Enzyme-linked Immunosorbent Assay Reagents Research Articles

Blood serum biomarkers in the dynamics of treatment for cerebrovascular pathology in post-COVID-19 patients

Neurological manifestations after COVID-19 infections can be attributed to chronic immune and inflammatory reactions induced by SARS-CoV-2, as well as endothelial dysfunction and other vascular issues. This study included 24 patients with cerebrovascular disease (CVD) who had contracted COVID-19 1 – 1.5 years before the examination (experimental group) and 20 patients with CVD who had not previously been infected with the coronavirus (comparison group). Both groups were representative in terms of age and gender. Blood serum biomarkers, including interleukin (IL)-6, endothelin-1 (ET-1), and vascular endothelial growth factor A (VEGF-A), were tested before and after a 14-day standard complex therapy in the hospital at our institute using appropriate enzyme-linked immunosorbent assay reagent kits. Increases in the levels of IL-6 (by 20%), ET-1 (by 200%), and VEGF-A (by 100%) were observed in patients with CVD who had contracted COVID-19 1 – 1.5 years earlier compared to the comparison group. The effectiveness of a 2-week therapeutic intervention in patients with a history of COVID-19 was found only for VEGF-A: its serum level decreased twofold, leading to the normalization of this indicator. The concentrations of IL-6 and ET-1 were not affected by the treatment. In addition, the serum levels of ET-1 and VEGF-A in unvaccinated individuals exceeded those in vaccinated patients.

Clinical trial and performance evaluation of the Wantai HBsAg (CMIA) diagnostic kit for screening blood donors in China

In China, according to the ‘Technical Operating Procedures for Blood Stations (2019 Edition),’ blood stations are authorized to utilize Chemiluminescence Immunoassay (CLIA) to detect pathogen markers linked with transfusion-transmissible infections. However, currently, there is no approved CLIA reagent for the screening of blood-borne diseases in China, specifically for the detection of Hepatitis B surface antigen. The objective of this research project is to conduct a comprehensive evaluation of the performance of the Wantai Chemiluminescent Microparticle Hepatitis B surface antigen reagent. This study evaluates the performance of the Wantai Chemiluminescent Microparticle Immunoassay (CMIA) on the Wan200 + analyzer in screening for Hepatitis B Surface Antigen (HBsAg) in blood samples. The clinical trial component of this evaluation is included as part of the documentation submitted to the National Medical Products Administration (NMPA) of China for the approval of blood screening reagents. The evaluation plan of this study encompasses two main components: clinical trials and performance assessment. We adopted a controlled trial design, utilizing the WanTai Chemiluminescent Microparticle Immunoassay (CMIA) on the Wan200 + analyzer and the Enzyme-Linked Immunosorbent Assay (ELISA) to screen for Hepatitis B Surface Antigen (HBsAg) in routine blood donor samples and reference serum panel samples. To ensure the accuracy of the screening, we additionally employed Abbott's ELISA reagents and HBV DNA for validation. The assessment primarily focused on key performance indicators such as sensitivity, specificity, and analytical sensitivity. Moreover, this clinical trial data has been included as part of the submission to China's National Medical Products Administration (NMPA). In the clinical trials of this study, a total of 10,470 blood donor samples underwent simultaneous testing using both CMIA and ELISA methods. Across two clinical trials, there was remarkable concordance between CMIA and the two ELISA reagents, with Kappa values exceeding 0.82. Among the 269 samples that were double-reactive in the enzyme immunoassay (ELISA) tests, CMIA exhibited a 100% reactivity detection rate. However, CMIA produced 14 and 6 false-positive results in the respective clinical trials, resulting in specificities of 99.73% and 99.89%. In contrast, the specificities for Wantai ELISA and Xin Chuang ELISA were both greater than 99.94%.When testing samples in the gray zone serum plates, CMIA's detection limit significantly exceeded that of the two ELISA assays. CMIA had a detection cutoff of 0.05 IU/mL, while the two ELISA reagents had cutoffs of 0.1 IU/mL and 0.09 IU/mL, respectively. CMIA's detection limits for the adr and adw subtypes were 0.05 IU/mL, and for the ay subtype, it was 0.1 U/mL. The detection limit for 10 HBV mutant samples was 0.5 U/mL. In 165 cases where ELISA tested negative but HBV DNA tested positive, CMIA detected 5 HBsAg-positive samples. This study evaluated the performance of the Wantai CMIA in screening for HBsAg among blood donors. The results demonstrate outstanding performance of CMIA in both clinical trials and performance assessments, detecting all true positive samples with a sensitivity of 100%. It exhibits excellent concordance with the two ELISA assays. Of particular note is its superiority in early detection of HBsAg in the screening of early-stage hepatitis B infections, reducing the window period compared to ELISA. CMIA achieves a specificity exceeding 99.73% for negative blood donors, aligning with the European Union's standards for blood screening assay specificity. In summary, Wantai's CMIA displays high sensitivity and specificity in blood donor screening, making it suitable for screening blood donors in China.

Suggested blood donor deferral strategy regarding hepatitis B infections in China.

Hepatitis B virus (HBV) reactivity in individual immunologic and nucleic acid tests (NAT) tests does not represent the true infectious status of the blood donor. This study discusses the use of confirmatory tests to determine when deferral of blood donors is appropriate. HBsAg or HBV NAT reactive samples were confirmed via a neutralisation test. All the HBsAg reactive but neutralisation test negative samples were subjected to further anti-HBc testing. The receiver operating characteristic curve was used to obtain the best threshold value using signal-to-cut-off ratios of two HBsAg enzyme-linked immunosorbent assay reagents. Of the 780 HBV reactive samples collected, there were 467 HBsAg reactive but HBV DNA negative samples, of which 65 (13.92%) and 402 (86.08%) were neutralisation test positive and negative, respectively. Of the 402, 91 samples (30% of tested samples) were anti-HBc reactive. HBV DNA positive specimens negative by virus neutralisation were >80% HBcAg positive. A screening strategy was proposed for Chinese blood collection agencies. These findings suggest that adopting a screening algorithm for deferring HBV reactive blood donors based on HBsAg and NAT testing followed with HBsAg S/CO consideration and HBcAg testing can be both safe and feasible in China.

First Report of Beet western yellows virus Infecting Wheat in China.

Wheat (Triticum aestivum L.) is an important cereal crop widely cultivated worldwide. Viral disease is a major threat to wheat yield. In April 2022, fifteen winter wheat plants with yellowing and stunting symptoms were collected from wheat fields in Jingjiang, Jiangsu Province. Total RNA of each sample was extracted, and RT-PCR was performed using two pairs of degenerate luteovirus primers Lu-F (5'-CCAGTGGTTRTGGTC-3') and Lu-R (5'-GTCTACCTATTTGG-3'), Leu-F (5'-GCTCTAGAATTGTTAATGARTACGGTCG-3') and Leu-R (5'-CACGCGTCN ACCTATTTNGGRTTNTG-3'). Amplicons with the expected size were obtained from 10 of the 15 samples (using primers Lu-F/Lu-R) and 3 of the 15 samples (using primers Leu-F/Leu-R), respectively. These amplicons were cloned into the pDM18-T vector (TaKaRa) for sequencing. Blastn alignment showed that 10 amplicons (531 bp) from Lu-F/Lu-R primers were essentially identical to one another, which shared 99.62% nucleotide sequence identity to barley yellow dwarf virus-PAV (BYDV-PAV) isolate GJ1 from Avena sativa in South Korea (LC550014). Three amplicons (635 bp) from Leu-F/Leu-R primers had 99.68% nucleotide identity to the corresponding part of an isolate of beet western yellows virus (BWYV) from saffron (Crocus sativus) in China (MG002646). Among the 13 virus-positive samples, none were co-infected by BYDV-PAV and BWYV. Then, amplification using BWYV-specific primers (BWYV-F: 5'-TGCTCCGGTTTTGACTGGAGTGT-3', BWYV-R: 5'-CGTCTACCT ATTTTGGGTTGTGG-3') generated a 1409 bp product, corresponding to the partial sequence of the viral RNA-dependent RNA polymerase gene and complete sequence of the coat protein (CP) gene. The sequences (GenBank accession no. ON924175) of amplicons from 3 BWYV samples were identical to one another, which shared 98.41% nucleotide identity to BWYV isolate Hs from Japanese hop (Humulus scandens) in China (KC210049). The predicted coat protein of the BWYV wheat isolate had 99.51% nucleotide and 100% amino acid identity to BWYV isolate Hs. BWYV infection on wheat samples was also verified by dot-nucleic acid hybridization using a digoxigenin-labeled cDNA probe against the CP gene, as described previously (Liu et al. 2007). Further, RNA-positive samples were tested by enzyme-linked immunosorbent assay (ELISA) using the ELISA reagent kit for BWYV (Catalog No. KS19341, Shanghai Keshun Biotech, Shanghai, China); test results were also BWYV-positive, confirming that both BWYV nucleic and coat protein are present in these wheat samples. BYDV-PAV is a common wheat virus (Chay et al. 1996), while BWYV has never been reported to infect wheat. BWYV, an aphid-transmitted virus belonging to the Polerovirus genus, has an extensive host range, including over 150 plant species from 23 dicotyledonous families, such as Beta vulgaris, Spinacia oleracea, Lactuca sativa, and Brassica oleracea var. italica (Duffus 1964, 1973; Russell 1965; Beuve et al. 2008). In addition, BWYV was reported to infect a monocotyledonous plant, Crocus sativus (Iridaceae) (Zheng et al. 2018). To our knowledge, this is the first report of BWYV in wheat or any other Gramineae crop. The result also implies that BWYV has a potential risk to cereal crops in the field.

A New Insight Into Pathophysiological Mechanism of Abdominal Aortic Aneurysm With Novel Parameters Salusin-β and Arterial Stiffness

Abdominal aortic aneurysm (AAA) has risk factors similar to those of atherosclerosis. Salusin-β and arterial stiffness are novel parameters that have been shown to predict atherosclerosis and related cardiovascular disorders. However, their predictive value for detecting AAA remains unclear. Forty-eight patients with AAA and 47 age- and sex-matched participants without AAA were enrolled in the study. Arterial stiffness parameters were obtained via an oscillometric Mobil-O-Graph PWA Monitor device (IEM GmbH) with integrated ARCSolver software (Australian Institute of Technology). Plasma salusin-β levels were analyzed using an enzyme-linked immunosorbent assay reagent kit (Abbkine, Inc). The measured salusin-β levels and arterial stiffness parameters of the AAA and control groups were compared. Salusin-β levels were significantly lower in patients with AAA (P = .014). There was a significant negative correlation between salusin-β levels and abdominal aorta diameter. No significant difference was detected between AAA and control groups in terms of arterial stiffness parameters (P > .05). In backward multiple regression analysis, the presence of AAA, platelet count, and augmentation index were found to be independent predictors of salusin-β levels (P = .006 and P = .023, respectively). Arterial stiffness parameters were not found to be associated with AAA. Contrary to previous results regarding atherosclerosis and related cardiovascular disorders, salusin-β levels were found to be lower in patients with AAA. Although AAA is thought to have similar risk factors as atherosclerosis, the exact pathophysiologic mechanism remains unclear.

Selection of a SARS-CoV-2 antibody quantification method and development of an antibody reference standard for ELISA to test immunoglobulin preparations

The development of COVID-globulin, a COVID-19-specific human immunoglobulin preparation, involved choosing a method to quantify antibodies to SARS-CoV-2. Antibody titre determination by virus neutralisation (VN) is labour-intensive and unsuitable for large-scale application. To enable routine testing, it was necessary to develop a less demanding method; the enzyme-linked immunosorbent assay (ELISA) was the most appropriate of solutions. The lack of international and industry reference standards (RS) prompted the preparation and certification of an RS for COVID-globulin potency control.The aim of the study was to examine the possibility of substituting ELISA for VN and to develop an RS for SARS-CoV-2 antibody quantification in immunoglobulin preparations.Materials and methods: the authors used commercial ELISA kits by several manufacturers, COVID-globulin by Microgen (48 batches), and human plasma samples from multiple sources (1499 samples). The tests were performed by VN, ELISA, and chemiluminescent microparticle immunoassay.Results: the authors validated an ELISA method for SARS-CoV-2 antibody quantification with the selected reagent kits by the National Medical Research Center for Hematology (NMRC for Hematology) and Euroimmun AG. The authors demonstrated the possibility of using ELISA instead of VN (with a correlation coefficient of more than 0.9). They developed and characterised an in-house RS for SARS-CoV-2 antibody content in human immunoglobulin preparations. The RS was certified in newly introduced anti-COVID units (ACU) and in international binding antibody units (BAU) using the World Health Organisation (WHO) international reference panel (NIBSC code: 20/268). The RS's potency was measured in terms of its neutralising activity in ACU (320 ACU/mL) and BAU (2234.8 BAU/mL). The authors established the relationship between ACU and BAU units. For the selected ELISA reagent kits, the conversion factors were 6.4 (NMRC for Hematology) and 7.0 (Euroimmun AG).Conclusions: the ELISA method for SARS-CoV-2 antibody quantification and the RS for SARS-CoV-2 antibody content can be applied to determine the potency of human anti-COVID-19 immunoglobulins.

Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform.

Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.

Does interaction of monoclonal antibody charge variants with VEGF-A and ELISA reagents affect its quantification?

The ability of an enzyme linked immunosorbent assay (ELISA) to measure the concentration of charge variants of a monoclonal antibody (mAb) biotherapeutic has been investigated. Percentage recovery was found to be in the range of 80%–120% with inter and intra assay variation between 5% and 15%. Linear regression analysis of ELISA output at different dilutions yielded a good fit (R2 > 0.98). Assay output was not hindered by the presence of serum proteins and other analytes such as GCSF, demonstrating acceptable precision and specificity. It was concluded that the charge variants of the mAb can be accurately quantified by ELISA.

Two Potential Clinical Applications of Origami-Based Paper Devices.

Detecting small amounts of analyte in clinical practice is challenging because of deficiencies in specimen sample availability and unsuitable sampling environments that prevent reliable sampling. Paper-based analytical devices (PADs) have successfully been used to detect ultralow amounts of analyte, and origami-based PADs (O-PADs) offer advantages that may boost the overall potential of PADs in general. In this study, we investigated two potential clinical applications for O-PADs. The first O-PAD we investigated was an origami-based enzyme-linked immunosorbent assay (ELISA) system designed to detect different concentrations of rabbit IgG. This device was designed with four wing structures, each of which acted as a reagent loading zone for pre-loading ELISA reagents, and a central test sample loading zone. Because this device has a low limit of detection (LOD), it may be suitable for detecting IgG levels in tears from patients with a suspected viral infection (such as herpes simplex virus (HSV)). The second O-PAD we investigated was designed to detect paraquat levels to determine potential poisoning. To use this device, we sequentially folded each of two separate reagent zones, one preloaded with NaOH and one preloaded with ascorbic acid (AA), over the central test zone, and added 8 µL of sample that then flowed through each reagent zone and onto the central test zone. The device was then unfolded to read the results on the test zone. The three folded layers of paper provided a moist environment not achievable with conventional paper-based ELISA. Both O-PADs were convenient to use because reagents were preloaded, and results could be observed and analyzed with image analysis software. O-PADs expand the testing capacity of simpler PADs while leveraging their characteristic advantages of convenience, cost, and ease of use, particularly for point-of-care diagnosis.

Challenges of measuring accurate estradiol levels in aromatase inhibitor-treated postmenopausal breast cancer patients on vaginal estrogen therapy.

Breast cancer patients who are taking adjuvant Aromatase Inhibitor (AI) therapy typically have extremely low estradiol levels, which are undetectable by routine clinical laboratories. Thus, it becomes difficult to assess the safety of interventions such as low‐dose vaginal estrogen, which may increase estradiol levels. In this study, we aimed to assess the utility of enzyme‐linked immunosorbent assay (ELISA) to measure low estradiol concentrations in breast cancer survivors on AI therapy treated with either vaginal estrogen or lubricant for atrophic vaginitis as a part of clinical trial. The samples were tested using two independent ELISA kits. Some of the samples were also evaluated using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) for comparison. We found that while the results by ELISA were reproducible, they were not accurate when compared to LC‐MS/MS. It is possible that medications or supplements may cross‐react with the ELISA reagents and confound the assessment; however, those were often not the reason for the discrepancy. Our results highlight the need for developing novel, reliable, and clinically accessible assays to measure ultra‐low estradiol levels to improve care of breast cancer survivors. At this stage, based on our findings, we recommend using MS‐based assays for estradiol quantitation for breast cancer survivors, whenever necessary.

A fully integrated distance readout ELISA-Chip for point-of-care testing with sample-in-answer-out capability

Enzyme-linked immunosorbent assay (ELISA) is a popular laboratory technique for detection of disease-specific protein biomarkers with high specificity and sensitivity. However, ELISA requires labor-intensive and time-consuming procedures with skilled operators and spectroscopic instrumentation. Simplification of the procedures and miniaturization of the devices are crucial for ELISA-based point-of-care (POC) testing in resource-limited settings. Here, we present a fully integrated, instrument-free, low-cost and portable POC platform which integrates the process of ELISA and the distance readout into a single microfluidic chip. Based on manipulation using a permanent magnet, the process is initiated by moving magnetic beads with capture antibody through different aqueous phases containing ELISA reagents to form bead/antibody/antigen/antibody sandwich structure, and finally converts the molecular recognition signal into a highly sensitive distance readout for visual quantitative bioanalysis. Without additional equipment and complicated operations, our integrated ELISA-Chip with distance readout allows ultrasensitive quantitation of disease biomarkers within 2h. The ELISA-Chip method also showed high specificity, good precision and great accuracy. Furthermore, the ELISA-Chip system is highly applicable as a sandwich-based platform for the detection of a variety of protein biomarkers. With the advantages of visual analysis, easy operation, high sensitivity, and low cost, the integrated sample-in-answer-out ELISA-Chip with distance readout shows great potential for quantitative POCT in resource-limited settings.

Comprehensive analysis of single comparison and composite detection on samples with HBsAg around ELISA cut-off value

Objective To investigate the retesting borderline range of hepatitis B surface antigen (HBsAg) negative samples and the reexamination range of weakly positive samples above the critical value determined with enzyme-linked immunosorbent assay (ELISA), and the performance of single comparison with electrochemiluminescence immunoassay (ECLIA) qualitative detection and two half-and-half composite detection with latex immunochromatographic assay. Methods Serum HBsAg was detected with ELISA reagents made in China. Negative samples were retested with ELISA. Samples with HBsAg around ELISA cut-off value were compared with ECLIA qualitative detection and two half-and-half detections of the borderline samples were made with latex immunochromatographic assay. Results HBsAg determination around ELISA cut-off value samples were of a certain degree of false negative (positive) rate. Anti-HBs positive 682/1020 (66.86%) and all negative 345/1020 (33.82%) in one side of 0.073 5≤ A<0.105 were found in two half-and-half examinations with latex immunochromatographic test, and in another 0.105 ≤ A<1.000 side anti-HBe or/and anti-HBc positive 329/333 (including 36 cases with HBeAg positive) (99.80%) were major. Conclusions HBsAg ELISA borderline samples would be carried on qualitative detection with ECLIA as a comparison. If comprehensive analysis of two half-and-half examinations with latex immunochromatographic test were used, retesting of the most boardline samples would be omitted. Key words: Enzyme-linked immunosorbent assay; Hepatitis B surface antigens/AN

Use of Heavy Water (D2O) in Developing Thermostable Recombinant p26 Protein Based Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Equine Infectious Anemia Virus Infection

Thermostabilizing effect of heavy water (D2O) or deuterium oxide has been demonstrated previously on several enzymes and vaccines like oral poliovirus vaccine and influenza virus vaccine. In view of the above observations, effect of heavy water on in situ thermostabilization of recombinant p26 protein on enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of equine infectious anemia virus (EIAV) infection was investigated in the present study. The carbonate-bicarbonate coating buffer was prepared in 60% and 80% D2O for coating the p26 protein in 96-well ELISA plate and thermal stability was examined at 4°C, 37°C, 42°C, and 45°C over a storage time from 2 weeks to 10 months. A set of positive serum (n = 12) consisting of strong, medium, and weak titer strength (4 samples in each category) and negative serum (n = 30) were assessed in ELISA during the study period. At each time point, ELISA results were compared with fresh plate to assess thermal protective effect of D2O. Gradual increase in the stabilizing effect of 80% D2O at elevated temperature (37°C < 42°C < 45°C) was observed. The 80% D2O provides the thermal protection to rp26 protein in ELISA plate up to 2 months of incubation at 45°C. The findings of the present study have the future implication of adopting cost effective strategies for generating more heat tolerable ELISA reagents with extended shelf life.

Ring test evaluation of the repeatability and reproducibility of a Porcine reproductive and respiratory syndrome virus oral fluid antibody enzyme-linked immunosorbent assay

The precision of a Porcine reproductive and respiratory syndrome virus (PRRSV) oral fluid antibody enzyme-linked immunosorbent assay (ELISA) was evaluated by calculating reliability coefficients for assay repeatability (within laboratory) and assay reproducibility (between laboratories). Randomly ordered oral fluid samples of known (n = 39) and unknown (n = 224) PRRSV antibody status were tested in 12 diagnostic laboratories. Each laboratory tested the samples twice, first using an antibody ELISA kit and reagents provided to them (phase 1) and then using an ELISA kit and reagents configured in their respective laboratory (phase 2). Repeatability (within laboratory) reliability coefficients calculated using results from samples of known PRRSV antibody status ranged from 0.724 to 0.997 in phase 1 and from 0.953 to 0.998 in phase 2. Reproducibility (between laboratories) reliability coefficients were calculated for 3 conditions: case 1--samples of unknown status (n = 224); case 2--samples of known status (n = 39), and case 3--all samples (n = 263). Among the 3 cases, reliability coefficients ranged from 0.937 to 0.964 in phase 1 and from 0.922 to 0.935 in phase 2. For case 3, it was estimated that 96.67% of the total variation in phase 1 and 93.21% in phase 2 could be attributed to the oral fluid samples themselves. Overall, the PRRSV oral fluid antibody ELISA was highly repeatable and reproducible. The current study supports the routine use of this test in laboratories providing diagnostic service to pig producers.

A High-Throughput Microchromatography Platform for Quantitative Analytical Scale Protein Sample Preparation

Protein analysis (using either specific protein quantitation by methods such as HPLC and immunoassays or structural analysis by methods such as LC-MS) usually requires significant sample preparation, including quantitative purification of the target protein from complex sample matrices and potentially enzymatic treatment or labeling. We have developed platform for high-throughput microchromatography, capable of running 96 or more small volume samples in parallel, producing from 10 pg to 100 μg of purified protein from each sample. The platform is based on disposable cartridge devices with 5 μL packed bed of resin. The cartridges may be operated as spin columns or run on a modified 96-channel liquid handler with ultra-low dead volume syringes that directly connect to the cartridges, providing very precisely controlled positive-displacement flow control. A major application is quantitative purification of target proteins using affinity or physical chromatography. Using large diameter nonporous beads, standard microplate enzyme-linked immunosorbent assay reagents can be used to perform 30-min immunoassays. Enzymatic digestion methods have also been developed on the system for application in glycan profiling.

A simple approach to improve the sensitivity of enzyme-linked immunosorbent assay: Using the IMAPlate 5RC96 for result readout

In this article, we describe a new enzyme-linked immunosorbent assay (ELISA) setup to improve the sensitivity of commercial or homemade ELISAs. In the new ELISA setup, an IMAPlate 5RC96, a disposable multi-utility lab device developed by NCL New Concept Lab is used as a self-uptaking microcuvette array to read out the result of the ELISA that is performed in the normal 96-well plate with reduced substrate solution and stop solution. A commercial interleukin-6 (IL-6) ELISA reagent kit was used for the evaluation. Compared with the conventional ELISA setup, the new ELISA setup could easily increase the absorbance values by up to more than 10-fold. Therefore, the sensitivity (change in absorbance/change in concentration [DeltaAbs/DeltaConc]) is increased accordingly. In addition, methods to extend the upper detection limit of plate readers for the IMAPlate 5RC96 are described. This new ELISA setup may be more notable for the approach employed than for the specific analyte. It should generally be applicable to any conventional ELISA and should serve as an example of a simple solution that increases the detection sensitivity and/or detection range of other assays as well. We expect the approach to have a substantial practical impact on analytical methods and to accelerate discovery, research, and application of analytes at low concentration in life sciences and diagnostics.

Multiple Modes for Conferring Surface Expression of Homomeric β1 GABAA Receptors

The gamma-aminobutyric acid type A (GABA(A)) receptor assembles from individual subunits to form ligand-gated ion channels. Human (h) beta3 subunits assemble to form homomeric surface receptors in somatic cells, but hbeta1 subunits do not. We have identified three distinct sets of amino acid residues in the N-terminal extracellular domain of the hbeta1 subunit, which when mutated to the homologous residue in hbeta3 allow expression as a functional homomeric receptor. The three sets likely result in three modes of assembly. Mode 1 expression results from a single amino acid change at residue hbeta1 Asp-37. Mode 2 expression results from mutations of residues between positions 44 and 73 together with residues between positions 169 and 173. Finally, mode 3 results from the mutations A45V and K196R. Examination of homology-based structural models indicates that many of the residues are unlikely to be involved in physical inter-subunit interactions, suggesting that a major alteration is stabilization of an assembly competent form of the subunit. These mutations do not, however, have a major effect on the surface expression of heteromeric receptors which include the alpha1 subunit.

Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 microL hemolymph from an infected shrimp and differentiated it from a healthy shrimp. [figure: see text]

Acylation Determines the Toll-like receptor (TLR)-dependent Positive Versus TLR2-, Mannose Receptor-, and SIGNR1-independent Negative Regulation of Pro-inflammatory Cytokines by Mycobacterial Lipomannan

Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria.

An ELISA for detection of infectious bursal disease virus and differentiation of very virulent strains based on single chain recombinant chicken antibodies

Two chicken single-chain variable antibody fragments (scFv) designated scFv154 and scFv88, previously shown to react with either all or very virulent (vv) infectious bursal disease virus (IBDV) strains, respectively, were evaluated for use in an enzyme-linked immunosorbent assay (ELISA) for differentiation of vvIBDV. Specificity and sensitivity of the vvIBDV ELISA was assessed when scFv154 and scFv88 were expressed as soluble antibodies (sAb), phage antibodies (pAb) or hyper-phage antibodies (hpAb). The highest test sensitivity and specificity was obtained using hpAb154 to detect all IBDV and pAb88 to differentiate vvIBDV strains. Such an ELISA was eight to 16 times more sensitive for IBDV antigen detection than the mouse monoclonal antibody ELISA. Using field samples, the scFv ELISA was able to differentiate between flocks infected with vvIBDV and those infected with classical or variant IBDV. In one instance IBDV was detected in a flock found to be negative by the monoclonal antibody ELISA. The results showed that scFv can be utilized as highly specific and sensitive ELISA reagents for the detection and discrimination of avian pathogens.