Step by Step with ELISA: Mechanism of Operation, Crucial Elements, Different Protocols, and Insights on Immobilization and Detection of Various Biomolecular Entities

Hosseini, Samira; Vázquez-Villegas, Patricia; Rito‐Palomares, Marco; Martínez-Chapa, Sergio O.

doi:10.1007/978-981-10-6766-2_3

Cited by 4 publications

(5 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Section: Reasons For False Negatives In Antibody Immunoassaysmentioning

confidence: 99%

“…Then the unreacted protein reagents might not be thoroughly eliminated from the gel network by the conventional washing buffer, hence resulting in an unacceptable level of background noise originated from the substrate. 178 Antigens Used. To date, N and S proteins as well as their subunits have been used for developing antibody assays (Table S1).…”

Section: Immunoassaysmentioning

confidence: 99%

“…However, the polymers may change from the linear form to cross-linked gel after reacting with cross-linker reagents such as glutaraldehyde in the covalent immobilization. Then the unreacted protein reagents might not be thoroughly eliminated from the gel network by the conventional washing buffer, hence resulting in an unacceptable level of background noise originated from the substrate …”

Section: Reasons For False Negatives In Antibody Immunoassaysmentioning

confidence: 99%

See 2 more Smart Citations

COVID-19 Antibody Tests and Their Limitations

2021

View full text Add to dashboard Cite

COVID-19, caused by the SARS-CoV-2 virus, has developed into a global health crisis, causing over 2 million deaths and changing people’s daily life the world over. Current main-stream diagnostic methods in the laboratory include nucleic acid PCR tests and direct viral antigen tests for detecting active infections, and indirect human antibody tests specific to SARS-CoV-2 to detect prior exposure. In this Perspective, we briefly describe the PCR and antigen tests and then focus mainly on existing antibody tests and their limitations including inaccuracies and possible causes of unreliability. False negatives in antibody immunoassays can arise from assay formats, selection of viral antigens and antibody types, diagnostic testing windows, individual variance, and fluctuation in antibody levels. Reasons for false positives in antibody immunoassays mainly involve antibody cross-reactivity from other viruses, as well as autoimmune disease. The spectrum bias has an effect on both the false negatives and false positives. For assay developers, not only improvement of assay formats but also selection of viral antigens and isotopes of human antibodies need to be carefully considered to improve sensitivity and specificity. For clinicians, the factors influencing the accuracy of assays must be kept in mind to test patients using currently imperfect but available tests with smart tactics and realistic interpretation of the test results.

show abstract

Section: Reasons For False Negatives In Antibody Immunoassaysmentioning

confidence: 99%

Section: Immunoassaysmentioning

confidence: 99%

Section: Reasons For False Negatives In Antibody Immunoassaysmentioning

confidence: 99%

See 1 more Smart Citation

COVID-19 Antibody Tests and Their Limitations

2021

View full text Add to dashboard Cite

show abstract

“…The prominent types of biosensorsutilized in clinical diagnostic automated FIA/SIAare enzyme-based, immunological, and nucleic acid–based . For instance, immunological biosensorssuch as enzyme-linked immunosorbent assay (ELISA)require meticulous pipetting and manipulation of reagents and samples at every step: coating of sensors with antibodies, blocking, washing, introduction of samples, addition of detection antibodies, incubating and washing, addition of enzyme conjugates, inclusion of substrates, and introduction of stopping solution . During each individual step, analysts must ensure precise and accurate execution of steps to obtain reliable results.…”

Section: Different Types Of Sensing Systems With Elements Of Automationmentioning

confidence: 99%

“… 38 For instance, immunological biosensors—such as enzyme-linked immunosorbent assay (ELISA)—require meticulous pipetting and manipulation of reagents and samples at every step: coating of sensors with antibodies, blocking, washing, introduction of samples, addition of detection antibodies, incubating and washing, addition of enzyme conjugates, inclusion of substrates, and introduction of stopping solution. 39 During each individual step, analysts must ensure precise and accurate execution of steps to obtain reliable results. Automation of these steps by FIA/SIA reduces the variabilities caused by manual pipetting and manipulation of reagents and samples.…”

Section: Different Types Of Sensing Systems With Elements Of Automationmentioning

confidence: 99%

Automation and Computerization of (Bio)sensing Systems

Raju,

Elpa,

Urban

2024

ACS Sens.

View full text Add to dashboard Cite

Sensing systems necessitate automation to reduce human effort, increase reproducibility, and enable remote sensing. In this perspective, we highlight different types of sensing systems with elements of automation, which are based on flow injection and sequential injection analysis, microfluidics, robotics, and other prototypes addressing specific real-world problems. Finally, we discuss the role of computer technology in sensing systems. Automated flow injection and sequential injection techniques offer precise and efficient sample handling and dependable outcomes. They enable continuous analysis of numerous samples, boosting throughput, and saving time and resources. They enhance safety by minimizing contact with hazardous chemicals. Microfluidic systems are enhanced by automation to enable precise control of parameters and increase of analysis speed. Robotic sampling and sample preparation platforms excel in precise execution of intricate, repetitive tasks such as sample handling, dilution, and transfer. These platforms enhance efficiency by multitasking, use minimal sample volumes, and they seamlessly integrate with analytical instruments. Other sensor prototypes utilize mechanical devices and computer technology to address real-world issues, offering efficient, accurate, and economical real-time solutions for analyte identification and quantification in remote areas. Computer technology is crucial in modern sensing systems, enabling data acquisition, signal processing, real-time analysis, and data storage. Machine learning and artificial intelligence enhance predictions from the sensor data, supporting the Internet of Things with efficient data management.

show abstract

Universal scaling theory of electrochemical immunosensors: An analytical approach to define and compare performance metrics

Fratus

2023

Applied Physics Letters

View full text Add to dashboard Cite

Electrochemical immunosensors have emerged as a versatile, sensitive, and selective sensor technology of choice for a variety of applications, including detection of proteins, food pathogens, bacteria, viruses, and cancerous molecules. The combination of highly specific biorecognition elements and electrical readout systems facilitates the detection of antigens down to femtomolar concentrations. However, a lack of quantitative theoretical framework has made the design, optimization, and comparison of sensors difficult, without a clear and definitive understanding of the limits of detection, dynamic range, and sensitivity. In this paper, we integrate reaction-diffusion and effective media theories to derive a generalized scaling model for an arbitrary immunosensor that relates the relative change of redox current to the corresponding change in antigen concentration, through scaling exponents related to the geometry of biomolecules diffusion and the measurement resolution. Experimental data from dozens of immunosensors (for a variety of antigens, material systems, and sensor geometry) validate our sensor-agnostic scaling formula. Our results would allow cross-calibration of the emerging and traditional immunosensors reported across the literature and define a physics-based, standardized methodology to compare performance metrics, such as limits of detection, dynamic range, and sensitivity.

show abstract

Step by Step with ELISA: Mechanism of Operation, Crucial Elements, Different Protocols, and Insights on Immobilization and Detection of Various Biomolecular Entities

Cited by 4 publications

References 60 publications

COVID-19 Antibody Tests and Their Limitations

COVID-19 Antibody Tests and Their Limitations

Automation and Computerization of (Bio)sensing Systems

Universal scaling theory of electrochemical immunosensors: An analytical approach to define and compare performance metrics

Contact Info

Product

Resources

About