infection of the fetus. As a result, the control of the loss of teratogenicity inherent in «wild-type» virus strains is a necessary stage of a preclinical study of the vaccine strain for a live attenuated rubella vaccine.The purpose of the study is to comprehensively study the teratogenic properties of the vaccine strain of rubella virus «Orlov-V» in the experiment on rhesus macaques. Seronegative to rubella virus female rhesus macaques in early pregnancy at the age of 4-7 years (n = 13) were used in the experiment. Animals of the experimental group (n = 9) received single immunization intramuscularly with a preparation from the «Orlov-V» strain. The control group of the monkeys (n = 3) were immunized with a commercial vaccine containing Wistar RA27/3 strain. The female of the control group (n = 1) was injected with a solvent used in the rubella vaccine. Study of possible teratogenic properties of vaccine strains of rubella virus was carried out using a complex of clinical, immunological, pathomorphological and virological methods. Clinical observations were made within 3 months after the monkeys' birth. Determination of antibody titers in the blood serum of immunized monkeys was performed in HI test on the 28th-30th day after infection. The ELISA method was applied to determine IgM antibodies in the blood serum of newborns within the first month of life. Detection of rubella virus RNA was performed by PCR with electrophoretic detection of amplicons. No markers of congenital rubella infection were found in infants born from monkeys vaccinated during the pregnancy. It is shown that PCR can be an informative method to confirm the absence of teratogenic properties of vaccine strains of rubella virus. The obtained data demonstrated that vaccine strains of the «Orlov-V» rubella virus and Wistar RA27/3 have lost their teratogenic properties. The possibility of using an alternative strategy for preclinical assessment of specific safety of antiviral vaccines including a complex of clinical, immunological, pathologic and virological methods instead of the classical pathologic method is discussed. The results obtained in this study showed the absence of teratogenic properties and high immunogenic activity of the vaccine strain of rubella virus «Orlov-V».

A novel ELISA method to determine human MrgX2 in chronic urticaria

BackgroundMas-related G-protein coupled receptor member X2 (MrgX2) directly mediates drug-induced pseudo allergic reactions. Skin mast cell MrgX2 is upregulated in severe chronic urticaria (CU). Mast cells and leukocytes are key effector cells in allergic reactions and undergo degranulation upon stimulation. It is unknown whether circulating MrgX2 expression can be detected occurs in the whole blood of CU patients and reflects pseudo-allergic reaction. There is no effective method for its detection. Therefore, an enzyme-linked immuno-sorbent assay (ELISA) for MrgX2 was developed.MethodsMonoclonal and polyclonal MrgX2 specific antibodies were obtained from rabbits and mice immunized by MrgX2 peptides prepared. Indirect ELISA and Dot blot were used to determine antibody titers before a sandwich ELISA for MrgX2 was established. The whole blood from healthy subjects and CU patients was used to detect MrgX2 concentrations. The use of feasibility of this MrgX2-ELISA as a clinical detection tool was explored and diagnostic purposes was assessed.ResultsThe sandwich antibody ELISA method for MrgX2 was established with good linearity regression (R2 = 0.9910). The lowest detection limit was 3.125 ng/mL. The quantification limit was 6.25 ng/mL. The sandwich ELISA for MrgX2 have good stability and high specificity. The initial truncation value of MrgX2 was 60.91 ng/mL (95% confidence interval). The whole blood MrgX2 concentrations in CU patients (median 98.01 ± 4.317 ng/mL, n = 75) was significantly increased compared to healthy subjects (58.09 ± 1.418 ng/mL, n = 75), with significant difference (p < 0.0001) and higher accuracy of (AUC = 0.8795). Comprehensive the frequency analysis of MrgX2 expression in 75 CU patients reference frequency distribution and ROC curve analysis, determined the threshold for CU patients as 71.23 ng/mL, with 81.33% sensitivity and 90.67% specificity.ConclusionMrgX2-ELISA provides a useful and convenient method for detecting MrgX2 in whole blood samples. The MrgX2-ELISA will help improve the understanding of the role of MrgX2 in regulating chronic urticaria.

A COMPARATIVE STUDY OF WIDAL TEST WITH BLOOD CULTURE IN THE DIAGNOSIS OF TYPHOID FEVER IN FEBRILE PATIENTS ATTENDING GGH, ANANTHAPURAMU.

Introduction Typhoid fever is a systemic prolonged febrile illness caused by certain Salmonella serotypes including Salmonella typhi, S. paratyphi A, S. paratyphi B and S. paratyphi C. 1 Human beings are the only reservoir host for typhoid fever. It is transmitted is by feco-oral contamination of water and food in endemic areas especially by carriers handling food. The World Health Organization (WHO) estimates about 21 million cases of typhoid fever with &gt;600,000 deaths annually. 2 The cases are more likely to be seen because of rapid population growth, increased urbanization, and limited safe water, limited infrastructure and health systems. Aims &amp; Objectives The main aim of this study is to compare the result of Widal test and blood culture in the diagnosis of typhoid fever in febrile patients. Material &amp; Methods: This is a prospective study conducted in the GGH Anantapuramu, for a period of 18 months .i.e. from May 2018 to Oct 2019. Blood samples were collected from 542 febrile patients with symptoms clinically similar to typhoid fever. Blood culture was used to isolate S.typhi and S.paratyphi. Slide agglutination test and tube agglutination tests were used for the determination of antibody titer. An antibody titer of ≥1:80 for anti TO and ≥1:160 for anti TH were taken as a cut of value to indicate recent infection of typhoid fever. Results: A total of the 542 patients were the study population, among which 384 (70.8%) were females, and 158 (29.1%) were males. Widal and blood culture was performed, 11 (2.02%) cases of S. typhi and 5 (0.9%) cases of S. paratyphi were identified using blood culture. The total prevalence of typhoid fever is 2.95%. The specificity, sensitivity, Positive Predictive Value, and Negative Predictive Value of the Widal test with O Ag is 70%, 81.25%, 10.93%, and 96%, respectively, and with H Ag is 40%, 96%, 18.1%, 98.8% respectively.

Ataxia associated with anti-glutamic acid decarboxylase antibodies

Anti-glutamic acid decarboxylase (GAD) antibody-associated ataxia is a rarely diagnosed but potentially curable disease associated with autoimmune damage to and death of Purkinje cells in the cerebellar cortex. In Russia, the authors have provided for the first time descriptions of three own observations of this disease, which had a number of clinical features, such as slow progression, mild ataxia, stroke-like episodes with stem symptoms, concomitant gluten sensitivity, onset of ataxia after hepatitis C with cerebellar hemiataxia and hemiatrophy. In the all patients, the diagnosis was verified based on the determination of high anti-GAD antibody titers in serum and cerebrospinal fluid. All the patients lacked intrathecal synthesis of oligoclonal antibodies; protein levels and cytosis were normal. Pulse therapy with methylprednisolone at a total dose of 3–5 g led to a slight reduction in ataxia in one case (a female patient with subacute onset of the disease); the treatment was ineffective in two other cases (patients with a primary chronic course). The paper analyzes the literature covering the pathogenesis and clinical presentations of this type of ataxia, and difficulties in its diagnosis and treatment.

Seroprevalence and associated risk factors of Toxoplasma gondii infection in stray cats in Algiers urban area, Algeria

Toxoplasmosis is a zoonotic parasitic disease caused by the protozoan Toxoplasma gondii. Human infections are common and generally asymptomatic, but they can become very dangerous in immunosuppressed and HIV-positive patients. The infection can also be serious if it is transmitted to the fetus during pregnancy. Infection in untreated mothers can lead to abortion, birth defects and blindness of the fetus. The aim of this study is to determinethe seroprevalence of Toxoplasma IgT antibodies in cats in the urban area of Algiers. From December 2017 to August 2018, blood samples from 184 stray cats were collected and analyzed for IgG antibodies against T. gondii using an ELISA method. Overall, the prevalence of T. gondii infection in stray cats was 58.15% (107/184). There was no statistically significant difference between male and female cats. The rate of seropositivity of T. gondii increased with age (p &lt;0.05). There was no statistically significant difference between the different regions from which the samples were taken. The results of the present study showed the high seroprevalence of toxoplasmosis in Algiers rather than in other countries. Due to the high seroprevalence of Toxoplasma IgG antibodies in cats, it is recommended to include in the prenatal evaluation, together with the adoption of a screening test and the determination of the IgG antibody titer in the high-risk populations (young girls, pregnant women) public information programs on the disease and measures that can contribute to prevention

Ezh2-mediated epigenetic modification is required for allogeneic T cell-induced lupus disease

BackgroundThe mechanisms involved in the pathogenesis of autoimmune disorders, including systemic lupus erythematosus (SLE), have not been fully elucidated. Some of these mechanisms involve epigenetic regulation of gene expression. The histone methyltransferase Ezh2 contributes to epigenetic regulation of gene expression, is highly expressed in germinal center (GC) B cells and follicular T helper (TFH) cells, and may be involved in lupus pathogenesis.MethodsThe murine bm12 model of lupus-like chronic graft versus host disease (cGVHD) was induced by intra-peritoneal injection of negatively isolated allogeneic CD4+ T cells. Lupus-like disease development was monitored by ELISA determination of serum anti-dsDNA and anti-chromatin antibody titers. Immune cell activation and Ezh2 expression were evaluated by flow cytometry and Western blotting.ResultsDecreased autoantibody production and GC formation are observed when Ezh2-deficient CD4+ T cells are used instead of wild-type (WT) to induce cGVHD and when mice that receive allogeneic WT donor T cells to induce cGVHD are treated with GSK503, an Ezh2-specific inhibitor. In the bm12 cGVHD model, WT donor T cells are normally fully activated 1 week after infusion into an allogeneic host, exhibit a TFH cell (PD-1hi/CXCR5hi) phenotype with upregulated Ezh2, and activate B cells to form germinal centers (GCs). In contrast, Ezh2-deficient donor T cells generate fewer TFH cells that fail to activate B cells or promote GC formation. Despite similar T-independent, LPS-induced B cell responses, OVA-immunized CD4.Ezh2-KO mice had a skewed low-affinity IgM phenotype in comparison to similarly treated WT mice. In addition, early after OVA immunization, more CD4+ T cells from B6.CD4.Ezh2-KO mice had a CD44lo/CD62Llo phenotype, which suggests arrested or delayed activation, than CD4+ T cells from ovalbumin-immunized B6.WT mice.ConclusionEzh2 gene deletion or pharmacological Ezh2 inhibition suppresses autoantibody production and GC formation in bm12 lupus-like cGVHD and decreases affinity maturation and isotype switching in response to immunization with a T cell-dependent antigen. Ezh2 inhibition may be useful for the treatment of lupus and other autoimmune disorders.

The expression and purification of LpxA of Chlamydia trachomatis and preparation of its polyclonal antibody.

The purpose of this study is to purify the LpxA protein of Chlamydia trachomatis (Ct) and prepare the polyclonal antibody against LpxA protein, so as to lay a foundation for studying the function of LpxA protein. The LpxA gene was amplified by PCR. The expression plasmid pET28a-LpxA was constructed by using pET28a as the vector. The fusion protein containing 6 histidine tag was induced by IPTG and purified by Ni2+ chromatography gel. The purified His-LpxA protein was used as an immunogen to immunize New Zealand rabbits subcutaneously through the back to prepare polyclonal antibody. Immunoblotting was used to detect the reaction between the antibody and His-LpxA. The determination of polyclonal antibody titer was detected by ELISA. The relative molecular weight of His-LpxA was 32.8 kDa, and it could be expressed in Escherichia coli. The purity of the purified protein was about 95%. After immunizing New Zealand rabbits, the antiserum was able to recognize the recombinant His-LpxA protein with a titer greater than 1:10240. In this study, LpxA protein was successfully purified and antiserum was prepared, which provided an experimental basis for studying the function of LpxA protein.

Comparison of influenza-specific neutralizing antibody titers determined using different assay readouts and hemagglutination inhibition titers: good correlation but poor agreement

Determination of influenza-specific antibody titers is commonly done using the hemagglutination inhibition assay (HAI) and the viral microneutralization assay (MN). Both assays are characterized by high intra- and inter-laboratory variability. The HAI assay offers little opportunity for standardization. For the MN assay, variability might be due to the use of different assay protocols employing different readouts. We therefore aimed at investigating which of the MN assay readout methods currently in use would be the most suitable choice for a standardized MN assay that could serve as a substitute for the HAI assay. For this purpose, human serum samples were tested for the presence of influenza specific neutralizing antibodies against A/California/7/09 H1N1 (49 sera) or A/Hong Kong/4801/2014 (50 sera) using four different infection readout methods for the MN assay (cytopathic effect, hemagglutination, ELISA, RT qPCR) and using the HAI assay. The results were compared by correlation analysis and by determining the level of agreement before and after normalization to a standard serum. Titers as measured by the 4 MN assay readouts showed good correlation, with high Person’s r for most comparisons. However, agreement between nominal titers varied with readouts compared and virus strain used. In addition, Pearson’s correlation of MN titers with HAI titers was high but agreement of nominal titers was moderate and the average difference between the readings of two assays (bias) was virus strain-dependent. Normalization to a standard serum did not result in better agreement of assay results. Our study demonstrates that different MN readouts result in nominally different antibody titers. Accordingly, the use of a common and standardized MN assay protocol will be crucial to minimize inter-laboratory variability. Based on reproducibility, cost effectiveness and unbiased assessment of results we elected the MN assay with ELISA readout as most suitable for a possible replacement of the HAI assay.

Analysis of determination of rabies virus neutralizing antibody titres in the sera of vaccinated humans.

Rabies is an infectious disease that is always fatal following the onset of clinical symptoms. The only way to prevent the cases of rabies in humans is timely carried out the rabies post-exposure prophylaxis in accordance with the recommended schedule. The aim of the study was to characterize the level of immune response in persons that received a post-exposure prophylaxis against rabies, to consider the role of the factors of the formation immune responses to rabies vaccines. In the laboratory of viral vaccines of the Scientific Centre for Expert Evaluation of Medicinal Products, the 48 sera of patients that received the post-exposure prophylaxis of rabies after wounds from a rabid or suspected rabid animal has been studied. The titer of virus neutralizing antibodies (VNA) to the rabies virus in the sera of the vaccinated not less than 1:64 (corresponding to a level of VNA at least 0,5 IU /ml) in the mouse neutralization test indicates the effective vaccination. Our data confirm the absence of statistically significant differences in the level of VNA in the vaccinated persons that received a complete and incomplete (5 doses) course of post-exposure vaccination against rabies. Depending on the level of VNA, all patients are divided into groups with conditionally low, medium and high content of antibodies in sera. It has been shown that in most cases properly administered vaccination contributed to the formation of effective immune response. The lack of a protective level of BHA requires additional administration of the vaccine and analysis of the factors that influenced the ineffectiveness of vaccination. In some patients the determination of rabies virus neutralizing antibody titres is necessary.

High-Throughput Quantification and Glycosylation Analysis of Antibodies Using Bead-Based Assays.

A novel version of bead -based assays with fluorescence detection enables the high-throughput analysis of antibodies and proteins. The protocols are carried out in special 384-well plates, require very few manual interventions, and are easy to automate. Here we describe how the technology can be used to determine antibody titers and screen for product glycosylation, a critical quality attribute, early in cell line and bioprocess development.