Mucin 13 (MUC13) as a candidate biomarker for ovarian cancer detection: potential to complement CA125 in detecting non-serous subtypes.

OBJECTIVES: Ovarian cancer is the most lethal gynecological malignancy in developed countries. One of the key associations with the high mortality rate is diagnosis at late stages. This clinical limitation is primarily due to a lack of distinct symptoms and detection at the early stages. The ovarian cancer biomarker, CA125, is mainly effective for identifying serous ovarian carcinomas, leaving a gap in non-serous ovarian cancer detection. Mucin 13 (MUC13) is a transmembrane, glycosylated protein with aberrant expression in malignancies, including ovarian cancer. We explored the potential of MUC13 to complement CA125 as an ovarian cancer biomarker, by evaluating its ability to discriminate serous and non-serous subtypes of ovarian cancer at FIGO stages I-IV from benign conditions. METHODS: We used our newly developed, high sensitivity ELISA to measure MUC13 protein in a large, well-defined cohort of 389 serum samples from patients with ovarian cancer and benign conditions. RESULTS: MUC13 and CA125 serum levels were elevated in malignant compared to benign cases (p<0.0001). Receiver-operating characteristic (ROC) curve analysis showed similar area under the curve (AUC) of 0.74 (MUC13) and 0.76 (CA125). MUC13 concentrations were significantly higher in mucinous adenocarcinomas compared to benign controls (p=0.0005), with AUC of 0.80. MUC13 and CA125 showed significant elevation in early-stage cases (stage I-II) in relation to benign controls (p=0.0012 and p=0.014, respectively). CONCLUSIONS: We report the novel role of MUC13 as a serum ovarian cancer biomarker, where it could complement CA125 for detecting some subtypes of non-serous ovarian carcinoma and early-stage disease.

Evaluation of efficacy of various immunochromatographic rapid tests for dengue diagnosis.

OBJECTIVE: The immunochromatographic rapid tests facilitate the early diagnosis of dengue by providing evidence of the presence of virus specific proteins (antigens/ antibody) in human blood. Many products for rapid dengue diagnosis are available in the market; the performance of few selected products was evaluated and compared with enzyme linked immuno sorbent assays (ELISA). METHODS: Sera from a large number of patients (n=184) admitted to National Institute of Blood Diseases & Bone Marrow Transplantation (NIBD) were used to determine the efficiency of non-structural (NS) 1, IgA, IgG and IgM based rapid test devices for dengue diagnosis. RESULTS: The dengue NS1 antigen based device was least efficient while among the antibody based devices the dengue IgA rapid test (RDT) was comparatively better (specificity: 80.95%; sensitivity: 85.21%). This device could detect both primary and secondary dengue infection and was found to be the most sensitive device at all point of sample collection. CONCLUSION: The dengue IgA RDT could be a cost effective and efficient rapid test device for timely dengue diagnosis at all levels of healthcare settings.

Serological Comparison of Native Antigen ELISAs with Rapid ICT Test Kits for the Diagnosis of Human Alveolar and Cystic Echinococcosis in China.

BACKGROUND: The aim of this study was to compare the diagnostic performance of native antigen ELISAs and ADAMU-AE/CE commercial ICT test kits in subjects either exposed to Echinococcus infection or with clinically diagnosed alveolar (AE) or cystic (CE) echinococcosis. METHODS: A total of 370 subjects with a previous clinical confirmation of CE or AE from northwestern China were recruited. Serum samples were also obtained from 3923 children/teenagers during a community survey. All sera were tested using native antigen ELISAs. The ADAMU-AE/CE test kits were subsequently used for the serology of the 370 clinically confirmed individuals and of 251 children/teenagers that were ELISA antibody-positive for both Echinococcus species but ultrasound-negative during baseline survey. An analysis of the association between the serological tests and ultrasound classification was carried out amongst 89 AE and 164 CE cases. A Kappa consistency analysis was undertaken to compare the diagnostic performance of the native antigen ELISAs and the ADAMU kits and the ultrasound imaging results. The χ² test was also used for a comparison of the different seropositivity rates between the groups. FINDINGS: There was poor consistency (Kappa = 0.26 and 0.28 for AE and CE respectively) between the native antigen ELISAs and the ADAMU kits for the diagnosis of AE and CE among the cases and the surveyed children/teenagers, but a relatively good consistency (Kappa = 0.63) between the ADAMU-AE kit and ultrasound observations for the AE cases. Additionally, of the 251 teenagers co-positive for both AE and CE antibodies by the native antigen ELISAs, only one was found positive by the ADAMU-AE kit, verified as a new AE case on subsequent ultrasound follow-up. The remainder (N = 250) were negative by serology using the ADAMU-AE/CE kits and by ultrasound examination. The two native antigen ELISAs did not discriminate well between cases of clinically diagnosed AE and CE. In contrast, ADAMU-AE and ADAMU-CE commercial ICT test kits readily differentiated cases of AE from CE with specificities of 99% for AE and 100% for CE. CONCLUSIONS: The ADAMU-AE/CE kits proved reliable, accurate, and amenable diagnostic tools in the clinical setting for confirmation of suspected AE/CE cases. The native antigen ELISAs tests can provide useful information on the level of human exposure to Echinococcus infection.

Development of in vitro assays for measuring the relative potency of leptospiral bacterins containing serogroups canicola, grippotyphosa, icterohaemorrhagiae, and pomona.

Historically, potency testing of bacterins containing Leptospira involved a hamster vaccination-challenge assay. The United States Department of Agriculture (USDA) has long recognized that an in vitro system has several inherent advantages over the animal model. This is a review of the work performed at the USDA to replace the hamster vaccination-challenge model used to test Leptospira bacterins. The work covered a span of approximately 20 years and resulted in the development of USDA monoclonal antibody based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of antigen in bacterins containing Leptospira serogroups canicola, icterohaemorrhagiae, pomona, and grippotyphosa. The monoclonal antibodies used in the assay a) recognize lipopolysaccharide-like epitopes on the surface of the whole cell, b) agglutinate the homologous leptospiral serovars but do not agglutinate heterologous leptospiral serovars or heterologous bacterial species, and c) passively protect hamsters against a homologous challenge but fail to protect hamsters against heterologous challenges. Once developed, the performance of each ELISA was evaluated at the USDA followed by industry evaluation. Serials that passed the hamster vaccination-challenge assay yielded ELISA relative potency values of 1.0 or greater. These ELISAs have been shown to be a reproducible, sensitive, specific, and inexpensive alternative to the current Codified hamster potency assay.

Generation of a highly reactive chicken-derived single-chain variable fragment against Fusarium verticillioides by phage display.

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10(-2) µg/mL and contaminating mycelium at a quantity of 10(-2) mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples.

A Novel Full-length IgG Recombinant Antibody Highly Specific to Clothianidin and Its Application in Immunochromatographic Assay.

The toxicity of clothianidin to non-target organisms has gradually attracted world-wide attention. It is essential to develop reliable methods for the on-site detection of clothianidin residue. In this study, analogue-based heterologous ic-ELISAs were designed to rapidly screen desirable hybridomas, which could be used for the construction of recombinant antibodies (RAbs) against clothianidin. Based on the antibody variable region genes, two full-length IgG RAbs (1F7-RAb and 5C3-RAb) were produced by the mammalian cell expression system. The performance of the two RAbs was characterized and compared by heterologous ic-ELISAs and non-competitive surface plasmon resonance (SPR) assays. Using heterologous ic-ELISAs, the 1F7-RAb exhibited highly specific and sensitive recognition to clothianidin with an IC50 of 4.62 µg/L, whereas the 5C3-RAb could bind to both clothianidin and dinotefuran. The results of the non-competitive SPR assay further verified that the 1F7-RAb had a higher specificity and affinity to clothianidin than the 5C3-RAb. Finally, a gold immunochromatographic assay based on the novel antibody, 1F7-RAb, was developed for rapid detection of clothianidin with high sensitivity (visual detection limit of 2.5 µg/L), specificity, and good reproducibility, which can be used as an effective supervision tool for clothianidin residue in agricultural and environmental samples.

The Effects of Pathogen-Associated Molecular Patterns on Peripheral Blood Monocytes in Patients with Non-alcoholic Fatty Liver Disease.

Background: Innate immune responses to gut-derived pathogen-associated molecular patterns (PAMPs) have been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether NAFLD patients have increased sensitivity to PAMP exposure has yet to be reported. Methods: Peripheral blood mononuclear cell (PBMC)/monocytes were exposed to lipopolysaccharide (LPS), Pam3CSK4, or BSA conjugated palmitate in vitro. Changes in toll-like receptors (TLR), cytokines, and chemokine receptors (CR) expressions were documented by flow cytometry and/or enzyme-linked immunoabsorbent assays (ELISAs). Results: TLR2 and TLR4 expression were similar at baseline and increased to a similar extent (TLR2) or remained unchanged (TLR4) following PAMP exposure in NAFLD and healthy control (HC) monocytes. Proinflammatory IL-1ß and IL-6 levels were similar at baseline but increased in a concentration-dependent manner to a greater extent in NAFLD PBMCs. CCR1 and CCR2 expressions at baseline were similar and decreased to a similar extent in NAFLD and HC monocytes. The extent of PAMP-induced proinflammatory cytokine release correlated with evidence of hepatocyte injury (CK18M30 levels). Discussion: NAFLD patients have increased proinflammatory cytokine responses following exposure to PAMPs relative to HC subjects. This response is concentration-dependent and correlates with the extent of hepatic injury.

Mutations in the Amyloid-ß Protein Precursor Reduce Mitochondrial Function and Alter Gene Expression Independent of 42-Residue Amyloid-ß Peptide.

BACKGROUND: Dominant missense mutations in the amyloid-ß protein precursor (AßPP) cause early-onset familial Alzheimer's disease (FAD) and are associated with changes in the production or properties of the amyloid-ß peptide (Aß), particularly of the 42-residue variant (Aß42) that deposits in the Alzheimer's disease (AD) brain. Recent findings, however, show that FAD mutations in AßPP also lead to increased production of longer Aß variants of 45-49 residues in length. OBJECTIVE: We aimed to test neurotoxicity of Aß42 vis-á-vis longer variants, focusing specifically on mitochondrial function, as dysfunctional mitochondria are implicated in the pathogenesis of AD. METHODS: We generated SH-SY5Y human neuroblastoma cells stably expressing AßPP mutations that lead to increased production of long Aß peptides with or without Aß42. These AßPP-expressing cells were tested for oxygen consumption rates (OCR) under different conditions designed to interrogate mitochondrial function. These cell lines were also examined for expression of genes important for mitochondrial or neuronal structure and function. RESULTS: The mutant AßPP-expressing cells showed decreased basal OCRs as well as decreased OCRs associated with mitochondrial ATP production, even more so in the absence of Aß42 production. Moreover, mutant AßPP-expressing cells producing longer forms of Aß displayed altered expression of certain mitochondrial- and neuronal-associated genes, whether or not Aß42 was produced. CONCLUSION: These findings suggest that mutant AßPP can cause mitochondrial dysfunction that is associated with long Aß but not with Aß42.

Saliva-Based ELISAs for Effective SARS-CoV-2 Antibody Monitoring in Vaccinated Individuals.

In March 2020, the World Health Organization (WHO) declared a global health emergency-the coronavirus disease 2019 (COVID-19) pandemic. Since then, the development and implementation of vaccines against the virus amidst emerging cases of re-infection has prompted researchers to work towards understanding how immunity develops and is sustained. Serological testing has been instrumental in monitoring the development and persistence of antibodies against SARS-CoV-2 infection, however inconsistencies in detection have been reported by different methods. As serological testing becomes more commonplace, it is important to establish widespread and repeatable processes for monitoring vaccine efficacy. Therefore, we present enzyme linked immunosorbent assays (ELISAs) compatible for antibody detection in saliva as highly accurate, efficacious, and scalable tools for studying the immune response in individuals vaccinated against SARS-CoV-2.

Serum Samples from Co-Infected and Domestic Cat Field Isolates Nonspecifically Bind FIV and Other Antigens in Enzyme-Linked Immunosorbent Assays.

We evaluated enzyme-linked immunosorbent assay (ELISA) specificity for measuring seroantibody responses to two types of retroviral infections in domestic cats: feline immunodeficiency virus (FIV) and feline foamy virus (FFV). We compared the seroreactivity of specific pathogen-free (SPF) cat sera, sera from SPF cats inoculated with either FIV or FFV, and field isolates (e.g., shelter or privately owned cats). Sera from SPF cats experimentally infected with the cognate virus had significantly lower background in both FIV and FFV ELISAs compared to sera from negative field isolates. ELISA values for SPF cats exposed to either FIV or FFV tended to have higher OD values on the opposite ELISA antigen plate. FIV nonspecific background absorbance was greater than that of FFV, and 10 of 15 sera samples from FIV seronegative field samples were measured in the indeterminant range. These findings highlight that exposure to off-target pathogens elicit antibodies that may nonspecifically bind to antigens used in binding assays; therefore, validation using sera from SPF animals exposed during controlled infection results in the setting of a cutoff value that may be inappropriately low when applied to field samples. Our work also suggests that infection of domestic cats with pathogens other than FIV results in antibodies that cross-react with the FIV Gag antigen.

SARS-CoV-2 Specific IgG Antibodies Persist Over a 12-Month Period in Oral Mucosal Fluid Collected From Previously Infected Individuals.

Background: Developing an understanding of the antibody response, seroprevalence, and seroconversion from natural infection and vaccination against SARS-CoV-2 will give way to a critical epidemiological tool to predict reinfection rates, identify vulnerable communities, and manage future viral outbreaks. To monitor the antibody response on a larger scale, we need an inexpensive, less invasive, and high throughput method. Methods: Here we investigate the use of oral mucosal fluids from individuals recovered from SARS-CoV-2 infection to monitor antibody response and persistence over a 12-month period. For this cohort study, enzyme-linked immunosorbent assays (ELISAs) were used to quantify anti-Spike(S) protein IgG antibodies in participants who had prior SARS-CoV-2 infection and regularly (every 2-4 weeks) provided both serum and oral fluid mucosal fluid samples for longitudinal antibody titer analysis. Results: In our study cohort (n=42) with 17 males and 25 females with an average age of 45.6 +/- 19.3 years, we observed no significant change in oral mucosal fluid IgG levels across the time course of antibody monitoring. In oral mucosal fluids, all the participants who initially had detectable antibodies continued to have detectable antibodies throughout the study. Conclusions: Based on the results presented here, we have shown that oral mucosal fluid-based assays are an effective, less invasive tool for monitoring seroprevalence and seroconversion, which offers an alternative to serum-based assays for understanding the protective ability conferred by the adaptive immune response from viral infection and vaccination against future reinfections.

New Diagnostic Assays for Differential Diagnosis Between the Two Distinct Lineages of Bovine Influenza D Viruses and Human Influenza C Viruses.

Influenza D virus (IDV), a novel orthomyxovirus, is currently emerging in cattle worldwide. It shares >50% sequence similarity with the human influenza C virus (HICV). Two clades of IDV are currently co-circulating in cattle herds in the U.S. New assays specific for each lineage are needed for accurate surveillance. Also, differential diagnosis between zoonotic human influenza C virus and the two clades of IDV are important to assess the zoonotic potential of IDV. We developed an enzyme-linked immunosorbent assay (ELISA) based on two different epitopes HEF and NP and four peptides, and fluorescent focus neutralization assay to differentiate between IDV bovine and swine clades. Calf sera were obtained, and bovine samples underwent surveillance. Our results highlight the importance of position 215 with 212 in determining the heterogeneity between the two lineages. We needed IFA and FFN for tissue culture-based analysis and a BSL2 facility for analyzing virus interactions. Unfortunately, these are not available in many veterinary centers. Hence, our second aim was to develop an iELISA using specific epitopes to detect two lineages of IDVs simultaneously. Epitope-iELISA accurately detects neutralizing and non-neutralizing antibodies against the IDV in non-BSL2 laboratories and veterinary clinics and is cost-effective and sensitive. To differentiate between IDVs and HICVs, whole antigen blocking, polypeptides, and single-peptide ELISAs were developed. A panel of ferret sera against both viruses was used. Results suggested that both IDV and ICV had a common ancestor, and IDV poses a zoonotic risk to individuals with prior or current exposure to cattle. IDV peptides IANAGVK (286-292 aa), KTDSGR (423-428 aa), and RTLTPAT (448-455 aa) could differentiate between the two viruses, whereas peptide AESSVNPGAKPQV (203-215 aa) detected the presence of IDV in human sera but could not deny that it could be ICV, because the only two conserved influenza C peptides shared 52% sequence similarity with IDV and cross-reacted with IDV. However, blocking ELISAs differentiated between the two viruses. Diagnostic tools and assays to differentiate between ICV and IDV are required for serological and epidemiological analysis to clarify the complexity and evolution and eliminate misdiagnosis between ICV and IDV in human samples.

The 2014 autochthonous dengue fever outbreak in Tokyo: A case series study and assessment of the causes and preventive measures.

OBJECTIVE: In 2014, an autochthonous dengue fever outbreak occurred around the Yoyogi Park in Japan for the first time in 70 years. Despite no local cases reported since then, the risk of another outbreak remains high. This study reviews the autochthonous dengue fever cases of the outbreak, investigates its causes, and delineates preventive measures against autochthonous dengue epidemics. METHODS: We conducted a case series study of 15 patients who visited our institution during the 2014 outbreak. We collected and evaluated data on the surveillance of vector mosquitoes, weather, pest control, travelers' origins and destinations, and imported dengue fever cases using reports made by public institutions. RESULTS: All patients recovered with supportive treatments and none met the diagnostic criteria for severe dengue infection. Twelve patients with positive real-time polymerase chain reactions were confirmed as having dengue virus-1 infections. We found no obvious associations between the number of mosquitoes and the weather, or between the number of imported dengue fever cases and that of travelers. Insect growth regulator (IGR) against vector mosquitoes has been used since 2014 for pest control, but the number of larvae has not declined in the Yoyogi Park, although that of imagoes has been relatively suppressed. CONCLUSION: The 2014 outbreak emerged without particularly favorable climate conditions for vector mosquitoes. We found no obvious associations between the number of travelers or the imported dengue fever cases and the outbreak, but the increasing number of travelers may contribute to another outbreak. Pest control, including IGR, remains essential for infection control.

Diagnostic performance of a fully automated chemiluminescent enzyme immunoassay for Alzheimer's disease diagnosis.

The variability of Alzheimer's disease (AD) cerebrospinal fluid (CSF) biomarkers (Aß42, t-Tau and p-Tau) undermines their full-fledged introduction into routine diagnostics and clinical trials. The introduction of automatic systems can improve the diagnostic performance promoting standardization and reducing the impact of preanalytical and analytical factors. Here we assessed the diagnostic performance of a fully automated chemiluminescent enzyme assay (LUMIPULSE) and compared it with that obtained by using the classical manual enzyme-linked immunosorbent assays (ELISAs). Patients were clinically diagnosed as AD (n = 42) and non-AD (n = 38). Clinical diagnosis was confirmed at follow-up. LUMIPULSE Aß42 was reduced in AD (969.4 ±â€¯329.6 pg/mL vs. 1625.9 ±â€¯745.9 pg/mL, p <0.001), whereas LUMIPULSE t-Tau was increased in AD (768.2 ±â€¯281.0 pg/mL vs. 337.5 ±â€¯159.1 pg/mL, p < 0.001) compared to non-AD patients. Both LUMIPULSE Aß42 (AUC = 0.78, spec. = 0.74, sens. = 0.76) and t-Tau (AUC = 0.94, spec. = 0.93, sens. = 0.87) showed good accuracy in distinguish AD from non-AD and a high correlation with the manual ELISAs (r = 0.87, p < 0.001 and r = 0.92, p < 0.001, respectively). LUMIPULSE improves clinical accuracy in AD diagnosis, promoting the use of standardized values for CSF biomarkers with a good correlation with classical manual assays.

Expression and Function of Chemokines CXCL9-11 in Micturition Pathways in Cyclophosphamide (CYP)-Induced Cystitis and Somatic Sensitivity in Mice.

Changes in urinary bladder function and somatic sensation may be mediated, in part, by inflammatory changes in the urinary bladder including the expression of chemokines. Male and female C57BL/6 mice were treated with cyclophosphamide (CYP; 75 mg/kg, 200 mg/kg, i.p.) to induce bladder inflammation (4 h, 48 h, chronic). We characterized the expression of CXC chemokines (CXCL9, CXCL10 and CXCL11) in the urinary bladder and determined the effects of blockade of their common receptor, CXCR3, at the level urinary bladder on bladder function and somatic (hindpaw and pelvic) sensation. qRT-PCR and Enzyme-Linked Immunoassays (ELISAs) were used to determine mRNA and protein expression of CXCL9, CXCL10 and CXCL11 in urothelium and detrusor. In urothelium of female mice treated with CYP, CXCL9 and CXCL10 mRNA significantly (p ≤ 0.01) increased with CYP treatment whereas CXC mRNA expression in the detrusor exhibited both increases and decreases in expression with CYP treatment. CXC mRNA expression urothelium and detrusor of male mice was more variable with both significant (p ≤ 0.01) increases and decreases in expression depending on the specific CXC chemokine and CYP treatment. CXCL9 and CXCL10 protein expression was significantly (p ≤ 0.01) increased in the urinary bladder with 4 h CYP treatment in female mice whereas CXC protein expression in the urinary bladder of male mice did not exhibit an overall change in expression. CXCR3 blockade with intravesical instillation of AMG487 (5 mg/kg) significantly (p ≤ 0.01) increased bladder capacity, reduced voiding frequency and reduced non-voiding contractions in female mice treated with CYP (4 h, 48 h). CXCR3 blockade also reduced (p ≤ 0.01) hindpaw and pelvic sensitivity in female mice treated with CYP (4 h, 48 h). CXC chemokines may be novel targets for treating urinary bladder dysfunction and somatic sensitization resulting from urinary bladder inflammation.

Development of a pharmacodynamic biomarker to measure target engagement from inhibition of the NGF-TrkA pathway.

BACKGROUND: NGF signaling through TrkA triggers pathways involved in a wide range of biological effects. Clinical trials targeting either NGF or TrkA are ongoing to treat various diseases in the areas of oncology, neuroscience, and for pain, but there is no described measure of target engagement of TrkA in these studies. NEW METHOD: We have developed custom ELISA assays to measure NGF-induced phosphorylation of TrkA specific for rodent and human receptors. Optimized tissue processing methods allow for detection in both the brain and in skin. In addition, TrkB and TrkC assays have been in established to evaluate selectivity against other neurotrophin receptors. RESULTS: In a preclinical NGF-induced pain model, we show that pre-dosing with a TrkA inhibitor prevents phosphorylation of TrkA in the skin at a dose that is efficacious in reversal of thermal hypersensitivity. In addition, we show data in non-human primate and human skin supporting the potential use of this approach to enable translational target engagement. Comparison with existing methods: Existing methods involve animal models expressing TrkA tumors or injection of over-expressing TrkA recombinant cells into animals. Our method can measure target engagement in both normal and disease tissues in preclinical animal models and human skin. CONCLUSIONS: We have developed methods to assess target engagement for drug programs aimed at disrupting NGF-induced TrkA signaling. This includes preclinical determination of selectivity against other neurotrophin receptors and estimation of functional peripheral restriction. Preliminary data supports this method can be translated into a clinical pharmacodynamic readout using human skin biopsies.

Development and evaluation of indirect ELISAs for the detection of IgG, IgM and IgA1 against duck hepatitis A virus 1.

Duck hepatitis A virus 1 (DHAV-1) is the principal pathogen that causes duck viral hepatitis (DHV), a highly fatal infectious disease in ducklings. Given the importance of the humoral immune response in the clearance of DHAV-1, indirect enzyme-linked immunosorbent assays (I-ELISAs) to detect immune indices, including IgG, IgM and IgA1, were developed and evaluated in this study. The optimal concentrations of coating-antigen were 1.79µg/ml, 2.23µg/ml and 2.23µg/ml for IgG, IgM and IgA1, respectively. Meanwhile, the optimal dilutions of sera were 1:80, 1:40 and 1:40, respectively; and of the conjugates were 1:300, 1:1800 and 1:800, respectively. Based on these conditions, three linear regression equations, y=1.363+1.954x (r2=0.983), y=1.141+2.228x (r2=0.970) and y=1.103+1.559x (r2=0.995) were derived for IgG, IgM and IgA1, respectively. Analytical sensitivities of the new methods were 1:2560, 1:1280 and 1:640 for IgG, IgM and IgA1, respectively. The concordances between the I-ELISAs and serum-neutralization were 95.2% for IgG and IgA1, and 75% for IgM. Although there was a weak cross-reaction with DHAV-3 positive serum for the IgG and IgA1 tests, it didn't affect the ability to detect DHAV-1 specific antibodies. Thus, these new I-ELISAs were shown to be potentially convenient methods to survey the status of humoral immune response to DHAV-1.

Development and evaluation of baculovirus-expressed Chikungunya virus E1 envelope proteins for serodiagnosis of Chikungunya infection.

Population-based serosurveillance studies provide critical estimates on community-level immunity and the potential for future outbreaks. Currently, serological assays, such as IgG enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence tests (IIFT) based on the inactivated whole virus are used to determine past Chikungunya virus (CHIKV) infection. However, these commercially available tests have variable sensitivities. To develop and evaluate recombinant based CHIKV-specific IgG antibody capture ELISAs (GAC-ELISAs), baculoviruses carrying wild-type (E1-A226, named WT) or mutant (E1-A226V, named MUT) E1 envelope protein genes of CHIKV were generated. The seroreactivity of recombinant CHIKV WT and MUT envelope proteins were determined using residual blood, collected from CHIKV-confirmed patients. The sensitivities of both recombinant CHIKV envelope proteins were 83.0% as measured by GAC-ELISAs. The specificities of both recombinant proteins were 87.8%. These GAC-ELISAs were also able to detect the persistence of anti-CHIKV IgG antibodies up to 6 months after the disease onset, together with rise in sensitivities with increasing time. These results suggest that the baculovirus purified recombinant CHIKV envelope proteins react with anti-CHIKV IgG antibodies and may be useful in population-based seroprevalence surveys. In addition, these GAC-ELISAs offer good diagnostic value to determine the recent/past CHIKV infection status in non-endemic populations.

Adaptive Focused Acoustics (AFA) Improves the Performance of Microtiter Plate ELISAs.

We investigated the use of Adaptive Focused Acoustics (AFA) technology to improve the performance of microtiter plate enzyme-linked immunosorbent assays (ELISAs). Experiments were performed with commercially available AFA instrumentation and off-the-shelf 96-well microtiter plate sandwich ELISAs. AFA was applied over a range of acoustic energies, temperatures, and durations to the antigen/antibody binding step of an ELISA for measuring HIV-1 p24 in tissue culture samples. AFA-mediated antigen/antibody binding was enhanced up to 2-fold over passive binding at comparable temperatures and was superior or comparable at low temperature (8-10 °C) to passive binding at 37 °C. Lower nonspecific binding (NSB), lower inter- and intra-assay coefficients of variation (CVs), higher Z' factors, and lower limits of detection (LODs) were measured in AFA-mediated assays compared with conventional passive binding. In a more limited study, AFA enhancement of antigen/antibody binding and lower NSB was measured in an ELISA for measuring IGFBP-3 in human plasma. We conclude from this study that application of AFA to antigen/antibody binding steps in microtiter plate ELISAs can enhance key assay performance parameters, particularly Z' factors and LODs. These features render AFA-mediated binding assays potentially more useful in applications such as high-throughput screening and in vitro diagnostics than assays processed with conventional passive antigen/antibody binding steps.