Nucleic Acid Amplification Techniques in Immunoassay: An Integrated Approach with Hybrid Performance

Abstract

An immunoassay is mostly employed for the direct detection of food contaminants, and a molecular assay for targeting nucleic acids employs amplification techniques for distinguishing genes. The integration of an immunoassay with nucleic acid amplification techniques inherits the direct and rapid performance of an immunoassay and the ultrasensitive merit of a molecular assay. Enthusiastic attention has been attracted in recent years on the utilization of isothermal amplification techniques in an immunoassay, as well as the employment of a lateral flow immunoassay in a molecular assay. Thus, this Review discussed these kinds of approaches from two categories: immuno-nucleic acid amplification (I-NAA) and nucleic acid amplification-immunoassay (NAA-I). The advantages, drawbacks, and future developments were discussed for a comprehensive understanding.

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe

“…The tags can be low molecular, such as fluorescein (FAM), biotin, digoxygenin, etc., or a single-stranded (ss) DNA tail. The tags are affinely captured by receptor molecules at LFT that are located at specific zones and interact with tag-labeled amplicons that pass through the test strip [ 5 , 6 , 7 ].…”

Simultaneous Determination of Antibiotics, Mycotoxins, and Hormones in Milk by an 8–17 DNAzyme-Based Enzyme-Linked Immunosorbent Assay

“…However, the classical immunoassays such as enzyme-linked immunosorbent assay (ELISA), is restricted to limited sensitivity and can only realize single-target detection because of the undifferentiated catalytic behavior of horseradish peroxidase (HRP). Due to the development of DNA signal amplification technologies such as PCR, as well as some isothermal amplification techniques, − ultrasensitive and multiplex testing based on fluorescence immunoassays is becoming possible and popular.…”

CRISPR/Cas12a-based fluorescence immunoassay: combination of efficient signal generation with specific molecule recognition

“…The streptavidin-biotin interaction can effectively bind the oligonucleotide to the antibody through a simple process, 25,26 and was used for the conjugation of the DNA activator to Ab2. In this method, the Ab2 is employed for target recognition, and the DNA acts as a signal unit.…”

“…So the sensitivity of this method can be further improved through combination with some nucleic acid amplification techniques. 25,30 Next, the specificity of this sensing method for IgG detection was studied. Bovine serum albumin (BSA), D-dimer, alpha fetoprotein (AFP), vascular endothelial growth factor (VEGF), and two closely related antibodies (IgA and IgM) were selected as the possible interferences.…”

“…Considering that each streptavidin has four biotin-binding sites, streptavidin was used to modify Ab2, and biotin was used to modify the DNA activator, which may achieve multilevel amplification. 25 In order to reduce the steric-hindrance effect, we added ten T bases (T10, grey region in dsDNA activator) as a spacer sequence between biotin and the DNA sequence. As shown in Fig.…”