Enzyme-linked Immunosorbent Assay System Research Articles

Digital Quantification and Ultrasensitive Detection of Single Influenza Virus Using Microgel-in-Droplet Enzyme-Linked Immunosorbent Assay.

Detection and quantification of viral particles (VPs) facilitate both diagnostics of pathogenic viruses and quality control testing of virus-based products. However, existing technologies fail to afford concurrent ultrasensitive detection and large-scale absolute quantification of VPs. Here, we propose a digital Microgel-in-Droplet enzyme-linked immunosorbent assay (ELISA) system that enables the processing and monitoring of millions of ELISA reactions at the single-VP level by incorporating droplet microfluidics with sandwich ELISA. Upon validating the microfluidic workflow and optimizing ELISA parameters, we demonstrate ultrasensitive VP detection at a limit of detection of 56 PFU/test. Leveraging a fluorescence-based screening platform, we further realize high-throughput digital counting of VPs with a linear detection range of 500-64 000 PFU/test. The precision is comparable to that of the gold standard, the plaque assay, across a wide range of virus concentrations. We anticipate that our system will provide a novel paradigm for the absolute enumeration of various types of viral particles.

Optimizing antigen preparation for oxalyl-CoA decarboxylase enzyme diagnostic kit and ELISA system cutoff determination.

The prevalence of kidney stone disease is increasing globally, with calcium oxalate stones being the most common type. Oxalyl-CoA decarboxylase (OXC), an enzyme produced by the gut bacterium Oxalobacter formigenes, plays a crucial role in oxalate metabolism. Deficiencies in OXC activity can lead to the accumulation of oxalate, contributing to kidney stone formation. This study aimed to develop a reliable diagnostic assay for OXC by optimizing antigen production and establishing a cutoff value for an enzyme-linked immunosorbent assay (ELISA). We cloned, expressed, and purified recombinant OXC protein in Escherichia coli BL21(DE3), and generated specific polyclonal antibodies in rabbits. The ELISA system was optimized and validated using serum samples from 40 healthy individuals and 6 patients with oxalate-related disorders. The cutoff value was determined using the formula (M + 2SD), where (M) is the mean and (SD) is the standard deviation of the healthy sample results. The calculated cutoff value of 0.656750 effectively distinguished between healthy and affected individuals, with a sensitivity of 97.5% and a specificity of 83.3%. These findings provide a valuable tool for the early detection and management of oxalate-related disorders, with significant implications for clinical practice.

Development of two novel ELISAs based on Prussian blue nanoparticles for ultrasensitive detection of norfloxacin in milk

The development of traditional enzyme-linked immunosorbent assay (ELISA) systems for the detection of small-molecule residues in foods is limited because of the poor stability of biological enzymes and the lower sensitivity of absorption-based signals. Herein, two ELISAs based on Prussian blue nanoparticles (PBNPs) were developed to establish ultrasensitive and stable methods for detecting norfloxacin (NOR) in milk. The results show that the detection limit (IC15) of NOR was 0.77 μg L−1 and the sensitivity (IC50) was 18.28 μg L−1 in the standard solution using the PBNPs-based nano-ELISA. When the fluorescence quenching ELISA based on PBNPs was used, the detection limit was 0.06 μg L−1 and the sensitivity was 4.21 μg L−1 in the standard solution. The recoveries and precision were good, as confirmed by analysis of real milk samples. The results were consistent with those of commercial ELISA kits, indicating the high accuracy of these two methods.

Monoclonal Antibodies for Rift Valley Fever Virus Nucleocapsid: Application in IgG/IgM ELISA for Sero-Diagnosis.

Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley fever (RVF). Currently, RVFV has spread from Africa to Asia, and due to its ability to cause high mortality rates, it has significantly impacted human health and economic development in many societies. Highly specific and sensitive systems for sero-diagnosis of RVFV infection are needed for clinical use. BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of IgG and IgM using the new MAbs were established and evaluated. Serum samples from 96 volunteers and 93 patients of suspected RVF from Kenya were tested compared with the ELISA systems based on inactivated viruses and the rabbit polyclonal antibody. Three monoclonal antibodies against rRVFV-N protein were established. The performance of the MAb-based sandwich IgG ELISA and the IgM capture ELISA perfectly matched the ELISA systems using the inactivated virus or the polyclonal antibody. Recombinant RVFV-N protein-specific MAbs were developed and they offer useful tools for RVFV studies. The MAb-based ELISA systems for detecting IgG and IgM offer safe and useful options for diagnosing RVFV infections in humans.

Hsa_Circ_002144 Promotes Glycolysis and Immune Escape of Breast Cancer Through miR-326/PKM Axis.

Background: Breast cancer is a leading cause of cancer-related deaths in women worldwide, posing a significant threat to female health. Therefore, it is crucial to search for new therapeutic targets and prognostic biomarkers for breast cancer patients. Method: Bioinformatics analysis, quantitative real-time PCR (qRT-PCR), and fluorescence in situ hybridization (FISH) were employed to investigate the expression of hsa_circ_002144 in breast cancer. Transwell assay, Western blotting, and cell viability assay were utilized to assess the impact of hsa_circ_002144 on the proliferation, migration, and invasion of breast cancer cells. Additionally, a mouse model was established to validate its functionality. Flow cytometry, WB analysis, enzyme-linked immunosorbent assay (ELISA), qRT-PCR, exosomes isolation, and co-culture system were employed to elucidate the molecular mechanism underlying macrophage polarization. Result: we have discovered for the first time that hsa_circ_002144 is highly expressed in breast cancer. It affected tumor growth and metastasis and could influence macrophage polarization through the glycolytic pathway. Conclusion: This finding provides a new direction for breast cancer treatment and prognosis assessment.

A novel enzyme-linked immunosorbent assay tool to evaluate plasma soluble CD226 in primary Sjögren's syndrome

CD226 is an important receptor constitutively expressed on most immune cells, performing vital functions in immune responses. However, the levels of soluble CD226 (sCD226) and its roles in primary Sjögren syndrome (pSS) remain unclear. In this study, we developed two novel mouse anti-human CD226 monoclonal antibodies (mAbs) and established a novel sandwich enzyme-linked immunosorbent assay (ELISA) system, which proved to be highly effective in detecting human sCD226. We then analyzed the expression of sCD226 in the plasma of pSS patients. Our results showed that the levels of sCD226 were significantly lower in patients with pSS compared to healthy controls. The significant decline was also observed in active group and the patients with high levels of IgG or positive anti-SSB. Additionally, reduced sCD226 was found to be negatively correlated with the disease activity of pSS and several clinical manifestations, including arthralgia, fatigue, decayed tooth and interstitial lung disease (ILD). Furthermore, receiver operator characteristics (ROC) curve analysis showed that sCD226 displayed outstanding capacity in discriminating pSS and predicting the disease activity. Altogether, plasma sCD226 emerges as a promising candidate for diagnostic markers in the context of pSS.

Influence of Abutments Surface (machined vs. laser-microgrooved) in Soft-tissue Response During One Year of Function: Clinical and Biochemical Outcomes of a RCT with Split-Mouth Design

The purpose of this study was to evaluate peri-implant soft tissue response by assessing IL-6, IL-1b and MMP-8 levels in peri-implant crevicular fluid (PICF) around machined vs. laser-microgrooved implants/healing/prosthetic abutments during 1 year of function. Twenty-four patients each received 2 one-stage implants in a split mouth design on the same jaw. In each patient, one implant, one immediate healing, and one prosthetic abutment with a machined surface (M group), and one implant, one immediate healing abutment and one prosthetic abutment with a laser-microgrooved surface (LMS group) were used. PICF sampling, pocket probing depths (PPD) and bleeding on probing (BOP) were assessed at 1, 3, and 12 months. IL-6, IL-1b and MMP-8 levels were determined by specific enzyme-linked immunosorbent assay systems (ELISA). Repeated measure ANOVA was used to run comparisons with groups and between groups months at 1, 3, and 12 months. At 3 and 12 months, the LMS group showed significantly lower PD, BOP and IL-6, IL-1β and MMP-8 levels than the M group (P<.05). This study suggests the presence of more remodeling and/or inflammatory phenomena around implants/abutments with a machined surface than around implants/abutments with a laser-microgrooved surface.

The Potential Mechanism of the Anti-Liver Fibrotic Effect of Curcumin in the Gut-Liver Axis.

This study aimed to explore the curative effect of curcumin on liver fibrosis and its correlation with the gut-liver axis in animal models. Histological staining was utilized to conduct histological analysis of the liver and intestine. An automatic biochemical analyzer or enzyme-linked immunosorbent assay system was utilized for analyzing the biochemical indexes in mice. Western blotting was employed to examine the level of relevant proteins. Furthermore, 16S rRNA high-throughput sequencing was performed to explore the impact of curcumin on intestinal microorganisms in rats with liver fibrosis. Ultrahigh-performance liquid chromatography with quadrupole-orbitrap mass spectrometry was utilized to analyze the effect of curcumin on rat feces metabolites. Our results showed that curcumin reduced the formation of collagen fibers caused by carbon tetrachloride in a dose-dependent manner. In addition, curcumin was able to restore intestinal permeability in rats with liver fibrosis. By adopting α diversity analysis (Chao 1 index, Shannon index, and Simpson index), we observed that both the diversity and the abundance of intestinal flora in rats with liver fibrosis were increased. The principal component analysis diagram demonstrated that curcumin could enhance the abundance and diversity of intestinal flora, and also restore the composition of model rat flora, which was similar to that in normal rats, thereby correcting the imbalance of flora in rats with liver fibrosis. In addition, curcumin regulated feces metabolites and their signaling pathways, including glycerophospholipid metabolism, pantothenate and CoA biosynthesis. Our findings suggest that curcumin exhibits antiliver fibrosis effects, and its antiliver fibrosis effects might correlate with gut-liver axis.

Development of an indirect ELISA system for diagnosis of porcine edema disease using recombinant modified Stx2e antigen.

This study aimed to develop a sensitive and specific recombinant antigen protein indirect enzyme-linked immunosorbent assay (ELISA) kit to detect the Shiga toxin (Stx)-producing Escherichia coli (STEC) antibodies against porcine edema disease (ED). The recombinant antigen was co-expressed with the STEC-derived Stx2e A2-fragment and Stx2e B protein in E. coli BL21(DE3) pLysS cells and purified using maltose-binding protein open columns. We used a Shiga-like toxin 2 antibody to test the specificity of the recombinant antigen in an indirect ELISA, which was detected in antigen-coated wells but not in uncoated wells. We tested the indirect ELISA system using samples from the STEC-immunized pig group, the commercial swine farm group, and healthy aborted fetal pleural effusion group; five and twenty samples, respectively, were positive for STEC in the former, whereas all three samples were negative for STEC in the latter. This newly developed indirect ELISA may be a specific method for diagnosing STEC infections in pigs.

Automatic microdispenser-integrated multiplex enzyme-linked immunosorbent assay device with autonomously driven centrifugal microfluidic system.

In this study, we established the control and design theory of an autonomously driven dispenser at a steady rotation speed and proposed a dispenser-integrated multiplex enzyme-linked immunosorbent assay (ELISA) device. In establishing the theory of the dispenser, we estimated the flow rate in the dispenser and the applied pressure onto the passive valves, so that the suitable burst pressure of the valves and flow rate could be designed. The dispenser-integrated multiplex ELISA device has the potential to perform flow control for executing an ELISA of 6 samples/standards per chip or 18 samples/standards per compact disk by just steadily rotating a chip. In the immunoassay evaluation of the device using mouse IgG detection, it was confirmed that the device could assay 5 μL of several standards in just 30 min without nonspecific reactions, and although this system has a high limit of detection (LOD, 63.4-164 pg mL-1) it is equal to that of manual assay with a titer plate. The device can be fabricated by transferring the microchannel pattern from a mold without complex assembly or alignment, and it can control the liquid operation by just steadily rotating. Thus, the device system developed will contribute to reducing the cost of fabricating chips and control equipment for ELISA systems. Consequently, a compact, portable, and low-cost ELISA system for point-of-care testing is expected to be realized.

Establishment and characterization of noro-VLP measurement by digital ELISA.

Highly sensitive viral analytical techniques are essential tools for preventing the spread of infections. In this study, we established a digital enzyme-linked immunosorbent assay (ELISA) system to quantify norovirus proteins with high sensitivity. We used norovirus-like particles (noro-VLPs) as a surrogate for norovirus and constructed two digital ELISA systems using two different antibody pairs. The quantitative performance of the noro-VLP measurement using each digital ELISA system was evaluated. Both assay systems exhibited high sensitivity, good linearity, and high stability. The first system exhibited a limit of detection (LOD) of 87 pg mL-1 and correlation coefficient (R2) of 0.9984. Analysis of samples containing 5 ng per mL noro-VLP confirmed inter-assay variation of 5.5%, and intra-assay variation of 5.2%. The second system exhibited an LOD of 19 pg mL-1 and R2 of 0.9984. Analysis of samples containing 5 ng per mL noro-VLP confirmed inter-assay variation of 4.5%, and intra-assay variation of 2.5%. Comparison of the two systems using the same calibrant for unpurified and fractionated noro-VLPs revealed that the quantitative values for unpurified noro-VLPs were the same, whereas those for fractionated noro-VLPs were dramatically different. Our findings indicate that the reactivity to various components in the noro-VLP solution was altered depending on the different antibodies. Furthermore, our study highlights the importance of using appropriate calibrants, which contain the same ratio of components as the noro-VLP analyte, to afford accurate measurements.

Enzyme-Linked Immunosorbent Assay for Antibodies Against the Tumor-Associated Antigen-Derived Cytotoxic T-Lymphocyte Epitope.

Antibodies serve as crucial indicators of the immune system in clinical tests. In therapeutic cancer vaccines, IgG antibodies against target antigens are vital for immune monitoring. Additionally, assessing baseline antigen-specific immune responses before cancer vaccine administration is possible by measuring IgM and IgG antibodies against the target antigen. To this end, we have developed an enzyme-linked immunosorbent assay (ELISA) system that detects and quantifies serum levels of IgG and IgM antibodies against the WT1 cytotoxic T-lymphocyte epitope peptide. The assay immobilizes the epitope peptide in a microplate to capture antigen-specific antibodies. Here, this article presents the details of our ELISA system to detect and measure antibodies against a tumor-associated antigen-derived cytotoxic T-lymphocyte epitope with high reproducibility. Detecting these antibodies has novel significance in the context of emerging critical roles of B lineage-cells in tumor immunity.

Functionalized Carbon Nanotube - Modified ELISA for Early Detection of Heart Attack

A warning issue of heart attacks in young adults needs immediate attention lately. Enzyme-linked immunosorbent assay (ELISA) is an easy and commonly used method for detecting early stages of heart attack. Cardiac troponin I (cTnI) is a responsible biomarker for acute myocardial infarction. However, the conventional ELISA system was only able to detect at 100 pM of cTnI. To improve the system, enhancements were introduced through the integration of functionalized carbon nanotube (fCNT) to amplify cTnI detection signals. By utilizing the advantage of fCNT, a noticeable improvement in results can be obtained. The detection limit was lowered down to an impressive 10 pM. Furthermore, the change of absorbance increased from 31.90% for conventional ELISA surge to 98.61 for modified ELISA system. This three-fold increase in sensitivity shows remarkable improvement through the introduction of fCNT in modified ELISA technique.

A single synthetic lipid antigen for improved serological diagnosis of Buruli ulcer

SETTING: The diagnosis of Buruli ulcer (BU) is frequently made by experienced health workers in rural regions. This leads to long turnaround times to confirm the diagnosis as it requires specialised laboratory infrastructure to perform confirmatory testing.BACKGROUND: Given the lack of success with protein antigens to detect BU in human sera, the aim of this study was to evaluate a range of single synthetic lipid antigens using an enzyme-linked immunosorbent assay (ELISA). The ELISA system used was initially developed to detect TB using single synthetic lipid antigens.METHODS: Thirty polymerase chain reaction (PCR) positive BU samples and 30 PCR-negative healthy contact samples collected from Asante Akim North and Ahafo Ano North Districts, Ghana, that are endemic for BU between 2013 and 2016 were used to evaluate the synthetic lipid antigen ELISA. A Quantikine ELISA was also conducted on a randomly blinded sub-set of 30 samples.RESULTS: The synthetic lipid ELISA evaluated here outperforms all other ELISA tests using protein antigens to detect BU to date and has shown potential as a fast (2 h) test for BU which may be adapted for use at the point of care. A sensitivity of 63% and specificity of 80% was observed for 30 BU-positive and 30 BU-negative samples, with significantly reduced interleukin-8 (IL-8) levels in a subset of patients with BU.CONCLUSION: A single lipid was shown for the first time to have the ability to distinguish between PCR-positive BU and negative sera using ELISA. The low lipid antibody load detected may be a result of immune suppression caused by the presence of mycolactone in patients with BU, given that levels of IL-8 were significantly reduced in patients with BU compared to the control serum samples.

B-052 Establishment of New Antibodies and ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine

Abstract Background Food poisoning by potato alkaloids, α-solanine (SO) and α-chaconine (CHA), currently occurs worldwide, however laboratory methods have not yet been established to detect these toxins in biological samples. In this study, we established new antibodies to detect SO and CHA and evaluated the practicality of enzyme-linked immunosorbent assays (ELISAs) using these antibodies in laboratory medicine. Methods Solanidine, a chemical compound found in both SO and CHA, was conjugated with bovine albumin, and this complex was repeatedly injected into two rabbits for two months. Two types of polyclonal solanidine antibodies were then purified from rabbit serum using affinity column chromatography. Using these antibodies, two forms of direct ELISAs (Sold ELISA-1, 2) were constructed. The basic detection performance of the ELISAs was confirmed by measuring the levels of SO and CHA diluted in phosphate-buffered saline (PBS). To simulate biological samples from patients with potato food poisoning, SO and CHA powders were mixed with commercially available human serum and urine, and SO and CHA levels were then measured using ELISA. Lastly, SO and CHA levels were detected in extracts from rotten potato (Irish Cobbler) sprouts, peels, and tubers. Results In samples of SO diluted in PBS, the limit of quantification (LoQ) was determined as 2.56 ng/mL and 9.55 ng/mL for Sold ELISA-1 and -2, respectively. In CHA diluted in PBS, the LoQs of Sold ELISA-1 and -2 were 1.36 ng/mL and 4.8 ng/mL, respectively. The LoQs of SO in the serum were calculated as 14.25 ng/mL and 19.41 ng/mL using Sold ELISA-1 and -2, respectively. In contrast, the LoQs of CHA in the serum were 12.48 ng/mL and 16.92 ng/mL using Sold ELISA-1 and -2, respectively. The LoQs of SO in the urine were determined as 30.28 ng/mL and 45.16 ng/mL using Sold ELISA-1 and -2, respectively. Conversely, the LoQs of CHA in the urine were 22.92 ng/mL and 38.15 ng/mL using Sold ELISA-1 and -2, respectively. Using Sold ELISA-1, both SO and CHA in the extracts from rotten potato tubers and peels were detected as approximately 1.7- and 2.3-fold higher, respectively, in each part than those in the extracts from fresh potatoes. When both SO and CHA were measured in the same samples using Sold ELISA-2, the levels were approximately 1.6- and 2.4-fold higher, respectively, in the tubers and peels than those in fresh potato samples. Furthermore, the potato sprout extract contained approximately 80-fold more SO and CHA than the tuber and 8-fold more than the peel. Discussion and Conclusion Based on our results, Sold ELISA-1 presented greater performance in detecting SO and CHA than Sold ELISA-2. The non-specific reactions in the serum samples and influence of urea in the urine samples may underlie the reduced detection performance of SO and CHA in ELISA, respectively. Although the sensitivity depends on the sample type, these ELISAs may be effective as future clinical and food testing methods.

Breast Cancers Secreting Sialyl-fibronectin Are Less Likely to Cause Epithelial-mesenchymal Transition and Have Good Prognoses.

Elevated blood fibronectin (FN) levels have been observed in various cancers; however, their significance is controversial. We measured sialyl-fibronectin (S-FN), a type of FN secreted by tumor cells in the blood, and investigated whether blood S-FN secretion is associated with cancer malignancy and recurrent metastases. We constructed an enzyme-linked immunosorbent assay (ELISA) system that recognizes S-FN as an antigen and measured the amount of S-FN secreted into the blood of 89 patients with breast tumors. The relationship between S-FN secretion and prognostic predictors was analyzed. Immunostaining was performed to identify the site of S-FN secretion in the breast tissue. Among the 82 patients, 21 (25.6%, 21/82) and 61 (74.4%, 61/82) were blood S-FN-positive and S-FN-negative, respectively. Regarding prognostic predictors, blood S-FN-positive and S-FN-negative patients showed significant difference in locoregional recurrence (p=0.026), remote metastases (p=0.049), and histological margins (p=0.001). Locoregional recurrence was associated with positive histological margins in S-FN-positive patients. However, remote metastases were associated with N-factor and histological classification (HC) in S-FN-negative patients. Furthermore, S-FN particles were detected in the cytoplasm of breast cancer cells through immunostaining. After the onset of recurrent metastases, two S-FN-positive and six S-FN-negative patients received anticancer drug treatment; however, further progression was observed in five S-FN-negative patients. S-FN-positive patients are less likely to develop distant metastases, have a better prognosis, and may be less resistant to therapeutic agents than S-FN-negative patients, which contain many epithelial-mesenchymal transition cells.

Urinary titin is not an early biomarker of skeletal muscle atrophy induced by muscle denervation in mice.

Early detection of skeletal muscle atrophy is important to prevent further muscle weakness. However, there are few non-invasive biomarkers for skeletal muscle atrophy. Recent studies have reported that the N-terminal fragment (N-titin) of titin, a giant sarcomeric protein, is detected in the urine of patients with muscle damage. In this study, we hypothesized that urinary N-titin would be a potential early biomarker of skeletal muscle atrophy in mice caused by sciatic nerve denervation. Male mice were randomly divided into control and denervation groups, and urinary N-titin levels were assessed daily for 9 days using an enzyme-linked immunosorbent assay system. Despite reduced titin protein levels in atrophic muscles 10 days after denervation, cleaved N-titin fragments were not increased in the urine of mice with denervation-induced muscle atrophy. Furthermore, we found no uptake of Evans blue dye from the extracellular space into the cytoplasm in atrophic muscles, suggesting that the sarcomeric membrane is intact in those muscles. The present results suggest that cleaved N-titin in the urine is not suitable as an early biomarker of skeletal muscle atrophy.

Development of a Novel Enzyme-Linked Immunosorbent Assay and Lateral Flow Test System for Improved Serodiagnosis of Visceral Leishmaniasis in Different Areas of Endemicity.

Visceral leishmaniasis (VL) is caused by protozoan parasites of the Leishmania donovani complex and is one of the most prominent vector-borne infectious diseases with epidemic and mortality potential if not correctly diagnosed and treated. East African countries suffer from a very high incidence of VL, and although several diagnostic tests are available for VL, diagnosis continues to represent a big challenge in these countries due to the lack of sensitivity and specificity of current serological tools. Based on bioinformatic analysis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed. The diagnostic performance of rKLi8.3 was evaluated by enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) on a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other diseases, including tuberculosis, malaria, and trypanosomiasis. The diagnostic accuracy of rKLi8.3 was compared with rK39 and rKLO8 antigens. The VL-specific sensitivity of rK39, rKLO8, and rKLi8.3 ranged from 91.2% over 92.4% to 97.1% and specificity ranged from 93.6% over 97.6% to 99.2%, respectively. In India, all tests showed a comparable specificity of 90.9%, while the sensitivity ranged from 94.7% to 100% (rKLi8.3). In contrast to commercial serodiagnostic tests, rKLi8.3-based ELISA and LFT showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer improved VL serodiagnostic efficiency in East Africa and other areas of endemicity. IMPORTANCE Reliable and field suitable serodiagnosis of visceral leishmaniasis (VL) in East Africa has until now been a big challenge due to low sensitivity and cross-reactivity with other pathogens. To improve VL serodiagnosis, a new recombinant kinesin antigen from Leishmania infantum (rKLi8.3) was developed and tested with a panel of sera from Sudanese, Indian, and South American patients diagnosed with VL or other infectious diseases. Both prototype rKLi8.3-based enzyme-linked immunosorbent assay (ELISA) and lateral flow test (LFT) showed improved sensitivity and no cross-reactivity with other parasitic diseases. Thus, rKLi8.3-based ELISA and LFT offer substantially increased diagnostic efficiency for VL in East Africa and other areas of endemicity, compared to currently commercially available serodiagnostic tests.

Development of New Antibodies and an ELISA System to Detect the Potato Alkaloids α-Solanine and α-Chaconine.

Food poisoning can be caused by the potato alkaloids α-solanine (SO) and α-chaconine (CHA). Therefore, this study aimed to establish new enzyme-linked immunosorbent assays (ELISAs) for detecting these two toxins in biological samples and potato extracts. Two antibodies that bind to solanidine, a chemical compound found in both SO and CHA, were newly developed, and two types of ELISAs (Sold1 ELISA and Sold2 ELISA) were constructed. We measured SO and CHA diluted in phosphate-buffered saline (PBS), serum, and urine. The detection performance of the two ELISAs for SO and CHA in PBS was higher than in serum and urine, and the sensitivity of Sold2 ELISA was lower than that of Sold1 ELISA. Thus, we used these ELISAs to measure SO and CHA in potato part extracts and found that potato sprouts contained approximately 80-fold more SO and CHA than tubers and 8-fold more SO and CHA than peels. Although the detection sensitivity of SO and CHA depends on the sample types, these ELISAs may be effective as future clinical and food testing methods after further improvements.

Antibiotic-enzyme-inorganic nanoflowers based immunoassay for the ultrasensitive detection of Staphylococcus aureus

In this work, we constructed a moderate and convenient approach for the determination of staphylococcus aureus (S. aureus) by using organic-inorganic flower-like hybrid nanoflowers and Pig IgG together in an enzyme-linked immunosorbent assay (ELISA) system. To ensure efficient capture, the hybrid nanoflowers were prepared by encapsulating horseradish peroxidase (HRP) and vancomycin (VAN) in the inorganic nanocrystal composites (calcium ion solution), just like the mimic biomineralization process. Owing to the self-assembly technique, the synthesized VAN-HRP-CaHPO4 nanoflowers (NFs) can not only retain the ability to particularly capture the gram-positive bacteria but also enhance the stability and enzymatic activity to achieve the signal output amplification. Then, taking advantage of the integration of signal amplification elements (HRP) and biorecognition unit (VAN), the VAN-HRP-CaHPO4 NFs were utilized as a new kind of capture & signal regent in the procedure of S. aureus detection. Based on this ELISA system, S. aureus could be clearly detected within the concentration ranging from 1.0 × 102 to 1.0 × 107 CFU mL−1. The detection limit was defined as 4.3 CFU mL−1, which performance is superior to some commercial ELISA kits. Additionally, this system detected the S. aureus in food samples and showed an acceptable recovery. As a cost-effective and sensitive platform, this proposed assay was enable to fulfill the requirement of a quick and effective detection of S. aureus.