Antibody-based Enzyme-linked Immunosorbent Assay Research Articles

Novel antibody- and aptamer-based approaches for sensitive detection of mycotoxin fusaric acid in cereal

This study presents the first successful generation of polyclonal antibodies (pAbs) and oligonucleotide aptamers specifically targeting fusaric acid (FA). Utilizing these pAbs and aptamers, three highly sensitive and specific assays were developed for the detection of FA in cereals with limits of detection (LOD) ranging from 5 to 50 ng/g: an antibody-based enzyme-linked immunosorbent assay (ELISA), an aptamer-based enzyme-linked aptamer-sorbent assay (ELASA), and a hybrid enzyme-linked aptamer-antibody sandwich assay (ELAAA). The recovery rates of FA in spiked cereal samples ranged from 87 % to 112 % across all assays. Analysis of 15 cereal feed samples revealed FA contamination levels of 459 to 1743 ng/g (ELISA), 427 to 1960 ng/g (ELASA), and 381 to 1987 ng/g (ELAAA). These results were further validated by HPLC analysis, confirming high consistency within developed assays. Overall, the ELISA, ELASA, and ELAAA are promising tools for the rapid detection of FA, significantly contributing to food safety monitoring.

DNA Meets Protein─Development, Characterization, and Application of Aptamers against Peanut Allergen.

Allergen detection methods support food labeling and quality assessment at the allergen component level of allergen preparations used for allergy diagnosis and immunotherapy (AIT). Commonly applied enzyme-linked immunosorbent assay (ELISA) requires animal antibodies but potentially shows batch variations. We developed synthetic aptamers as alternative binders in allergen detection to meet the replacement, reduction, and refinement (3R) principle on animal protection in science. ssDNA aptamers were specifically selected against the major peanut allergen Ara h 1 and identified by next-generation sequencing. Application in various detection systems (ELISA-like assays, western blot, and surface plasmon resonance) was demonstrated. The ELISA-like assay comprised a sensitivity of 10 ng/mL Ara h 1, comparable to published antibody-based ELISA, and allowed Ara h 1 detection in various peanut flours, similar to those used in peanut AIT as well as in processed food. This ELISA-like aptamer-based assay proofs antibody-free allergen detection for food labeling or quality assessment of diagnostic and therapeutic allergen products.

Monoclonal antibody-based enzyme-linked immunosorbent assay for quantification of majonoside R2 as an authentication marker for Nngoc Linh and Lai Chau ginsengs

BackgroundRecent years have witnessed increasing interest in the high amount of ocotillol-type saponin in Panax vietnamensis, particularly in relation to majonoside R2 (MR2). This unique 3%–5% MR2 content impart Ngoc Linh and Lai Chau ginsengs with unique pharmacological activities. However, in the commercial domain, unauthentic species have infiltrated and significantly hindered access to the authentic, efficacious variety. Thus, suitable analytical techniques for distinguishing authentic Vietnamese ginseng species from others is becoming increasingly crucial. Therefore, MR2 is attracting considerable attention as a target requiring effective management measures. MethodsAn enzyme-linked immunosorbent assay (ELISA) was developed by producing monoclonal antibodies against MR2 (mAb 16E11). The method was thoroughly validated, and the potential of the immunoassay was confirmed by high-performance liquid chromatography with ultraviolet spectroscopy. Furthermore, ELISA was applied to the assessment of the MR2 concentrations of various Panax spp., including Korean, American, and Japanese ginsengs. Results and conclusionsAn icELISA using mAb 16E11 exhibited linearity between 3.91 and 250 ng/mL of MR2, with detection and quantification limits of 1.53 and 2.50 − 46.6 ng/mL, respectively. Based on this study, the developed icELISA using mAb 16E11 could be a valuable tool for analyzing MR2 level to distinguish authentic Ngoc Linh and Lai Chau ginsengs from unauthentic ones. Furthermore, the analysis of the samples demonstrated that Ngoc Linh and Lai Chau ginsengs exhibit a notably higher MR2 value than all other Panax spp. Thus, MR2 might be their ideal marker compound, and various bioactivities of this species should be explored.

A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples.

Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN-BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC50 and LOD of the ELISA for ZEN were 0.37 ng mL-1 and 0.04 ng mL-1, respectively, which were 1.6-308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB1, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, β-zearalanol, zearalanone, α-zearalenol, β-zearalenol), the CR values of the mAb with the homologs were within 3.59%-105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46-105.48% with RSDs of 4.87-10.11%. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation y = 1.0589x + 1.43815 (R2 = 0.998, n = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.

An Enzyme-Linked Immunosorbent Assay (ELISA) for Quantification of Circulating Pi*Z Alpha1-Antitrypsin Polymers.

Enzyme-linked immunosorbent assay (ELISA) is a sensitive immunoassay based on specific antigen-antibody reaction that is used for quantitative/qualitative analysis of various analytes in serum, plasma, saliva, cell and tissue lysates, and urine. ELISAs are typically performed in multi-well plates and depending on the design require coating antibody/antigen, analyte, detection antibodies, buffer, wash solution, and substrate/chromogen. Here we describe highly specific monoclonal antibody-based ELISA that detects circulating polymers formed by Pi*Z variant of human alpha-1-antitrypsin (Z-AAT). The circulating Z-AAT polymers are present in all individuals with inherited Pi*Z AAT deficiency. Thus, our assay provides a useful tool to examine the clinical significance and utility of Z-AAT polymers.

Development of Methyl Ester Antibody-Based Competitive Indirect ELISA for Quantitative Detection of Mitragynine in Human Urine.

Mitragynine is the main psychoactive compound of Mitragyna speciosa Korth. (kratom). This alkaloid could render psychotropic effects and is often misused as a substitute for commercial drugs. Nowadays, the increasing popularity of kratom has led to the development of a rapid and effective detection method. The detection of mitragynine in a biological sample such as urine requires a highly sensitive and specific method due to the complex nature of mitragynine in urine. Enzyme-linked immunosorbent assay (ELISA) is well known as a rapid screening method for biological samples. In this study, a competitive indirect ELISA was successfully developed using MG-22-OCH3 IgG as a detection antibody for mitragynine in human urine. The mitragynine immunoassay showed a limit of detection and a limit of quantification of 0.412 and 1.25 μg/mL, respectively. The measurement range was between 0.01 and 100.0 μg/mL, with a minimal inhibition (IC50) value of 0.152 μg/mL. The developed ELISA was validated using a gold method such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). The percentage of recovery and the coefficient of variation (CV) for the ELISA and LCMS/MS analyses were 84.0-95.70%, 99.20-112.0%, 7.69-9.78%, and 2.86-6.62%, respectively. This indicates that the developed ELISA is a reliable method that can be used as a rapid approach for quantifying mitragynine content in biological samples.

A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma.

Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis. Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10-9 M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard. Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays. These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas.

A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies.

Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodies derived from camelids with naturally missing light chains can overcome these deficiencies and are an excellent alternative to conventional antibodies. In this study, a nanobody (Nb)-AviTag fusion protein was constructed, and the feasibility of its use as a high-sensitivity probe in a blocking ELISA (bELISA) for classical swine fever virus (CSFV) was investigated. The CSFV E2 recombinant protein expressed by the CHO expression system exhibited good reactogenicity and immunogenicity and induced the production of high CSFV antibody levels in rabbits. Three different clones of Nbs were successfully isolated using a phage display system in alpaca, and an Nb1-AviTag fusion protein was successfully expressed using an Escherichia coli expression system. The purified Nb1-AviTag fusion protein was then biotinylated in vitro to obtain Nb1-biotin. A novel bELISA was developed for the detection of CSFV antibodies in clinical serum using Nb1-biotin as a probe. The cutoff value of bELISA was 32.18%, the sensitivity of bELISA was higher than that of the bELISA kit with IDEXX antibody, and the coincidence rate was 94.7%. A rapid, low-cost, highly sensitive and highly specific CSFV E2 antibody-based bELISA method was successfully established and can be used for the serological evaluation of CSFV E2 subunit vaccines and the ELISA-based diagnosis of CSFV infection. IMPORTANCE Currently, the epidemic situation of classical swine fever (CSF) is sporadic, and cases of atypical swine fever are on the rise in China. Therefore, it is necessary to accurately eliminate suspected cases by using highly sensitive and specific diagnostic techniques. In our study, a rapid, low-cost, highly sensitivity, highly reliable and reproducible, and highly specific classical swine fever virus (CSFV) E2 antibody-based blocking ELISA method was successfully established by using the phage display system and the Nb1-AviTag fusion expression platform. It provides a new technique for serological evaluation of CSFV vaccines and ELISA-based diagnosis of CSFV infection.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for determination of vomitoxin (DON) in Coicis Semen

Coicis Semen is a common bulk medicinal material used for both medicine and food, which has the effect of promoting diuresis, draining dampness, invigorating the spleen and checking diarrhea. It is derived from Coix lacryma-jobi var. ma-yuen of the family Poaceae, and is easily contaminated by fungi such as Fusarium graminearum and F. flavum due to climate reasons to produce vomitoxin. The guiding principles for determination of vomitoxin in Chinese medicinal materials in Chinese Pharmacopoeia are mainly HPLC and LC-MS, which have long detection period and are time-consuming and laborious, and thus cannot meet the requirements of on-site quality inspection of drugs. The complete antigen of vomitoxin-protein was obtained by chemical derivatization of vomitoxin. The monoclonal antibody against vomitoxin was prepared by classic monoclonal antibody preparation technology, and enzyme-linked immunosorbent assay(ELISA) method for detection of vomitoxin in Coicis Semen was established through methodological investigation. The IC_(50) based on the ELISA for vomitoxin in Coicis Semen was 3.88 μg·L~(-1), and the average recoveries and the RSD were 77.32%-93.73% and 4.6%-9.7%, respectively. With the established ELISA method, the vomitoxin residue in 14 batches of Coicis Semen samples were determined and validated by LC-MS, and the correlation between the two assays was found to be 0.997 8, indicating that the established ELISA method could be used for quantitative determination of vomitoxin residue in Coicis Semen and could achieve the rapid quantitative determination of the vomitoxin residue.

Preparation of Monoclonal Antibodies against the Viral p54 Protein and a Blocking ELISA for Detection of the Antibody against African Swine Fever Virus.

African swine fever virus (ASFV) causes a highly contagious viral disease in domestic and wild pigs, leading to serious economic losses. As there are no vaccines or drugs available, early accurate diagnosis and eradiation of infected animals are the most important measures for ASFV prevention and control. Therefore, improvement of available diagnostic assays and development of novel effective techniques are required. This study is devoted to generating a new detection platform of blocking monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) against ASFV p54 protein. Seven monoclonal antibodies against recombinant p54 protein were produced and four epitopes were identified. Three blocking ELISAs were developed with 6A5 and 6F9 mAbs labeled with HRP, respectively, of which the 6A5/6F9-based blocking ELISA displayed the best detection performance, with an AUC of 0.986, sensitivity of 98.36% and specificity of 92.36% in ROC analysis. Moreover, it has an excellent agreement at 96.59% (198/205) when compared to the commercial blocking ELISA (kappa value = 0.920). The method also has high repeatability, with CV <10%, and no cross reaction with the serum antibodies against PRV, PRRSV, CSFV, PCV2 or SVA. This indicates that the 6A5/6F9-based blocking ELISA has high accuracy with good sensitivity and specificity, suitable for viral detection, field surveillance and epidemiological studies.

Establishment of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay to Measure Soluble B7-H5 in Patients with Cancer.

B7-H5, an immune checkpoint molecule, is markedly upregulated in multiple cancers and plays an important role in tumor progression and immune escape. However, the expression and significance of soluble B7-H5 (sB7-H5) in cancer remain unclear. Herein, we generated two novel mouse anti-human B7-H5 monoclonal antibodies (mAbs) 2E5 and 7B10, which had different epitopes. Based on the two mAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) system was developed. Using this ELISA, we found that compared with healthy controls (HCs), sB7-H5 levels were significantly increased in the serum of patients with gastric cancer (GC), colorectal cancer (CRC), and lung cancer (LC) and were associated with TNM stage and metastasis. Receiver operating characteristic (ROC) curve analysis showed that sB7-H5 has diagnostic value for GC, CRC, and LC. Collectively, our findings delineate that sB7-H5 may be used as a predictor for diagnosis of cancer and a potential therapeutic target for cancer treatment.

A highly salt-tolerant monoclonal antibody-based enzyme-linked immunosorbent assay for the rapid detection of phenylethanolamine A in urine

ABSTRACT Phenylethanolamine A (PEAA), as a typical β2-adrenoceptor agonist (β-AA), was widely illegally used in feed to improve the lean meat ratio. The detection performance of immunoassay, which is one of the effective methods to monitor PEAA residues in animal urine, has been hindered by high concentration of salt. Herein, we produced one highly salt-tolerant monoclonal antibody (mAb) 3E2 by hybridoma technology. Homologous modelling and molecular docking were used to analyse the antibody salt tolerance mechanism. Results show that the tight hydrophobic binding cavity is the key to high tolerance. Then the mAb 3E2 was used for specific detecting PEAA by enzyme-linked immunosorbent assay (ELISA) with the IC50 value of 0.36 ng mL−1and the LOD value of 0.065 µg L−1. Benefiting from the high salt tolerance of antibodies, swine urine and sheep urine spiked with PEAA can be detected directly by ELISA, and the acceptable recovery rates of 80.1–108.8% were obtained.

Plasma Matrix Metalloproeteinase-9 Is Associated with Seizure and Angioarchitecture Changes in Brain Arteriovenous Malformations

Both angiogenesis and inflammation contribute to activation of matrix metalloproeteinase-9 (MMP-9), which dissolves the extracellular matrix, disrupts the blood-brain barrier, and plays an important role in the pathogenesis of brain arteriovenous malformations (BAVMs). The key common cytokine in both angiogenesis and inflammation is interleukin 6 (IL-6). Previous studies have shown elevated systemic MMP-9 and decreased systemic vascular endothelial growth factor (VEGF) in BAVM patients. However, the clinical utility of plasma cytokines is unclear. The purpose of this study is to explore the relationship between plasma cytokines and the clinical presentations of BAVMs. Prospectively, we recruited naive BAVM patients without hemorrhage as the experimental group and unruptured intracranial aneurysm (UIA) patients as the control group. All patients received digital subtraction angiography, and plasma cytokines were collected from the lesional common carotid artery. Plasma cytokine levels were determined using a commercially available, monoclonal antibody-based enzyme-linked immunosorbent assay. Subgroup analysis based on hemorrhagic presentation and angiograchitecture was done for the BAVM group. Pearson correlations were calculated for the covariates. Means and differences for continuous and categorical variables were compared using Student's t and χ2 tests respectively. Plasma MMP-9 levels were significantly higher in the BAVM group (42,945 ± 29,991pg/mL) than in the UIA group (28,270 ± 17,119pg/mL) (p < 0.001). Plasma MMP-9 levels in epileptic BAVMs (57,065 ± 35,732; n = 9) were higher than in non-epileptic BAVMs (35,032 ± 28,301; n = 19) (p = 0.049). Lower plasma MMP-9 levels were found in cases of BAVM with angiogenesis and with peudophlebitis. Plasma MMP-9 is a good biomarker reflecting ongoing vascular remodeling in BAVMs. Angiogenesis and pseudophlebitis are two angioarchitectural signs that reflect MMP-9 activities and can potentially serve as imaging biomarkers for epileptic BAVMs.

Development of a highly sensitive and specific monoclonal antibody-based ELISA coupled with immuno-affinity extraction for the detection of anticancer drug 5-fluorouracil in blood samples

5-Fluorouracil (5-FU) is an effective anticancer drug widely used in cancer treatment. In this study, two 5-FU derivatives containing a spacer arm with the carboxylic group at the end were synthesized, which were linked to the carrier proteins to form 5-FU-protein conjugates used as the immunogens for the production of monoclonal antibody (mAb). Based on the produced mAb, the highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for 5-FU detection was established. The IC50 and LOD values of the assay were found to be 19.5 ng mL−1 and 0.5 ng mL−1, respectively. There was no cross-reactivity (CR) of the ELISA with cytosine, thymine and uracil, which avoided the interference from inherent pyrimidines. The CR values of the assay with three substitutes of 5-FU (tegafur, 5-fluoro-2′-deoxyuridine, carmofur) were within 9.7%–17.6%. The produced mAb was also applied in sample extraction. The immuno-affinity column capable specific capturing 5-FU was prepared by immobilizing the mAb on Sepharose-4B gel and filling into a SPE column. The recoveries of 5-FU in spiked samples measured by ELISA were 72.4%–90.7% with RSD of 3.6%–8.3%. Five blood samples collected from patients were extracted by immuno-affinity column, then measured by ELISA and confirmed by HPLC-MS/MS. There was a good correlation between HPLC-MS/MS and ELISA. It is demonstrated that the developed ELISA combined with immuno-affinity extraction can be a powerful alternative method for the detection of 5-FU in blood samples.

Monoclonal antibody-based enzyme-linked immunosorbent assay and lateral flow immunoassay for the rapid screening of paraquat in adulterated herbicides

The use of the herbicide paraquat has been banned in many countries due to its high toxicity. However, there is an issue that illegal addition of paraquat in herbicide products. Therefore, a rapid, sensitive, and portable method will be useful to detect paraquat in adulterated herbicides for regulation purpose.In the present study, an indirect competition enzyme-linked immunosorbent assay (icELISA) was developed with a new paraquat-specific monoclonal antibody (mAb) for paraquat detection. The icELISA results showed a 50% inhibitory concentration (IC50) of 7.63 ng/mL with a working range (IC20-IC80) of 1.68–33.1 ng/mL. In addition, a lateral flow immunoassay (LFIA) with a cut-off value of 20.0 ng/mL was also developed. The LFIA showed no detection of the herbicides diquat, glyphosate and glufosinate up to 50 µg/mL. Nine herbicide samples were collected across Hainan province, China and analyzed by the LFIA dipsticks. One out of the nine samples was tested positive, which was confirmed with icELISA and HPLC. The accuracy of the LFIA was further verified through onsite tests in different places in China. Forty-seven commercial herbicide products were tested via the LFIA dipsticks, of which 9 samples were positive and confirmed with HPLC analysis.The paraquat LFIA exhibited the most promising potential as the point of care (POC) device for the rapid screening of paraquat in adulterated herbicide products.

Evaluation of poly- and monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) for their performance to detect crustacean residues in processed foods

Poly- and monoclonal antibody-based (PcAb and mAb) sandwich ELISA formats were developed to detect tropomyosin, a major crustacean allergen. Their performance was accessed by testing crustacean residue in commercial processed and model foods. Results obtained showed the constructed mAb ELISA have lower LOD (0.004 mg/kg) and LOQ (0.006 mg/kg), and higher specificity for crustacean tropomyosin than the PcAb ELISA (LOD: 0.008 mg/kg and LOQ: 0.01 mg/kg). Despite this, the PcAb ELISA system with fewer matrix interferences could discriminate against all commercial foods on the label and show higher tropomyosin recovery than the same positive samples based on a mAb ELISA test. Additionally, both ELISA systems possess adequate repeatability and precision, and can detect as low levels as 0.01% and 0.05% of incurred shrimp powder in cookies and sausages, respectively. Nevertheless, mAb ELISA system shows lower tropomyosin recovery in the same incurred foods, and fish sauce even when supplemented with 5% shrimp powder addition could not be detected by the mAb ELISA test. These findings indicated that the recovery of tropomyosin decreased with the intensity of thermal processing applied to food, while PcAb ELISA showed better matrix-tolerance and detectability in processed foods.

Development of a highly sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the detection of avilamycin in feed

ABSTRACT An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed using a highly sensitive and specific monoclonal antibody against avilamycin. The immunising antigen synthesised through the carbonyldiimidazole (CDI) method was used to prepare antibodies. The established ic-ELISA, after optimisation of conditions, possessed a half maximum inhibitory concentration (IC50) value of 7.44 ng mL−1 and the detection limit of 0.21 ng mL−1 in the standard curve. The spiked experiments indicated that the limits of detection were 1.86 µg kg−1 and 2.31 µg kg−1 in swine feed and chicken feed, respectively. In addition, the average recoveries ranged from 74.7 to 105.4% with the coefficient of variation less than 11%. The good correlation (R2 = 0.9818) between the result of ic-ELISA and HPLC demonstrated the reliability of the developed ic-ELISA, which showed that the ic-ELISA developed here achieves the aim of strengthening the monitoring of avilamycin residues and the inspection of import and export related products.

Evaluation of recombinant glutathione transferase 26 kDa, thioredoxin-1, and endophilin B1 of Taenia solium in the diagnosis of human neurocysticercosis

Neurocysticercosis caused by Taenia solium larvae is a neglected disease that persists in several countries, including Mexico, and causes a high disability-adjusted life year burden. Neuroimaging tools such as computed tomography and magnetic resonance imaging are the most efficient for its detection, but low availability and high costs in most endemic regions limit their use. Serological methods such as lentil lectin-purified glycoprotein enzyme-linked immunoelectrotransfer blot antibody detection and monoclonal antibody-based enzyme-linked immunosorbent assays for HP10 antigen detection have been useful in supporting the diagnosis of this disease. We evaluated three T. solium recombinant antigens: glutathione transferase of 26 kDa (Ts26GST); thioredoxin-1 (TsTrx-1), and endophilin B1 (TsMEndoB1) by EITB. These are antigenic proteins antigenic, abundant in excretion/secretion products of the parasite, and do not cross-react with homologous host proteins. Ts26GST and TsTrx-1 showed sensitivity of 79 and 88%, specificity of 86 and 97%, PPV of 83 and 97% and NPV of 82 and 91%, respectively, for neurocysticercosis diagnosis. The recombinant antigens allowed the diagnosis of 70% (Ts26GST) and 80% (TsTrx-1) of patients having only one cysticercus. Further studies on specific regions of these proteins could improve T. solium diagnostics.

Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). A sensitive and reliable serological diagnostic assay is required, so laboratories can effectively and quickly detect ASFV infection. The p30 protein is abundantly expressed early in cells and has excellent antigenicity. Therefore, this study aimed to produce and characterize p30 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Three monoclonal antibodies against p30 protein that were expressed in E. coli were generated, and their characterizations were investigated. Furthermore, a blocking ELISA based on a monoclonal antibody was developed. To evaluate the performance of the assay, 186 sera samples (88 negative and 98 positive samples) were analyzed and a receiver-operating characteristic (ROC) analysis was applied to determine the cutoff value. Based on the ROC analysis, the area under the curve (AUC) was 0.997 (95% confidence interval: 99.2 to 100%). Besides, a diagnostic sensitivity of 97.96% (95% confidence interval: 92.82 to 99.75%) and a specificity of 98.96% (95% confidence interval: 93.83 to 99.97%) were achieved when the cutoff value was set to 38.38%. Moreover, the coefficients of inter- and intra-batches were <10%, indicating the good repeatability of the method. The maximum dilution of positive standard serum detected by this ELISA method was 1:512. The blocking ELISA was able to detect seroconversion in two out of five pigs at 10 Dpi and the p30 response increasing trend through the time course of the study (0–20 Dpi). In conclusion, the p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity. The assay could be a useful tool for field surveillance and epidemiological studies in swine herd.

Clinical value of serum biomarkers, squamous cell carcinoma antigen and apolipoprotein C-II in follow-up of patients with locally advanced cervical squamous cell carcinoma treated with radiation: A multicenter prospective cohort study.

There are currently no reliable, established serum biomarkers to predict the prognosis of radiotherapy for advanced cervical cancer. We aimed to identify serum biomarkers for survival after radiotherapy for cervical cancer. In this multicenter prospective cohort study, the usefulness of pre- and posttreatment serum protein levels of potential biomarkers, including squamous cell carcinoma antigen (SCC-Ag), apolipoprotein C-II (ApoC-II), matrix metalloproteinase (MMP)1, and MMP2, were evaluated together with clinical factors in 145 cervical cancer patients in order to determine their suitability to predict survival. Progression-free survival (PFS) was the primary endpoint, and overall survival (OS), pelvic PFS (PPFS), and distant metastasis-free survival (DMFS) were the secondary endpoints. Blood samples were collected before and 1 month after radiotherapy to measure serum biomarker levels. ApoC-II was measured using a monoclonal antibody-based enzyme-linked immunosorbent assay, which was developed for this purpose. Kaplan-Meier method, log-rank test, and univariate and multivariate Cox proportional hazards models were used for statistical analyses. In multivariate analysis, larger tumor size was independently associated with shorter PFS, OS, PPFS, and DMFS, while longer overall treatment time was independently associated with shorter PPFS. Higher pretreatment SCC-Ag (P < 0.001) was associated with shorter DMFS. Higher posttreatment SCC-Ag (P = 0.017) was also associated with shorter DMFS. Pretreatment ApoC-II was associated with PPFS in univariate analysis (P = 0.048), but not in multivariate analysis. Patients with pretreatment ApoC-II levels ≤ 25.8 μg/ml had shorter PPFS than those with pretreatment ApoC-II levels > 25.8 μg/ml (P = 0.023, log-rank test). Pre- and posttreatment serum SCC-Ag and pretreatment serum ApoC-II levels may be important biomarkers to predict survival outcomes of patients with cervical cancer after radiotherapy. Pre- and posttreatment SCC-Ag and pretreatment ApoC-II might be useful in clinical settings for screening patients to improve treatment strategies in cervical cancer.