Owing to an excellent sensitivity and specificity, immunoassays have been widely used for rapid high throughput assays of a variety of substances including viruses bacteria, disease-associated proteins, food toxins, and environmental contaminants.1-5
Immunoassays can be categorized into two different assay formats, non-competitive sandwich and competitive format. The non-competitive sandwich type assays are mostly used for the detection of large molecules possessing more than two antibody-binding sites for which one antibody captures the target analyte and a second antibody conjugated to a signal producing molecule, binds to a second site on the analyte producing a quantitative readout. On the other hand, competitive assays are needed for the detection of small analytes because the small molecule bound to the surrogate antibody is unlikely to provide the recognizable portion for a secondary antibody. Thus, prior to the development of PHAIA, a non-competitive sandwich format was very difficult to apply to small molecules. Non-competitive assays are superior to competitive assays in terms of sensitivity, dynamic linear range, and easy adaptability into other formats, including immunochromatography and biosensors.6
Due to their superior performance, there have been efforts to develop non-competitive assays for small molecules by producing anti-immune complex antibodies.7,8
recombinant antibodies that form analyte-associated complexes9-11
, or employing modified assay procedures that convert competitive formats to non-competitive formats.12, 13
However, these methods are cumbersome and laborious, and success has been mostly case specific, which may explain why almost all assays reported for small size analytes have a competitive format.
To overcome this shortcoming, we have recently introduced the PHAIA (phage anti-immunocomplex assay) in which we use small peptide loops displayed on phage M13 as innovative elements for the specific detection of immunocomplexes. The M13 bacteriophage is a filamentous virus with a diameter of 6 nm and length of 0.9 μm containing single strained DNA packed in a few thousands of major and minor coat proteins. By modification of the phage genome it is possible to efficiently express polypeptides fused to its coat proteins, and build libraries of enormous complexity. The physical linkage between the phage phenotype (displayed peptide) and its genotype (peptide encoding sequence) allows the efficient selection of polypeptide ligands virtually for any selector molecule, and constitutes the working principle of the phage display technology.14
In order to generate more complex libraries and control the valence of the displayed peptide, phagemid libraries composed by hybrid viral particles have been introduced. Phagemids are plasmid vectors that encode the peptide fused to the gene of the coat protein used for display but lack all other phage proteins. These vectors can be propagated in bacteria and be packaged as single strand DNA in viral particles upon hyperinfection with a helper phage.15
In the PHAIA method, a phage borne cyclic peptide selected from phage display peptide libraries forms a trivalent antibody-analyte-peptide complex by specific recognition of the conformational change of the antibody binding pocket upon binding of the analyte.16-18
This method accelerates the development of non-competitive two site assays using a well known in vitro
selection method, “biopanning”, resulting in dramatically improved assay sensitivity and increased specificity.
In this paper we demonstrate that it is possible to combine the advantageous characteristics of PHAIA with the power of amplification of IPCR to develop a highly sensitive detection method for small molecules. IPCR was first reported by Sano et al.19
and has been used for ultrasensitive detection of virus and biomarkers.20-22
In the classical application, a DNA sequence is chemically conjugated to the detecting molecule. Upon binding, this tracer reagent can be detected with high sensitivity by PCR amplification.19
More recently, Guo et al.23
demonstrated that the DNA tracer could be substituted by a detecting molecule displayed on the surface of the M13 phage. In their application, they used phage particles expressing single chain antibody fragments (scFv) that served simultaneously as detection reagent and DNA template, allowing the ultrasensitive detection of viral particles and prion proteins in two-site sandwich formats. In spite of its advantages, IPCR could not be applied to the analysis of a critical group of analytes that due to their small size can not be simultaneously detected by two antibodies. This group includes most drugs, environmental pollutants, explosives, hormones, food additives, toxins, metabolites, etc., for which there is a growing need of rapid yet highly sensitive detection methods.
As a proof of concept, two PHAIA-PCRs for 3-phenoxybenzoic acid (3-PBA), a major human urinary metabolite of pyrethroid insecticides and the herbicide molinate were developed. The assay conditions were optimized using magnetic beads to separate the reacted phage, with allowed a 10-fold increase in sensitivity. The robustness of the PHAIA-PCR methods was validated with real samples, using agricultural drain water and human urine.