scholarly journals Development of Real-Time Immuno-PCR Based on Phage Displayed an Anti-Idiotypic Nanobody for Quantitative Determination of Citrinin in Monascus

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 572 ◽  
Author(s):  
Huang ◽  
Tu ◽  
Ning ◽  
He ◽  
Li
Keyword(s):  
Real Time ◽  
Rice Flour ◽  
Cross Reactivity ◽  
Amplification Efficiency ◽  
Elisa Method ◽  
Phage Elisa ◽  
Ic50 Value ◽  
Significant Difference ◽  
Antibody Phage

Citrinin (CIT) is a mycotoxin that has been detected in agricultural products, feedstuff, and Monascus products. At present, research has been performed to develop methods for CIT detection, mainly through TLC, HPLC, biosensor, and immunoassay. The immunoassay method is popular with researchers because of its speed, economy, simplicity, and ease of control. However, mycotoxins are inevitably introduced during the determination. Immunoassays require the use of toxins coupled to carrier proteins or enzymes to make competitive antigens. In this study, anti-idiotypic nanobody X27 as CIT mimetic antigen was used as non-toxic surrogate reagents in immunoassay. Therefore, the X27-based real-time immuno-PCR (rtIPCR) method had been established after optimal experiments of annealing temperature and amplification efficiency of real-time PCR, concentration of coating antibody, phage X27, and methyl alcohol. The IC50 value of the established method in the present study is 9.86 ± 2.52 ng/mL, which is nearly equivalent to the traditional phage ELISA method. However, the linear range is of 0.1–1000 ng/mL, which has been broadened 10-fold compared to the phage ELISA method. Besides, the X27-based rtIPCR method has no cross-reactivity to the common mycotoxins, like aflatoxin B1 (AFB1), deoxynivalenol (DON), ochratoxin A (OTA), and zearalenone (ZEN). The method has also been applied to the determination of CIT in rice flour and flour samples, and the recovery was found to be in the range of 90.0–104.6% and 75.8–110.0% respectively. There was no significant difference in the results between the rtIPCR and UPLC–MS. The anti-idiotypic nanobody as a non-toxic surrogate of CIT makes rtIPCR a promising method for actual CIT analysis in Monascus products.

Development and Characterization of Anti-Estradiol Polyclonal Antibody

2012 ◽  
Vol 461 ◽  
pp. 67-70 ◽  
Author(s):  
Chao Ying Li ◽  
Jin Qing Jiang
Keyword(s):  
Polyclonal Antibody ◽  
Dynamic Range ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Uv Absorbance ◽  
Standard Curve ◽  
Ic50 Value ◽  
New Zealand White Rabbits

This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.

Diarrhetic Shellfish Poisoning: Evaluation of Enzyme-Linked Immunosorbent Assay Methods for Determination of Dinophyslstoxin-2

1995 ◽  
Vol 78 (6) ◽  
pp. 1403-1407 ◽  
Author(s):  
Eoin P Carmody ◽  
Kevin J James ◽  
Seán S Kelly
Keyword(s):  
Immunosorbent Assay ◽  
Cross Reactivity ◽  
Regulatory Control ◽  
Enzyme Linked Immunosorbent Assay ◽  
Shellfish Poisoning ◽  
Diarrhetic Shellfish Poisoning ◽  
Elisa Method ◽  
Multiple Samples ◽  
Liquid Chromatographic

Abstract Dinophysistoxin-2 (DTX-2), an isomer of okadaic acid (OA), recently has been found in Irish waters. DTX-2 was the predominant toxin during prolonged infestations in cultivated mussels along the southwest coast of Ireland. Substantial variations in toxin levels may exist both horizontally and vertically in the water column. The need to take multiple samples and the ethical concern about the use of mammals for routine quality control of shellfish prompted examination of 2 commercially available enzyme-linked immunosorbent assay (ELISA) methods, designed to detect OA, for determination of both OA and DTX-2. One ELISA method (DSPCheck, Sceti Co. Ltd., (Tokyo, Japan) showed good cross-reactivity (40 ± 5%) with standard DTX-2. This study showed that both ELISA methods show good correlation with the liquid chromatographic analysis of 9-anthryldiazomethane derivatives when OA is the predominant toxin present. The sensitivity was also good for OA determination using both methods, which allowed toxin measurement at 10 ng/mL (0.5 ng/well). This level is equivalent to 0.03 μg/g mussel meat. Blank mussel samples spiked with DTX-2 standards gave a good linear correlation (r = 0.997) with this ELISA method when toxin levels were 0.03-0.3 μg/g mussel meat. This range is appropriate for regulatory control of diarrhetic shellfish poisoning.

Synthesis and Identification of Norfloxacin Artificial Antigen

2012 ◽  
Vol 433-440 ◽  
pp. 697-702 ◽  
Author(s):  
Jun Wei Liu ◽  
Jin Qing Jiang ◽  
Hai Tang Zhang ◽  
Guo Ying Fan ◽  
Zhi Xing An
Keyword(s):  
Dynamic Range ◽  
Food Samples ◽  
Cross Reactivity ◽  
Assay Performance ◽  
Ic50 Value ◽  
Artificial Antigen ◽  
Sds Page ◽  
Visible Spectra ◽  
Ic50 Values

A multiresidue immunoassay method for determination of Fluoroquinolones (FQs) residues has been developed. For this purpose, NHS ester technology was employed to synthesize the immunogen and coating antigen of Norfloxacin (NFLX). SDS-PAGE, UV-visible spectra and Infrared spectra identification showed that the artificial antigen was conjugated successfully. Based on the square matrix titration, an icELISA method was established. The dynamic range in assay buffer was from 0.038 to 112.8 ng/mL, with LOD and IC50 value of 0.02 ng/mL and 1.2 ng/mL, respectively. This assay showed a high cross-reactivity to Ciprofloxacin (86%), Enrofloxacin (75%), Difloxacin (63%), Sarafloxacin (57%) and Pefloxacin (33.8%). The chemical effects on assay performance showed that the physiological pH (7.4) in assay buffer pursued the maximum absorbance (Amax) and the most sensitive IC50 values. The results suggest the artificial antigen was synthesized successfully, and the established immunoassay could be used for simultaneous detecting of Norfloxacin, Ciprofloxacin, Enrofloxacin, Difloxacin, Sarafloxacin and Pefloxacin residues in animal-original food samples.

scholarly journals Evaluation of a duplex reverse-transcription real-time PCR assay for the detection of encephalomyocarditis virus

2018 ◽  
Vol 30 (4) ◽  
pp. 554-559 ◽  
Author(s):  
Shaomin Qin ◽  
Darren Underwood ◽  
Luke Driver ◽  
Carol Kistler ◽  
Ibrahim Diallo ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Reverse Transcription ◽  
Real Time Pcr ◽  
Virus Isolation ◽  
Cross Reactivity ◽  
Encephalomyocarditis Virus ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Clinical Specimens

We evaluated a fluorogenic probe–based assay for the detection of encephalomyocarditis virus (EMCV) by comparing a set of published primers and probe to a new set of primers and probe. The published reagents failed to amplify a range of Australian isolates and an Italian reference strain of EMCV. In contrast, an assay based on 2 new sets of primers and probes that were run in a duplex reverse-transcription real-time PCR (RT-rtPCR) worked well, with high amplification efficiency. The analytical sensitivity was ~100-fold higher than virus isolation in cell culture. The intra-assay variation was 0.21–4.90%. No cross-reactivity was observed with a range of other porcine viruses. One hundred and twenty-two clinical specimens were tested simultaneously by RT-rtPCR and virus isolation in cell culture; 72 specimens gave positive results by RT-rtPCR, and 63 of these were also positive by virus isolation. Of 245 archived cell culture isolates of EMCV that were tested in the RT-rtPCR, 242 samples were positive. The new duplex RT-rtPCR assay is a reliable tool for the detection of EMCV in clinical specimens and for use in epidemiologic investigations.

scholarly journals Highly Specific Chemiluminescence Immunoassay for the Determination of Chloramphenicol in Cosmetics

2019 ◽  
Vol 2019 ◽  
pp. 1-6
Author(s):  
Qiyan Li ◽  
Riran Zhu ◽  
Jun Li ◽  
Xiaobing Wang ◽  
Lihua Xu ◽  
...  
Keyword(s):  
Quality Control ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Chemiluminescence Immunoassay ◽  
Detection Range ◽  
Direct Immunoassay

A direct and highly specific chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) method for monitoring chloramphenicol (CAP) in cosmetics has been developed. The anti-chloramphenicol antibody (mAb) adopted in this work for direct immunoassay could bind to CAP specifically, with negligible cross-reactivity (CR) (less than 0.01%) with most CAP analogues, including structurally related thiamphenicol (TAP) and florfenicol (FF). The limit of detection (LOD), measured by IC10, was 0.0021 ng mL−1. The detection range (IC20-IC80) was ranged from 0.00979 to 0.12026 ng mL−1. In spiked cosmetics samples, mean recoveries ranged from 82.7% to 99.6%, with intraday and interday variation less than 9.8 and 8.2%, respectively. Moreover, with the help of HRP-labeled anti-CAP mAb, the method could be processed in fast direct immunoreaction mode. This CL-ELISA method could be applied for specific, rapid, semiquantitative, and quantitative detection of CAP in cosmetics, facilitating the precise quality control of CAP contamination.

The predominance of Hepatitis C Virus Genotypes and Its Association with the Viral Load in Bangladeshi Population

2021 ◽  
Vol 7 (1) ◽  
pp. 955-959
Author(s):  
Abu Sufian ◽  
Ashish Kumar Majumder ◽  
M Abu Taher ◽  
Md Abul Khaleque ◽  
Sharif Akhteruzzaman ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Viral Load ◽  
Hepatitis C ◽  
Real Time ◽  
Genotype 3 ◽  
Hcv Genotype ◽  
Significant Difference ◽  
Pre Treatment ◽  
Type 3

Determination of hepatitis C virus (HCV) genotype and viral load are two significant prognostic and assessment markers of treatment decisions. The study aimed to determine the predominant HCV types or subtypes and any association with the viral load in Bangladeshi chronic HCV infected patients. A total of 359 anti-HCV positive patients underwent investigation to estimate viral load and determination of genotype and subtype using real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). Among 306 detectable viral loads containing individuals, 278 (90.85%) genotyped successfully, and 28 (9.15%) had unknown genotypes. Among typable genotypes, 1a accounted for 14 (5.03%), 1b for 14 (5.03%), 3 for 247 (88.85%), 4 for 2 (0.72%) and genotype 6 for 1 (0.36%). Based on pre-treatment viral load levels, study subjects classified into three categories such as low (<50000 IU/mL), intermediate (50000-500000 IU/mL), and high (>500000 IU/mL). The majority of HCV other types (1a, 1b, 4, 6) infected patients (96.4%) had intermediate to high viral load compared to those infected with genotype 3 (77.7%) and unclassified types (55.0%) (χ2 =15.41; p = 0.004). HCV type 3 was prevalent (68.4%) in the above 40 years of group compared to less than 40 years group (31.6%). HCV genotype 3 was the predominant genotype circulating in Bangladesh. Pre-treatment viral load demonstrated significant difference among individuals having HCV other types and type 3. However, sequencing the HCV genome analysis would determine the exact types and subtypes among all possible HCV strains available in Bangladesh. Bioresearch Commu. 7(1): 955-959, 2021 (January)

scholarly journals Determination of Azadirachtin in Agricultural Matrixes and Commercial Formulations by Enzyme-Linked Immunosorbent Assay

2001 ◽  
Vol 84 (4) ◽  
pp. 1001-1010 ◽  
Author(s):  
Kalidindi Hemalatha ◽  
Namburi B K Venugopal ◽  
Namburi B K Venugopal ◽  
Beedu S Rao
Keyword(s):  
Immunosorbent Assay ◽  
Cross Reactivity ◽  
Competitive Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Agricultural Commodities ◽  
Indirect Competitive Elisa ◽  
Capture Assay ◽  
Ic50 Value ◽  
Antibody Capture

Abstract An enzyme-linked immunosorbent assay (ELISA) was developed for azadirachtin (aza), a biopesticide from the neem tree (Azadirachta indica A. Juss). The immunogen was synthesized by epoxidation using the furan ring in the aza molecule. Rabbits were immunized with either bovine serum albumin (BSA)-azadirachtin or ovalbumin (OA)-azadirachtin conjugate. Evaluation of the antisera by antibody capture assay showed that the antibody titer of antisera raised against OA-aza was 1:30 000. An indirect competitive ELISA was developed with BSA-azadirachtin as coating antigen and aza-specific antibodies raised against OA-aza immunogen. The immunoassay showed an inhibitory concentration (IC50) value of 75 ppb, with a range of detection from 0.5 to 1000 ppb for azadirachtin [based on regression analysis, y = 85.87 (−18.89x); r2 = −0.97]. Cross-reactivity of the antibodies with 2 aza- derivatives (22,23-dihydro-23β-methoxy azadirachtin and 3-tigloylazadirachtol) was 33 and 29%, respectively. The indirect competitive ELISA was validated and evaluated by quantitating aza in spiked agricultural commodities and from neem formulations. Azadirachtin was spiked into 5 different agricultural commodities: tomato, brinjal, coffee, tea, and cotton seed at 500 and 1000 ppb and recovered at 62–100%. In samples drawn from 6 lots, the aza content in neem-seed kernels ranged from 0.1 to 0.15%; in commercial neem formulations the content ranged from 200 to 2000 ppm. The method developed may be applied to environmental monitoring of aza and quality assurance studies of aza-based commercial formulations.

Detection of Circulating Tissue Factor and Factor VII in a Normal Population

1996 ◽  
Vol 75 (05) ◽  
pp. 772-777 ◽  
Author(s):  
Sybille Albrecht ◽  
Matthias Kotzsch ◽  
Gabriele Siegert ◽  
Thomas Luther ◽  
Heinz Großmann ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Normal Population ◽  
Mean Value ◽  
Factor Vii ◽  
Significant Difference ◽  
Age Dependent ◽  
The Mean ◽  
Highly Correlated ◽  
And Gender

SummaryThe plasma tissue factor (TF) concentration was correlated to factor VII concentration (FVIIag) and factor VII activity (FVIIc) in 498 healthy volunteers ranging in age from 17 to 64 years. Immunoassays using monoclonal antibodies (mAbs) were developed for the determination of TF and FVIIag in plasma. The mAbs and the test systems were characterized. The mean value of the TF concentration was 172 ± 135 pg/ml. TF showed no age- and gender-related differences. For the total population, FVIIc, determined by a clotting test, was 110 ± 15% and the factor VIlag was 0.77 ± 0.19 μg/ml. FVII activity was significantly increased with age, whereas the concentration demonstrated no correlation to age in this population. FVII concentration is highly correlated with the activity as measured by clotting assay using rabbit thromboplastin. The ratio between FVIIc and FVIIag was not age-dependent, but demonstrated a significant difference between men and women. Between TF and FVII we could not detect a correlation.

Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay

1979 ◽  
Author(s):  
Daniel Walz ◽  
Thomas Brown
Keyword(s):  
Blood Clotting ◽  
Factor Xa ◽  
Heart Association ◽  
Serum Levels ◽  
Cross Reactivity ◽  
Assay Procedure ◽  
A Chain ◽  
Prothrombin Activation ◽  
Theoretical Amount

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).

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