Immunological Tests for Diagnosis of Disease and Identification of Molecules
Julius P. Kreier in Infection, Resistance, and Immunity, 2022
The enzyme-linked immunosorbent assay (ELISA) technique is a highly versatile, sensitive, and quantitative procedure that requires little equipment and for which reagents are readily available. It was first introduced in 1971 by Engvall and Perlmann. The term enzyme immunoassay (EIA) is an alternative name for this type of test and is used for the many variants of the assay. Enzymes such as alkaline phosphatase or peroxidase can be linked to antibody without destroying either the antibody′s specificity or the enzyme′s activity. The enzyme acts as a label which makes detection of the antibody possible. Both monoclonal and polyclonal antibodies can be used in this type of test. Enzyme labels are cheaper, simpler to measure, and far more stable than radioactive labels. For these reasons, ELISA or EIA assays have in many cases replaced RIA and have done so while maintaining sensitivity and specificity. Many of the assays for hepatitis A and B in use today are based on ELISA. In addition to the use of enzymes in immunoassays, nonenzy-matic markers can also be used if they can be conjugated to the antibody (e.g., colloidal gold with silver enhancement, and biotination with avidine).
Diagnosis of COVID-19 Using Clinical Examination, Immunoassays, and Molecular Diagnostic Techniques
Srijan Goswami, Chiranjeeb Dey in COVID-19 and SARS-CoV-2, 2022
This serological method takes a much longer time as compared with the rapid test and is useful in gathering epidemiological data, as well as helping to develop treatments. ELISA stands for enzyme-linked immunosorbent assay. In this process, a small amount of serum sample is collected and put into the microtiter well, then IgG from recovered individuals (primary antibody) is added to it. The idea is that the primary antibody will bind to the spike protein antigen of SARS-CoV-2. After that, a secondary antibody tagged with the specific enzyme is added to the reaction mixture. The enzyme-linked secondary antibody binds to the primary antibody. A chromogenic chemical is added to the reaction mixture that acts as the substrate for the enzyme-linked secondary antibody. This enzyme substrate reaction generates a color that is sensed and analyzed by the ELISA reader. The generation of color means a positive test and the intensity of the color is directly proportional to the concentration of antigens. So these are the serology tests that are combined, in addition to RT-PCR (CDC, 2020; Center for Devices and Radiological Health, 2021; Uwamino et al., 2021; Kang et al., 2021).
Paragonimus
Dongyou Liu in Handbook of Foodborne Diseases, 2018
Many variants of ELISA have been described, such as indirect ELISA, two-site capture ELISA, and cystatin capture ELISA.40 Indirect ELISA is one of the most widely used methods. Various antigens have been used to detect specific antibodies in patient serum. These include crude antigen extracts of adult or juvenile worms, excretory-secretory products, recombinant peptides, and various purified or partially purified antigens including cysteine proteases or egg extracts of Paragonimus worms. Excretory-secretory antigens provided higher sensitivity than did somatic antigens (100% as opposed to 91.3%).74 Several cysteine proteases of P. westermani purified from crude somatic extracts of adult worms have been used for immunological tests, such as the cruzipain-like cysteine protease (Pw28CCP).75 Subsequently, Pw28CCP was cloned, and the recombinant protein was expressed. The protease is useful for differential diagnosis of active paragonimiasis from chronic calcified infection.75 Recently, a synthetic peptide has been applied for detection of IgG4 antibody.76
Unveiling the underpinnings of various non-conventional ELISA variants: a review article
Published in Expert Review of Molecular Diagnostics, 2022
Rajesh Ahirwar, Akanksha Bhattacharya, Saroj Kumar
Enzyme-linked immunosorbent assays are widely used tools in clinical medicine, dairy, forensic, environmental monitoring, research, and many other fields for the detection and quantification of specific antigens or antibodies in a given sample [13,43–45]. The present (conventional) ELISA is derived from the radioimmunoassay which uses radiolabelled ligands to compete and replace normal/unlabeled ligands from active binding sites of antibody [46]. In a bid to subside the dependence on hazardous radioactive isotopes, ELISA was introduced for the first time in the 1970s, with the concept of using antibodies (enzyme-linked and normal) to capture and quantify antigenic molecules [47]. A typical ELISA uses two antibodies, one either coated on microtitre plate surface to capture the antigen or detect it specifically, and a second antibody linked to an enzyme to provide detection and signal amplification [48]. ELISA protocols can be performed in different formats, viz. homogenous, heterogeneous, direct, indirect, sandwich or competitive as per assay requirements (Figure 1). The most often used protocols are based on heterogeneous platform that use solid supports to immobilize reaction components, and provide high surface area to volume ratio required for better sensitivity [49]. Contrarily, the homogenous assays involved performing the binding events in solution phase, thus benefitting the multiplexing and fast electrophoretic separations.
Development of a Novel Multiplex Bead-based Assay for Measuring Autoantibodies on Flow Cytometric Platform
Published in Immunological Investigations, 2022
Rajni Chauhan, Nikita Gupta, Aseem Kumar Tiwari, Vimarsh Raina, Shoma Paul Nandi
The multiplex assay can measure multiple antibodies simultaneously, which is one of the major advantages over ELISA which can measure only a single antibody at a time (Fulton et al. 1997). However, if the antigens in multiplex assay share common epitopes, the antibody competition may occur which can influence the results of the multiplex assay (Carson and Vignali 1999; Kellar 2002). The multiplex assay validated in the present study demonstrated that MFI values were similar when all five antigen coupled beads were analyzed in both “single” and “multiplex” formats suggesting that the multiplexing of beads did not affect the quality or sensitivity of NMBA . An additional problem associated with multiplex assays is the cross-reactivity and degree of interference among the different antigen coupled beads which may result in non-specific binding (De Voer et al. 2008; Lal et al. 2004). No remarkable differences were observed in the present study when the assay was run in a singleplex or multiplex format, further suggesting the specificity and stability of antigen-antibody complexes in NMBA.
Diagnosis, prevention, and treatment of coronavirus disease: a review
Published in Expert Review of Anti-infective Therapy, 2022
Manoj Kumar Sarangi, Sasmita Padhi, Shrivardhan Dheeman, Santosh Kumar Karn, L. D. Patel, Dong Kee Yi, Sitansu Sekhar Nanda
Repeated RT-qPCR with high-resolution CT is used for screening patients with negative RT-qPCR results. However, CT has a few limitations: it does not provide specific information to differentiate between COVID-19 pneumonia and other viral pneumonias. Enzyme-linked immunosorbent assay (ELISA) is still in trend for the diagnosis of SARS-CoV-2 infection, using IgM/IgG assay kits. ELISA is commonly used for serological testing and takes 2–5 h on average. It typically involves a surface coated with specific viral antigen(s), and corresponding patient antibodies (IgG, IgM, IgA) present in blood, plasma, or serum samples bind to these antigens. The antigen–antibody complex is then detected using a secondary antibody and substrate that produces a color or fluorescent signal. There are various types of ELISA, including direct, competitive, and double-antigen sandwich ELISA (used most commonly) [38].
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