Molecular zipper assays: a simple homosandwich with the sensitivity of PCR.
OBJECTIVE
The purpose of this study is to develop a simple and inexpensive method for detection of viral load or antigens present in the body fluids as for diagnosis or monitoring of infectious diseases. For example, in case of viral infection, nucleic acid based quantitative PCR/RTPCR are sensitive in measuring viral load to follow the course of therapy or infection. The key limitations of such assays include the need for sample extraction, susceptibility to inhibitors, and high cost.


METHODS
A molecular zipper assay based on the simple homosandwich concept for repeated epitopes was developed where the analyte or virus is sandwiched between the same antibodies for detection. A comparative study of the lower limit of detection of M13 model virus was performed with various substrates.


RESULTS
Homosandwich molecular zipper assay captured the model virus with high avidity resisting multiple rounds of washing. Detection of the virus by enzyme labeled MAb in combination with chemiluminescent substrates provided practical assay sensitivities of 7-15 phages and a theoretical detection sensitivity of one virus particle.


CONCLUSIONS
The significance of our results on the molecular zipper assay relates to the development of ultrasensitive pathogen assays at low cost. Such assays could be developed for pathogenic bacteria and viruses, especially HIV & HCV viruses, which are ravaging impoverished continents of Africa, Asia and Latin America.
Guttikonda
Sujatha
S
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada.
Wang
Welson
W
Suresh
Mavanur
M
D001431:Bacteriological Techniques; D017104:Bacteriophage M13 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D008967:Molecular Biology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity