scholarly journals Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Yi Ma ◽  
Chao Ni ◽  
Emmanuel E. Dzakah ◽  
Haiying Wang ◽  
Keren Kang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Early Diagnosis ◽  
Colloidal Gold ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Immunochromatographic Assay ◽  
Suitable Alternative ◽  
P24 Protein ◽  
Hybridoma Cell Lines ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed inE. colistrain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection.

scholarly journals Development of a Colloidal Gold Immunochromatographic Assay for Duck Enteritis Virus Detection Using Monoclonal Antibodies

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 365
Author(s):  
Fengli Liu ◽  
Yanxin Cao ◽  
Maokai Yan ◽  
Mengxu Sun ◽  
Qingshui Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Colloidal Gold ◽  
Preventive Intervention ◽  
Virus Detection ◽  
Cross Reactivity ◽  
Duck Enteritis Virus ◽  
Detection Sensitivity ◽  
Immunochromatographic Assay ◽  
Efficient Detection ◽  
Enteritis Virus

Duck viral enteritis is a highly contagious and fatal disease of commercial waterfowl flocks. The disease occurs sporadically or epizootically in mainland China due to insufficient vaccinations. Early and rapid diagnosis is important for preventive intervention and the control of epizootic events in clinical settings. In this study, we generated two monoclonal antibodies (MAbs) that specifically recognized the duck enteritis virus (DEV) envelope glycoprotein B and tegument protein UL47, respectively. Using these MAbs, a colloidal gold-based immunochromatographic assay (ICA) was developed for the efficient detection of DEV antigens within 15 min. Our results showed that the detection limit of the developed ICA strip was 2.52 × 103 TCID50/mL for the virus infected cell culture suspension with no cross-reactivity with other pathogenic viruses commonly encountered in commercially raised waterfowl. Using samples from experimentally infected ducks, we demonstrated that the ICA detected the virus in cloacal swab samples on day three post-infection, demonstrating an 80% concordance with the PCR. For tissue homogenates from ducks succumbing to infection, the detection sensitivity was 100%. The efficient and specific detection by this ICA test provides a valuable, convenient, easy to use and rapid diagnostic tool for DVE under both laboratory and field conditions.

scholarly journals Surface Plasmon Resonance Assay for Label-Free and Selective Detection of HIV-1 p24 Protein

Biosensors ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 180
Author(s):  
Lucia Sarcina ◽  
Giuseppe Felice Mangiatordi ◽  
Fabrizio Torricelli ◽  
Paolo Bollella ◽  
Zahra Gounani ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Covalent Binding ◽  
Hiv Infections ◽  
Selectivity Ratio ◽  
Selective Detection ◽  
Label Free ◽  
Self Assembled Monolayer ◽  
Label Free Detection ◽  
P24 Protein ◽  
Hiv 1

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10−9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10−5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.

scholarly journals Development of Vasoinhibin-Specific Monoclonal Antibodies

2021 ◽  
Vol 12 ◽  
Author(s):  
Nils Müller ◽  
Juan Pablo Robles ◽  
Magdalena Zamora ◽  
Johannes Ebnet ◽  
Hülya Markl-Hahn ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Serum Proteins ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Vascular Diseases ◽  
High Specificity ◽  
Cross Reactivity ◽  
Dimensional Structure ◽  
Serum Samples ◽  
Limit Of Quantitation

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

Development of a Sandwich ELISA to Detect Hepcidin in Human Serum.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2000-2000 ◽  
Author(s):  
Tara L Arvedson ◽  
George Doellgast ◽  
Hossein Salimi-Moosavi ◽  
Chadwick King ◽  
Ian Foltz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Polyclonal Antibody ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Alkaline Treatment ◽  
Detection Range ◽  
Antibody Preparation ◽  
Amino Acid Peptide ◽  
Class 1

Abstract Abstract 2000 Poster Board I-1022 Hepcidin is a 25 amino acid peptide that is the central mediator of iron metabolism. Iron excess, deficiency and maldistribution have been implicated in the etiology of many diseases including atherosclerosis, diabetes, neurodegeneration and the anemia of inflammation. Determination of hepcidin levels may be useful in diagnosis and treatment decisions for some or all of these diseases. Serum hepcidin measurement has so far been limited to a prohepcidin (60 amino acid hepcidin precursor) ELISA, mass spectrometry (MS)-based assays or competition ELISAs using polyclonal anti-hepcidin antibodies. The current work describes the generation of a sandwich ELISA using monoclonal antibodies to detect human hepcidin (hHepc) and optimization of assay conditions to resolve inconsistencies between MS- and ELISA-based detection. The ability of two anti-hHepc antibodies to sandwich (bind simultaneously) with hHepc was demonstrated using a rabbit polyclonal antibody preparation from hHepc-immunized animals. The same polyclonal antibody preparation was used for both hHepc capture and detection. The limit of detection achieved with this assay was O.D.450<1, suggesting that only a small proportion of the total antibodies could bind concurrently. To improve hHepc detection, a panel of monoclonal antibodies was screened for the ability to sandwich. Antibody epitope characterization studies using purified antibodies and >1000 hybridoma supernatants identified three classes of antibodies: classes 1 and 2 each recognized epitopes found in both full length mature hHepc (hHepc 25) and a shorter version (hHepc 20); class 3 bound a different epitope and demonstrated an increased affinity for hHepc 25 over hHepc 20. The majority of antibodies characterized were in class 1 while antibodies in classes 2 and 3 were rare (∼1% of antibody panel) highlighting the difficulty in achieving a sandwiching event. Antibodies 19D12 (class 1) and 23F11 (class 2) were identified as the optimal sandwich pair with a detection range of approximately 0.2-1000 ng/ml using synthetic hHepc. Initial comparisons of data generated using the sandwich ELISA and a fully-quantitative MS-based assay demonstrated a lack of consistent agreement. This issue was somewhat addressed by introduction of an alkaline treatment step to dissociate any protein/hHepc complexes in serum. Subsequent comparison of the two assays using sera from several different patient populations (anemia of cancer, chemotherapy-induced anemia, kidney disease) as well as healthy donors demonstrated good correlation (R2 range = 0.83-0.92; n=237). This sandwich ELISA may represent a tool for aligning the MS and ELISA-generated results in a format that has the potential to be high throughput and widely available. Disclosures: Arvedson: Amgen: Employment. Doellgast:Amgen: Employment. Salimi-Moosavi:Amgen: Employment. King:Amgen: Employment. Foltz:Amgen: Employment. Chen:Amgen: Employment. Li:Amgen: Employment. Sasu:Amgen: Employment.

scholarly journals Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 383
Author(s):  
Yanan Wang ◽  
Xiaofei Wang ◽  
Haitang Zhang ◽  
Hanna Fotina ◽  
Jinqing Jiang
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Limit Of Detection ◽  
Broad Class ◽  
Rapid Test ◽  
Competitive Elisa ◽  
Affinity Constant ◽  
High Affinity ◽  
Ic50 Values ◽  
Hybridoma Cell Lines

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

scholarly journals Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 533 ◽  
Author(s):  
Takeshi Tsumuraya ◽  
Masahiro Hirama
Keyword(s):  
Monoclonal Antibodies ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Food Poisoning ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fda Guidance ◽  
Fish Poisoning ◽  
Highly Sensitive ◽  
Taste And Smell ◽  
Reliable Methods

“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

scholarly journals Efficient detection of pre-proinsulin by double antibody sandwich ELISA

2020 ◽  
Author(s):  
Zhu Zhu ◽  
Guoliang Ma ◽  
Li Wang ◽  
Han Wang ◽  
Hengfang Tang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Insulin ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Insulin Production ◽  
Recombinant Human Insulin ◽  
Elisa Method ◽  
Efficient Detection ◽  
Sds Page ◽  
Conventional Cell

Abstract ObjectiveTo generate monoclonal antibodies against pre-proinsulin (PPI), and establish sandwich ELISA method to provide a basis for PPI detection in recombinant human insulin production.MethodsThe Balb /c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained by conventional cell fusion technique and ELISA screening.The antibody was purified using a Protein G gel column and identified for purity by SDS-PAGE. Pairing effect was found by the sandwich ELISA, and the specificity of the paired antibody was determined. A paired antibody with better specificity was selected to establish sandwich ELISA, and construct a quantitative curve, the accuracy and sensitivity of the method were evaluated.ResultsSix anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer. The sandwich ELISA method was established with P5 for plating and P2 for detection antibodie. The linear range of the quantitative curve of PPI by sandwich ELISA was 0. 645-82.5 pg/mL, the recovery was 89%–95%, and the limit of detection was 3.06 pg/mL.ConclusionSix monoclonal antibodies against PPI were generated and the sandwich ELISA method was established to detect PPI in process control and product release control for recombinant human insulin production.

Development of a Colloidal Gold-Based Immunochromatographic Assay for the Rapid Detection of Aflatoxin B1 in Food Samples

2013 ◽  
Vol 726-731 ◽  
pp. 1279-1282 ◽  
Author(s):  
Xue Qing Chen ◽  
Sha Sha Lu ◽  
Qing Sun ◽  
Jing Chen Yang ◽  
Guo Qing Shi
Keyword(s):  
Colloidal Gold ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Food Samples ◽  
Cross Reactivity ◽  
Immunochromatographic Assay ◽  
Rapid Testing ◽  
Elisa Method ◽  
Standard Solution ◽  
Aflatoxin B

A rapid and sensitive competitive immunochromatographic assay for aflatoxin B1 (AFB1) based on colloidal gold-antibody probe was developed. The limit of detection for AFB1 standard solution was 0.5ng/ml and for foodstuff sample was 10μg/Kg. Other aflatoxins including AFM1, AFB2, AFG1, and AFG2 have less cross-reactivity to the strip. The storge life of the strip was at least 12 monthes. The method was well agreement with the ELISA method by testing food samples. This method is suitable for rapid testing of AFB1 on site.

Application of Monoclonal Antibodies to Dulcitol 1-Phosphate Dehydrogenase for Rapid Detection of Salmonella

1995 ◽  
Vol 58 (8) ◽  
pp. 847-852 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
HUAIZE TIAN ◽  
KIYOSHI MATSUNO ◽  
RYOJI TAKATA ◽  
SHOJI HATANO
Keyword(s):  
Monoclonal Antibodies ◽  
Salmonella Typhimurium ◽  
Magnesium Chloride ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Inoculum Level ◽  
E Coli

Monoclonal antibodies raised against dulcitol 1-phosphate dehydrogenase of Salmonella typhimurium IFO 12529 were screened against 20 serotypes of Salmonella and 13 non-Salmonella bacteria. A sandwich-capture, enzyme-linked immunosorbent assay (sandwich ELISA) was developed for detection of Salmonella in food. The assay utilizes two monoclonal antibodies (DUI2 and DU28) which show no cross-reactions with non-Salmonella bacteria. The limit of detection of the sandwich ELISA was about 1 × 107 CPU/ml. After cultivation in a medium containing dulcitol at 37°C for 18 h followed by the sandwich ELISA. 1 CPU of Salmonella was detected. Although a high inoculum level of E. coli interfered with the detection of Salmonella, the interference was minimized by using a selective dulcitol-magnesium chloride-pyridinesulfonic acid medium for enrichment. The novel ELISA procedure detected Salmonella in chicken filtrates inoculated with 1.4 CPU/50 m1 and 1.3 × 107 CPU/50 ml of E. coli within 25 h.

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