Antibody-based Sandwich Enzyme-linked Immunosorbent Assay Research Articles

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.

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.

Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for porcine hemoglobin quantification

The major objective of this study was to establish a monoclonal antibody (mAb)-based sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of porcine hemoglobin (PHb) in raw meat products. Before assay development, two mAbs immunoreactive to PHb β subunit with different epitopes were characterized. The optimized immunoassay was specific to PHb and had a wide PHb working range from 15.6 µg/mL to 3,000 µg/mL and high reproducibility with low coefficient of variations (CV < 20%). Through this assay, the estimated PHb residuals in pork loin and shoulder meats were 0.4 mg/g and 1.1 mg/g, respectively. In addition, this immunoassay could effectively quantify PHb in laboratory-spiked meats (pork loin, pork shoulder, and turkey breast) with acceptable recovery. Overall, this is the first mAb-based sandwich ELISA that is suitable for the government, food industry, and third-party authority to monitor PHb residuals or porcine blood adulteration in raw pork and pork-free meat products.

Development of a sensitive monoclonal antibody-based sandwich ELISA to detect Vip3Aa in genetically modified crops

ObjectivesTo develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to detect Vip3Aa in genetically modified (GM) crops and their products.ResultsVegetative insecticidal proteins (Vips) are secreted by Bacillus thuringiensis (Bt) and are known to be toxic to Lepidoptera species. Vip3Aa family proteins, Vip3Aa19 and Vip3Aa20, were successfully applied in GM crops to confer an effective and persistent insecticidal resistance. A sensitive monoclonal antibody-based sandwich ELISA was developed to detect Vip3Aa in GM crops and their products. Two monoclonal antibodies were raised against the overexpressed and purified His-Vip3Aa20, were purified from mouse ascites and characterized. A sandwich ELISA method was developed using the 2G3-1D7 monoclonal antibody for capture and the biotin-labeled 1F9-1F5 monoclonal antibody for detection of Vip3Aa20. The linear detection range of the method was found to be approximately 31.25–500 pg/ml, with a sensitivity of 10.24 pg/ml.ConclusionsThe established ELISA was effective for detecting Vip3Aa family proteins other than Vip3Aa8, and was successfully applied in the detection of Vip3Aa20 and Vip3Aa19 expressed in transgenic maize and cotton.

Monoclonal antibody-based sandwich ELISA for the detection of mammalian meats

In order to (1) reduce the risk of intentional or unintentional contamination of foods, (2) better comply with food regulations, and (3) decrease economic loss to the food industry caused by recall, it is necessary to develop reliable methods for the detection of different food adulterants/contaminants. This study was conducted to characterize two mammalian skeletal troponin (sTn) specific monoclonal antibodies (mAbs 6G1 and 8F10), and use them to develop a mAb-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of mammalian meats. From our results, both mAbs were positive to porcine sTnI and sTnC but negative to sTnT. The extractability and antigenicity of target analytes in pork were enhanced by the addition of urea and β-mercaptoethanol into the extraction buffer. The optimized sandwich ELISA was specific to heated mammalian meats and was adequate to analyze samples subject to the most severe heat treatment (132 °C/2 bar/120 min). Mammalian fat (10–30%, g/g) did not significantly affect the assay signal. The optimized assay could detect as low as 1% (g/g) of heated mammalian meats adulterated in poultry meats. Overall, this assay has the potential to fight food fraud, comply with food regulations, and decrease food recalls, which may open up new diagnostic methods for the food industry and the food regulatory authorities.

Pecan (Carya illinoinensis) detection using a monoclonal antibody-based direct sandwich enzyme-linked immunosorbent assay

A commercially available (BioFront Technologies) monoclonal antibody-based direct sandwich enzyme-linked immunosorbent assay (ELISA) for pecan detection was evaluated. The assay can detect 0.5 ppm of pecan and reliably quantify >1.5 ppm of pecan with <14% intra- and inter-assay variabilities. The detection antibody did not exhibit cross-reactivity with 155 foods/ingredients tested at 100,000 ppm, although it registered 0.6% and 0.8% cross-reactivity with 10,000 ppm of English walnut and black walnut, respectively. The target antigen was stable against autoclaving, blanching, frying, microwaving, and roasting. The antigen was detected in a variety of food matrices with 80.5–111.6% and 22.2–154.5% recoveries for pecan-spiked and incurred samples, respectively. The assay did not yield any false negative results among tested commercial and in-house prepared samples, however, it recognized two commercial products with declared walnut and no declared pecan as positive. In conclusion, the ELISA was sensitive and robust for pecan detection and quantification and was specific other than the low level of cross-reactivity with walnut species.

Generation and Application of Rat Monoclonal Antibodies Specific for a Human Blood Coagulation Protein: von Willebrand Factor.

von Willebrand factor (VWF) is a glycoprotein that plays a central role in the initiation of blood coagulation. VWF performs two important functions: it acts as a molecular bridge between platelets and as a carrier for coagulation factor VIII (FVIII). von Willebrand disease (VWD) and acquired von Willebrand syndrome (AVWS) are caused by the absence of and/or abnormality in VWF. The pathophysiology of VWD and AVWS is complex and, therefore, it is difficult to diagnose them by conducting the same laboratory tests in all patients. To develop useful monoclonal antibodies (mAbs) for the diagnosis of VWD and AVWS, rat mAbs against human VWF were generated. Immunoblotting analysis revealed that mAbs recognized the reduced and nonreduced VWF protein and endogenous VWF in normal human plasma. Furthermore, we developed a highly sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay technique. In conclusion, the development of VWF-specific mAbs would be useful in the diagnosis of VWD and AVWS.

Dual monoclonal antibody-based sandwich ELISA for detection of in vitro packaged Ebola virus

BackgroundRapid transmission and high mortality of Ebola virus disease (EVD) highlight a urgent need of large scale, convenient and effective measure for Ebola virus screening. Application of monoclonal antibodies (mAbs) are crucial for establishment of an enzyme-linked immunosorbent assay (ELISA) with high sensitivity and specificity.MethodsThe traditional cell fusion technique was used to generate a panel of hybridomas. Two mAbs were characterized by SDS-PAGE, Western blot, Indirect immunofluorescence assay (IFA). A sandwich ELISA was established using the two mAbs. The detection capability of the ELISA was evaluated.ResultsIn the current study, we produced two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), the major viral transmembrane spike protein associated with viral attachment. It was shown that 6E3 and 3F21 recognized GP1 and GP2 subunits of the GP respectively. Furthermore, 6E3 and 3F21 bound to corresponding epitopes on GP without reciprocal topographical interpretation. Subsequently, a sandwich ELISA based on the two mAbs were established and evaluated. The detection limit was 3.6 ng/ml, with a linear range of 3.6–100 ng/ml. More importantly, Ebola virus like particles (eVLPs) were able to be detected by this established virus detection measure.ConclusionsWe produced and characterized two murine-derived mAbs (designated as 6E3 and 3F21) towards Zaire Ebola virus glycoprotein (GP), and established a sandwich ELISA based on the mAbs. It was suggested that the sandwich ELISA provided an alternative method for specific and sensitive detection of Ebola virus in the field setting.

Application of Monoclonal Antibodies Against Mouse Dermokine.

Dermokine is one of the most highly expressed proteins in differentiating keratinocytes. Mouse dermokine has been reported to be encoded by 22 exons, and its expression leads to three transcripts, β, γ, and α, which are transcribed from two different transcriptional start sites. The α isoform represents the carboxyl-terminal domain of the β isoform, whereas the γ isoform lacks this domain. To reveal the distributions and expression levels of each isoform in mice, we generated rat monoclonal antibodies against dermokine-β/γ and dermokine-β/α. In immunofluorescence studies, the expression levels of dermokine in the cytosol of the cultured mouse keratinocytes were significantly elevated by high levels of extracellular calcium. In Western blot analyses, the expression levels of dermokine-β and dermokine-α were increased in the presence of high calcium. Finally, we developed a monoclonal antibody-based sensitive sandwich enzyme-linked immunosorbent assay (ELISA) and showed that the secreted dermokine-β into the culture medium from mouse keratinocytes was significantly increased in a manner dependent on the extracellular calcium concentration. These dermokine-specific antibodies have allowed us to gain new insights into the role of each dermokine isoform in cutaneous homeostasis.

The performance of [-2]proPSA and prostate health index tumor markers in prostate cancer diagnosis

Abstract Background: Prostate-specific antigen (PSA) is an established tumor marker for the diagnosis of patients with prostate cancer. The aim of the study was to evaluate the performance of [-2]proenzyme PSA ([-2]proPSA) and prostate health index (PHI) tumor markers in the differential diagnosis between benign prostatic diseases and prostate cancer. Methods: Total PSA (tPSA), free PSA (fPSA) and [-2]proPSA were measured using antibody-based sandwich enzyme-linked immunosorbent assay with a chemiluminescent detection system in 110 patients, with a tPSA of 1.6–8.0 µg/L. The PHI and %[-2]proPSA were calculated from the PSA values mentioned above. The results were compared with histopathological examination results following a transrectal ultrasound-guided biopsy of the prostate. Results: For the prediction of a malignant histopathological result, the specificity at the 90% sensitivity level was 24.3% for [-2]proPSA, 32.4% for %[-2]proPSA, 28.4% for PHI, 18.9% for tPSA and 28.4% for the free-to-total PSA ratio. The area under the curve for [-2]proPSA, %[-2]proPSA, PHI, tPSA and the free-to-total PSA ratio was 0.663, 0.749, 0.742, 0.616 and 0.625, respectively. Conclusions: Our study found a moderate improvement over tPSA and %fPSA in detecting prostate cancer using the [-2]proPSA assay in patients with a tPSA range of 1.6–8.0 µg/L.

Specific determination of influenza H7N2 virus based on biotinylated single-domain antibody from a phage-displayed library

The unpredicted spread of avian influenza virus subtype H7N2 in the world is threatening animals and humans. Specific and effective diagnosis and supervision are required to control the influenza. However, the existing detecting methods are laborious, are time-consuming, and require appropriate laboratory facilities. To tackle this problem, we isolated VHH antibodies against the H7N2 avian influenza virus (AIV) and performed an enzyme-linked immunosorbent assay (ELISA) to detect the H7N2 virus. To obtain VHH antibodies with high affinity and specificity, a camel was immunized. A VHH antibody library was constructed in a phage display vector pMECS with diversity of 2.8 × 109. Based on phage display technology and periplasmic extraction ELISA, H7N2-specific VHH antibodies were successfully isolated. According to a pairing test, two VHH antibodies (Nb79 and Nb95) with good thermal stability and specificity can recognize different epitopes of H7N2 virus. The capture antibody (Nb79) was biotinylated in vivo, and the detection antibody (Nb95) was coupled with horseradish peroxidase (HRP). Based on biotin–streptavidin interaction, a novel sandwich immune ELISA was performed to detect H7N2. The immunoassay exhibited a linear range from 5 to 100 ng/ml. Given the above, the newly developed VHH antibody-based double sandwich ELISA (DAS–ELISA) offers an attractive alternative to other diagnostic approaches for the specific detection of H7N2 virus.

Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the Cronobacter sakazakii. A pair of monoclonal antibodies (mAbs) selected from mAbs produced by 11 murine hybridomas was selected for the sandwich ELISA procedure. Targets of two mAbs were 100 kDa and 42 kDa protein extracted from the bacteria, respectively, which were proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. The limit of detection of this method was established as 1 × 104 cfu/mL, and the linear range from 1 × 105 to 1 × 108 cfu/mL. In the real sample test, 1 cfu/g C. sakazakii was detected in artificially contaminated powdered infant formula with 4 h enrichment.

Development of monoclonal antibody-based sandwich ELISA for detection of dextran.

Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.

Correlation study of chronic nonbacterial prostatitis with the levels of COX-2 and PGE2 in prostatic secretion.

The prostatitis syndrome is a multifactorial condition of largely unknown etiology. This study is to analyze the relationship between cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) with the chronic nonbacterial prostatitis (CNBP). A total of 172 CNBP patients and 151 healthy males were recruited as CNBP and control group, respectively. The prostatic fluid was collected and tested by pre- and post-massage test. White blood cell (WBC) number was counted, and the contents of COX-2 and PGE2 were determined by double antibody-based sandwich enzyme-linked immuno-sorbent assay. The pain and discomfort of each patient were scored according to the National Institutes of Health chronic prostatitis symptom index. Compared with the control group, CNBP group displayed significantly higher WBC count, COX-2 level, and PGE2 level. Contents of COX-2 and PGE2 in prostatic secretion of CNBP group were positively correlated with pain scores (r = 0.855 and 0.675, respectively, P < 0.01) and total symptom scores (r = 0.674 and 0.566, respectively, P < 0.01). A significantly positive correlation between COX-2 and PGE2 levels was also discovered (r = 0.493, P < 0.05). The WBC number was not obviously correlated with the accumulations of COX-2 and PGE2 or the clinic symptoms of CNBP. Increase in PGE2 concentration caused by activated COX-2 pathway may contribute to the pain or discomfort symptom of the CNBP patients. Our results indicate that selective COX-2 inhibitors have application prospect in CNBP treatment.

Development of a monoclonal antibody TAS-ELISA assay for detection of Phaeomoniella chlamydospora

Summary. Phaeomoniella chlamydospora is an important tracheomycotic fungus involved in different syndromes of the esca disease complex affecting young and mature grapevine plants. Although grapevine planting material is frequently infected by this pathogen, no regular screening to detect the fungus is carried out, mainly due to the lack of fast, sensitive and affordable methods for large-scale application. A monoclonal antibody-based triple antibody sandwich (TAS) enzyme-linked immunosorbent assay (ELISA) was developed to specifically detect Pa. chlamydospora from grapevine wood tissues. This assay could be especially useful for large-scale application in nurseries, to ensure Pa. chlamydospora-free grapevine stocks, and thus contributing to the production of healthy propagation material.

A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of secreted α-defensin

Paneth cells at the base of small intestinal crypts secrete α-defensins, which contribute to innate immunity and shape composition of enteric microbiota. Efforts to establish a relationship between secreted α-defensins and disease have been hampered by a lack of sensitive assays to quantify luminal α-defensins. Here we report on a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for the mouse Paneth cell α-defensin cryptdin-4 (Crp4) in varied sources, including luminal contents rinsed from stomach to distal colon and fecal pellets. One pair of monoclonal antibodies (mAbs), selected from 10 rat hybridomas secreting Crp4-specific mAbs, was optimized for Crp4 detection and specificity in the sandwich ELISA. In CD1 mice, luminal Crp4 levels increased gradually from 6.8±5.2ng/ml in proximal small intestine to 54.3±10.3ng/ml in distal small intestine, and the peptide was detected in colonic lumen and feces. Secreted Crp4 was reduced significantly in feces of IL10 null mice, a model of inflammatory bowel disease (IBD) when compared with wild-type controls. This Crp4 sandwich ELISA enables accurate determinations of luminal α-defensins as biomarkers of Paneth cell function and enteric integrity in diverse disease states such as IBD, infectious disease, graft versus host disease, and obesity in association with dysbiosis of the intestinal microbiota.

Development of a Monoclonal Antibody-Based Sandwich ELISA for Peanut Allergen Ara h 1 in Food

We have established a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mAb) to measure the content of the major peanut allergen Ara h 1 in foods. Two mAbs were selected out of 12 murine hybridoma cells secreting Ara h 1-specific antibody. Using mAb 6 as the capture antibody and HRP-labelled mAb 4 as the detection antibody, the limit of detection (LOD) the assay was 0.34 ng/mL. Cross-reaction analysis showed that this method was strongly specific and had no cross-reactions with Ara h 2, pea protein or soy protein. Sample analysis showed that this ELISA was a useful tool to monitor peanut allergens in food products by measuring Ara h 1 content.

Monoclonal Antibody-Based Sandwich ELISA for the Detection of Staphylococcal Enterotoxin A

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established and validated for the detection of staphylococcal enterotoxin A (SEA). After routine fusion and selection, 10 monoclonal antibodies showed high affinity for SEA. An optimal pair for sandwich ELISA was selected by pairwise interaction analysis. After optimization, the limit of detection (LOD) and linear dynamic range of the method were established, and were found to be 0.0282 ng/mL and 0.06–2 ng/mL, respectively. The recovery in pure milk ranged from 82.67% to 111.95% and the intra- and inter-assay coefficients of variation ranged from 3.16% to 6.05% and from 5.16% to 10.79%, respectively. Cross-reactivity with staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), staphylococcal enterotoxin D (SED), and staphylococcal enterotoxin E (SEE) in this method were insignificant. These results indicate that the sandwich ELISA method developed in our study is effective for routine identification of SEA in food samples.

Development of a monoclonal antibody-based sandwich ELISA for the detection of ovalbumin in foods

Hen egg is one of the most frequent causes of food allergy in infants and adults. Ovalbumin (OVA) has been identified as a major egg allergen. In order to detect OVA in foods, a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mAbs) was established. The 2 mAbs were selected out of 17 murine hybridomas secreting OVA-specific antibody. Using mAb17 as the capture antibody and mAb15 as the detection antibody, the detection limit of the ELISA method was 0.51 ng/mL, and the linear dynamic range was between 1.95 and 500 ng/mL. The recovery ranged from 85.6 to 115.2%, whereas the intra- and inter-assay coefficients of variation were less than 8.6 and 13.9%, respectively. Sample analysis verified that the produced anti-OVA mAb and the developed ELISA may provide a valuable tool for the sensitive determination of OVA in processed foods and for future studies on the mechanism of how OVA functions in anaphylaxis.

Prevalence of porcine cysticercosis and associated risk factors in Homa Bay District, Kenya

BackgroundTaenia solium is an important zoonosis in many developing countries. Cysticercosis poses a serious public health risk and leads to economic losses to the pig production industry. Due to scarcity of data on the epidemiology of porcine cysticercosis in Kenya, the present study was conducted to determine the prevalence and risk factors for porcine cysticercosis within Homa Bay district. A cross-sectional survey was carried out in 2010, and a total of 392 pigs were recruited in a household survey, with all being tested by ante-mortem lingual palpation (together with questionnaire data on pig production, occurrence and transmission of porcine cysticercosis, risk factors and awareness of porcine cysticercosis collected from the households from which pigs were sampled). Sufficient serum was collected from 232 of the pigs to be tested for the presence of circulating parasite antigen using a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (Ag-ELISA).ResultsSeventy six pigs were found positive by the Ag-ELISA (32.8%, 95% C.I. 26.8-39.2%), while by tongue inspection cysticerci were detected in 22/ 392 pigs (5.6% 95% C.I. 3.6-8.4%).The most important risk factor for porcine cysticercosis in the Homa Bay area was for pigs to belong to a farm where latrine use by members of the household was not evident (OR = 1.9, 95% CI = 1.13–2.37).ConclusionThe present findings indicate that porcine cysticercosis is endemic in Homa Bay District, and that latrine provision, in conjunction with free-range pig keeping contributes significantly to porcine cysticercosis transmission.