Development Of Enzyme-linked Immunosorbent Assay Research Articles

Production of antibody and development of enzyme-linked immunosorbent assay for therapeutic drug monitoring of eravacycline

Eravacycline (ERC) was approved for clinical use in 2018. It is more potent than other tetracyclines and can overcome resistance, making it an attractive option for combating multidrug-resistant bacterial infections. Intensive pharmacokinetic (PK) studies are currently being conducted to ensure the effectiveness and safety of ERC in various groups of patients, including those undergoing extracorporeal therapies. This study is the first attempt to develop a simple, efficient, and high-throughput immunoassay for quantifying ERC in human or animal serum. BSA-ERC conjugate as immunogen elicited antibody production in rabbits. Monitoring of the immune response and comparison of homologous and heterologous coating antigens allowed selection of immunoreagents and development of an assay that was selective for ERC possessing sensitivity (IC50), dynamic range (IC20-IC80) and detection limit equal to 3.3 ng/mL, 0.27–54 ng/mL and 0.09 ng/mL, respectively. The developed ELISA showed acceptable recovery of ERC (85–105 %) from rabbit and human serum in the clinically relevant concentration range of 0.1–3.0 mg/L. The method was used to quantify serum ERC concentration in the pilot PK study in Soviet chinchilla rabbits. The results were confirmed by HPLC-MS/MS.

Development of enzyme-linked immunosorbent assay (ELISA) based on covalent immobilization of antibody on plate for measurement of digoxin

Objectives: The aim of this study was to design an enzyme immunoassay based on a modified ELISA with high sensitivity for detecting digoxin.
 Methods: The first step in development involved the conjugation of digoxin to the HRP (Horse Radish Peroxidase) enzyme via sodium metaperiodate oxidation. Surface modification, and thus assay modification, was achieved by the covalent immobilization of an anti-digoxin monoclonal antibody on a functional plate using 3-ATPES (3-aminopropyltriethoxysilane).
 Results: The developed ELISA demonstrated superior sensitivity (0.026μg/ml) and lower variability in measurements repeated throughout a day, compared to a conventional ELISA (0.051μg/ml). This assay detected the exact amount of digoxin. The sensitivity and specificity of the modified ELISA surpassed other methods, and measurements were performed within a few hours.
 Conclusion: An efficient ELISA kit was produced, characterized by its affordability, ease of learning, and absence of a hand-washing step.

Preparation of Monoclonal Antibody against Pyrene and Benzo [a]pyrene and Development of Enzyme-Linked Immunosorbent Assay for Fish, Shrimp and Crab Samples

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental and food pollutants that can cause cancer. In this work, a specific monoclonal antibody (mAb) to identify pyrene (PYR) and benzo [a]pyrene (BaP) was prepared, and an indirect competitive enzyme-linked immunoassay (ic-ELISA) was established to detect PYR and BaP residues in living aquatic products for the first time. The effects of complete antigens with different coupling ratios on the production of high-sensitivity mAb was explored. Under the optimal conditions, the IC50 value was 3.73 ± 0.43 µg/L (n = 5). The limits of detection (LODs) for PYR and BaP in fish, shrimp, and crab ranged from 0.43 to 0.98 µg/L. The average recoveries of the spiked samples ranged from 81.5–101.9%, and the coefficient of variation (CV) was less than 11.7%. The validation of the HPLC-FLD method indicated that the ELISA method set up in this experiment provided a trustworthy tool for PAHs residues detection in aquatic products.

Development of enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic assay for the rapid detection of gentamicin in chicken muscle and milk

• A specific monoclonal antibody against gentamycin was obtained. • Based on mAb, one-step ic-ELISA and GICA methods were developed. • Two immunoassays can be applied for the detection of gentamycin in real samples. A specific anti-gentamicin (GEN) monoclonal antibody (mAb) was used to develop an indirect competitive ELISA (ic-ELISA) and a colloidal gold immunochromatographic assay (GICA) for the determination of GEN residues in chicken muscle and milk. The result of ic-ELISA showed that the limits of detection (LOD) for GEN in chicken muscle and milk samples ranged from 0.54 µg kg −1 –0.57 µg kg −1 . As for the GICA method, the LOD of GEN in chicken muscle and milk were 10 µg kg −1 and 10 µg kg −1 , respectively. The GEN recovery of the two methods ranged from 83.8%–112.2%. In the actual samples detection, the ELISA and GICA methods had a good correlation with high-performance liquid chromatography (HPLC) results. These indicated that the methods established in this study could be a candidate tool for the rapid large-scale screening of GEN in animal-derived foods.

Універсальний метод очищення та концентрування вірусів на прикладі інноваційної розробки для парвовірусу гусей

A new method of purification and concentration of goose enteritis virus has been developed. For this purpose, three options for further purification of the virus were used and their comparative analysis was performed. The virus was purified without detergents, as well as with detergents – sarcosyl and nonide R-40. We obtained the best results using the mild nonionic detergent nonidet R-40, which was used in our further work. The virus was identified by electrophoretic studies in polyacrylamide gel, as well as electron microscopy. During purification and concentration of the virus, the infectious titer of the virus was 9.2-9.5 lg TCD50/cm3 suspension, in which the cell monolayer in 50% of the wells was affected by cytopathic action), which is 2 lg TCD50/cm3 higher than in the original material. The protein content in the test samples ranged from 200 to 500 mg/ ml. Thus, analyzing our results on the purification and concentration of goose enteritis virus, we can conclude that the antigen obtained by this method is suitable for the development of enzyme-linked immunosorbent assay (ELISA). Hyperimmune and negative sera for ELISA diagnostics were obtained, the optimal ratios of components for designing an ELISA test system were worked out, and the formula for recalculating antibody titers in geese blood sera when testing them in one dilution was derived. A positive-negative threshold was determined for this diagnosticum (which of the studied sera have a positive, doubtful or negative titer of antibodies to the causative agent of viral enteritis in geese). In the new conditions the spread of particularly dangerous viruses, this development, with the appropriate equipment, can be further used to purify and concentrate these viruses, study their biological properties, cultivate and use them in the development of new vaccines.

Detection of thioredoxin-1 using ultra-sensitive ELISA with enzyme-encapsulated human serum albumin nanoparticle

Many methods for early diagnosis of the disease use biomarker tests, which measure indicators of biological state in body fluids or blood. However, a limitation of these methods is their low sensitivity to biomarkers. In this study, human serum albumin (HSA) based nanoparticles capable of encapsulating excess horseradish peroxidase (HRP) are synthesized and applied to the development of enzyme-linked immunosorbent assay (ELISA) kit with ultra-high sensitivity. The size of the nanoparticles and the amount of encapsulated enzyme are controlled by varying the synthesis conditions of pH and protein concentration, and the surface of the nanoparticles is modified with protein A (proA) to immobilize antibodies to the nanoparticles by self-assembly. Using the synthesized nanoparticles, the biomarker of breast cancer, thioredoxin-1, can be measured in the range of 10 fM to 100 pM by direct sandwich ELISA, which is 105 times more sensitive than conventional methods.

Development of Enzyme-Linked Immunosorbent Assay with Tiramine Amplification for the Detection of Potato Virus X

A method is proposed for reducing the detection limit of enzyme-linked immunosorbent assay (ELISA) of potato virus X, based on the multiple introduction of tyramine into immune complexes and the subsequent detection of an enzyme label. During ELISA, a step by step formation of sandwich complex containing immobilized antibodies – potato virus X – horseradish peroxidase conjugate with antibodies to the virus was carried out. Peroxidase catalyzed the multiple insertion of a tyramine-biotin label into protein molecules, providing signal amplification upon the addition of the streptavidin-polyperoxidase conjugate. The conditions of the assay that ensure a high degree of amplification and a minimum background signal were established. The use of tyramine amplification made it possible to lower the detection limit by more than 30 times (from 100 to 3 ng/ml) when assayed in the buffer and extracts of potato leaves, slightly increasing its duration. Tyramine amplification is based on the use of universal reagents and can be used to reduce the detection limit of ELISA for other antigens.

Angiotensin II type 1 receptor antibodies in kidney transplantation

Angiotensin II type 1 receptor (AT1R) antibodies directly injure endothelial and vascular smooth muscle cells by activating transcription factors associated with proinflammatory responses. Previous studies have shown that AT1R antibodies are associated with allograft rejection and decreased graft survival in kidney transplantation. Development of enzyme-linked immunosorbent assay has facilitated semiquantitative detection of AT1R antibodies. Assessing AT1R antibodies along with donor specific human leukocyte antigen antibodies may have potential to identify patients with possible risk for allograft injury and improve overall outcomes. In this review, we summarize recent clinical studies about AT1R antibodies in kidney transplantation and provide perspectives for future research area.

Розробка імуноферментної тест-системи для діагностики лептоспірозу тварин

Розробка імуноферментної тест-системи для діагностики лептоспірозу тварин

Development of Enzyme-Linked Immunosorbent Assay for Analysis of Total Aflatoxins Based on Monoclonal Antibody Reactive with Aflatoxins B1, B2, G1 and G2.

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was developed for the determination of total amount of aflatoxin B1, B2, G1 and G2 (AFB1, AFB2, AFG1 and AFG2), using a mouse monoclonal antibody that shows similar reactivity to each of these AFs. The working range of the developed dc-ELISA was 50-230 pg/mL for AFB1, 50-270 pg/mL for AFB2, 60-390 pg/mL for AFG1 and 65-700 pg/mL for AFG2. The recovery of AFs from spiked roasted peanuts was 98％. Further, when 4 samples actually contaminated with AFB1, AFB2, AFG1 and AFG2 were examined, the results of dc-ELISA were highly correlated with the values assigned by the Food Analysis Performance Assessment Scheme. The developed dc-ELISA appears to be suitable for the determination of total AFs at concentrations around the maximum permitted level (10 μg/kg for all foods) in Japan.

Development of enzyme-linked immunosorbent assay for early pregnancy diagnosis in cattle

The prerequisite for enhancing reproductive efficacy and successful calving is an early and accurate diagnosis of pregnancy. A highly sensitive enzyme-linked immunosorbent assay (ELISA) utilizing the second antibody coating technique and the horseradish peroxidase conjugate as a label for the determination of Interferon-stimulated protein, 15 kDa (ISG15) in dairy cows was developed. To validate a neutrophil lysate based ELISA for early pregnancy diagnosis, blood samples from healthy multiparous Karan Fries (KF) cows were collected on day 0 (day of AI), 10, 14, 16 and 21 post artificial insemination. The pregnancy and non-pregnancy in cows were confirmed by plasma progesterone assay, ultrasonography and per rectal palpation. The detection range of the assay was 0.312 to 25 ng/ml with a sensitivity of 0.13 ng/ml. The inter-assay and intra-assay coefficient of variation was 12.1% and 10.1%, respectively. The parallelism for measured and expected concentrations had an r2 value of 0.90 and 0.93, respectively. It was observed that ISG15 concentration was greater (P < 0.01) in pregnant as compared to non-pregnant cows, the greatest being 9.36 ± 0.50 ng/ml on day 16 in pregnant cows. With the use of immunocytochemistry, there was a positive staining for ISG15 with greater staining in the neutrophils of day 16 pregnant cows. The study reveals that ISG15 protein in a neutrophil lysate can be utilized as a biomarker for early pregnancy diagnosis in cows.

Analysis of Carbamates Pesticides: Immunogical Technique by Local Development of Enzyme-linked Immuno-Sorbent Assay

Carbamates insecticides are used increasingly in agriculture as a replacement for environmentally more persistent organochlorine insecticides for the control of insect pests. It was the most used pesticides in Loukkous-Morocco. Conventional methods employed to detect/analyze carbamates residues are time consuming and require sophisticated equipment only available in well-equipped laboratories. In addition, the conventional methods usually require a lot of complex pre-treatment of samples. Therefore, convenient and rapid pesticide detection system is urgently needed. In this order, we aimed to develop a rapid ELISA (enzyme-linked immuno-Sorbent Assay) for detection of some carbamates such as carbendazim [methyl 2-benzimidazole carbamate] and carbofuran [2,3-dihydro-2,2-dimethyl-7-benzofuranylmethyl carbamate]. To develop an immunoassay for carbendazim and carbofuran we have synthesized molecules with acid function (haptens with 4 and 5 carbons) that are coupled with BSA protein and injected to the rabbits, collected antibodies are used for the achievements of the immunoanalytical assay.

Development of enzyme-linked immunosorbent assay for oxidized high density lipoprotein and its clinical application for cardiovascular risk assessment

Development of enzyme-linked immunosorbent assay for oxidized high density lipoprotein and its clinical application for cardiovascular risk assessment

Assessment of influenza A neuraminidase (subtype N1) potency by ELISA

Antibodies that inhibit neuraminidase (NA) activity of influenza virus provide resistance against disease and have been associated with milder epidemics. Although studies have demonstrated a correlation between NA inhibition antibody titers and vaccine efficacy, neither the quantity nor form of NA is measured in seasonal and pandemic influenza vaccines. In this report, we describe development of enzyme-linked immunosorbent assays (ELISAs) that are suitable for quantitation of the native form of NA of subtype N1. The assays use mouse monoclonal antibodies (mAbs) 1H5 and CD6 to capture NAs of viruses, and a different mAb 4E9 to detect bound antigen. The 1H5-capture ELISA detects NAs of seasonal and pandemic H1N1 viruses as well as H5N1 viruses and has a limit of quantitation (LOQ) of 5.5ng/mL for seasonal H1N1A/Brisbane/59/2007 NA. The CD6-capture ELISA is specific for NA of the 2009 pandemic viruses with a LOQ of 67ng/mL for A/California/07/2009 NA. The ELISA signals in both assays are proportional to NA enzymatic activity and correlate with NA immunogenicity. The ELISAs we describe may expedite the development of NA-based influenza vaccines by providing a practical assay to measure NA potency.

Monoclonal antibodies specific for Bacteroides fragilis enterotoxins BFT1 and BFT2 and their use in immunoassays.

We have developed 22 mouse IgG1 monoclonal antibodies (mAbs) against Bacteroides fragilis zinc metalloprotease toxins 1 and 2 (BFT1 and BFT2). Mice were immunized with recombinant BFT1 or BFT2 proteins with metalloprotease activity. Eight of the mAbs bind specifically to BFT1. One mAb, 2H6, binds specifically to BFT2. The remaining 13 mAbs bind to both BFT1 and BFT2. The eight BFT1-specific mAbs recognize at least five different epitopes on the toxin. Four of the BFT1-specific mAbs neutralized rBFT1 metalloprotease activity. Only one of these four mAbs, 1D9, neutralizes the cytotoxic effect of BFT1. Here, we describe the development of enzyme-linked immunosorbent assays (ELISAs) to detect BFT1 or BFT2 toxin in an isotype-specific manner. The sandwich ELISAs have a detection limit of 20 to 40 ng/ml when purified recombinant BFT protein is diluted into PBS. The sandwich ELISA can be used to distinguish and quantify levels of rBFT1 and rBFT2 in stool. This ELISA can be an important tool to investigate the association between BFT expression by enterotoxigenic B. fragilis and diseases such as diarrhea, inflammatory bowel disease and colorectal cancer.

Analytical Validation of the ReEBOV Antigen Rapid Test for Point-of-Care Diagnosis of Ebola Virus Infection.

Ebola virus disease (EVD) is a severe viral illness caused by Ebola virus (EBOV). The 2013-2016 EVD outbreak in West Africa is the largest recorded, with >11 000 deaths. Development of the ReEBOV Antigen Rapid Test (ReEBOV RDT) was expedited to provide a point-of-care test for suspected EVD cases. Recombinant EBOV viral protein 40 antigen was used to derive polyclonal antibodies for RDT and enzyme-linked immunosorbent assay development. ReEBOV RDT limits of detection (LOD), specificity, and interference were analytically validated on the basis of Food and Drug Administration (FDA) guidance. The ReEBOV RDT specificity estimate was 95% for donor serum panels and 97% for donor whole-blood specimens. The RDT demonstrated sensitivity to 3 species of Ebolavirus (Zaire ebolavirus, Sudan ebolavirus, and Bundibugyo ebolavirus) associated with human disease, with no cross-reactivity by pathogens associated with non-EBOV febrile illness, including malaria parasites. Interference testing exhibited no reactivity by medications in common use. The LOD for antigen was 4.7 ng/test in serum and 9.4 ng/test in whole blood. Quantitative reverse transcription-polymerase chain reaction testing of nonhuman primate samples determined the range to be equivalent to 3.0 × 105-9.0 × 108 genomes/mL. The analytical validation presented here contributed to the ReEBOV RDT being the first antigen-based assay to receive FDA and World Health Organization emergency use authorization for this EVD outbreak, in February 2015.

Development of a highly sensitive and specific monoclonal antibody based enzyme-linked immunosorbent assay for the detection of a new β-agonist, phenylethanolamine A, in food samples.

All β-agonists are banned as feed additives for growth promotion in animals due to toxic effects on humans after consuming the β-agonist contaminated meats. Phenylethanolamine A (PA) is a newly emerged β-agonist. Thus there is a need to develop highly sensitive and specific analytical methods for the detection of PA in food samples. In this study, the monoclonal antibody (mAb) against PA was produced by hybridoma technology and used for the development of enzyme-linked immunosorbent assay (ELISA). The IC50 values and limits of detection (LODs) of the ELISA using homogeneous combination of coating antigen/antibody for PA were 0.16 ng mL-1 and 0.011 ng mL-1 , respectively. The cross-reactive (CR) values of the assay with 14 structurally related β-agonists were lower than 0.59%. Swine liver and meat samples were spiked with PA at different content and analysed by ELISA. Acceptable recovery rates of 91.40-105.51% and intra-assay coefficients of variation of 1.56-9.92% (n = 3) were obtained. The ELISA for seven spiked samples was confirmed by LC-MS/MS with a high correlation coefficient of 0.9881. The proposed mAb-based ELISA was highly sensitive and specific for PA and could be used as a quantitative/screening method for PA analysis in food samples. © 2016 Society of Chemical Industry.

Development of Enzyme-Linked Immunosorbent Assay for Determination of Boldenone in Dietary Supplements

There is an increasing interest in the investigation and implementation of methods for the analysis of anabolic steroids in dietary supplements. In this study, a competitive indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of boldenone. For this purpose, an antiserum against boldenone was raised in a rabbit using boldenone-17-hemisuccinate-bovine serum albumin as an immunization conjugate. Based on the ELISA standard curve, the detection limit was as low as 0.014 ± 0.007 ng mL−1 with the IC50 value of 0.293 ± 0.084 ng mL−1 and linear working range of 0.065–1.529 ng mL−1. The intra- and inter-assay variations were found to be satisfactory. Relative standard deviations were calculated in the range of 3.8–10.5 and 7.3–12.9 %. The developed ELISA was applied in the analysis of extracts obtained from spiked samples of dietary supplements. Ethanol extracts were applied into the immunoassay without a cleanup procedure (only diluted) to minimize the effect of the matrix. Recoveries from spiked samples were from 86.0 to 115.7 %. An excellent correlation between ELISA and UHPLC-MS/MS was obtained with the linear equation of y = 1.0256 x − 0.7772 (R 2 = 0.9999). This ELISA as proposed here could be successfully applied for the simple monitoring of boldenone in dietary supplements.

Issues encountered in development of enzyme-linked immunosorbent assay for use in detecting Influenza A virus subtype H5N1 exposure in swine

A potential mechanism by which highly pathogenic avian Influenza A virus subtype H5N1 could more readily infect human beings is through the infection of and adaptation in pigs. To detect the occurrence of such infection, monitoring of pig populations through serological screening would be highly desirable. In the current study, hemagglutination inhibition assays were able to detect antibodies against H5N1 developed in pigs, but because of antigenic variation between clades, the use of multiple virus strains were required. Whole recombinant virus and recombinant hemagglutinin antigen enzyme-linked immunosorbent assays (ELISAs) were generated that could detect antibody against multiple H5N1 strains, but which also detected antibody against endemic swine influenza viruses. A recombinant hemagglutinin antigen-based ELISA was as effective as the whole virus antigen ELISAs in detecting antibody against the H5N1 virus strains used and eliminated nearly all of the cross-reactivity with non-H5N1 virus antibody. The current study also highlighted the difficulty in establishing a decision (cutoff) value that would effectively counterbalance nonspecific reactivity against sensitivity. The results provide important information and considerations for the development of serological screening assays for highly pathogenic avian H5N1 viruses.

Амперометрические моноаминоксидазные биосенсоры на основе графитовых электродов и оксида графена как модификатора поверхности для определения некоторых антидепрессантов

Амперометрические моноаминоксидазные биосенсоры на основе графитовых электродов и оксида графена как модификатора поверхности для определения некоторых антидепрессантов