PubMed · 7005271
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A G Maciver, B L Mepham. 1980. Immunoperoxidase techniques and controls.. https://doi.org/10.1136/jcp.33.12.1218
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One of the most widely used analytical techniques for sensitive detection of biologically and clinically significant analytes is the immunoassay. In recent years direct immunoprobes allowing label-free detection of the interaction between the antibody and the target analyte have proved their capabilities as fast, simple, and nevertheless highly sensitive methods. Cloned enzyme donor immunoassay (CEDIA) homogeneous assay is based on the bacterial enzyme beta-galactosidase, which has been genetically engineered into two inactive fragments, enzyme donor and enzyme acceptor. Reassociation of the fragments in the assay forms active enzyme, which acts on substrate to generate a colored product. A comprehensive kinetic model of CEDIA is developed to aid in understanding this method and to facilitate development of a truly homogeneous version, potentially applicable to a dipstick-type multianalyte point of care analytical device (ChemChip). Although the standard assay involves a two-step process, we also chose to model a single-combined process, which would be simpler to apply in a ChemChip device. From the modeling simulation, we obtain the time courses of the amounts of product and active enzyme, from which the dynamic ranges can be obtained as 10(-6)-10(-7) and 10(-5)-10(-7)M analyte concentration for two-step and single-combined processes under the conditions of the assumed parameters, respectively. A simple one-step immunoassay has the merit of reducing time and cost and has an improved dynamic range.
We briefly examined detection kit using the EIA method for Norwalk virus, and compared the results of the tests using the EIA method with those using RT-PCR method. In reproducibility, an amount of variation was observed in data obtained from positive controls and in lower values. The sensitivity obtained from the EIA method was about 300 times lower than that obtained from the RT-PCR method. Results accordance ratio between EIA method and RT-PCR method was 70%. This results discrepancy was presumably caused by a difference of sensitivity and specificity between these two methods. In conclusion, this detection kit using the EIA method is easily manually operated so that this kit can be considered as an effective and simple detection tool for Norwalk virus.
Astroviruses are small RNA viruses associated with pediatric gastroenteritis. A latex agglutination (LA) test is more convenient and rapid than electron microscopy, enzyme immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR) for detection of astroviruses in stool specimens was developed by using polyclonal antibody against cultured serotype 1 astrovirus, and tested on astrovirus positive- and negative- samples collected between 1985 and 1993 in Ehime, Japan. Cultured serotype 1 astrovirus was detected by the LA test, but serotype 2, 3, 4, 5, 6, and 7 viruses could not be detected, although they yielded high titer of the viruses. When tested on the 27 clinical samples, all 8 serotype 1 astroviruses determined previously by EIA were positive by the LA test, however, 9 other serotype astroviruses and 10 astrovirus negative-samples were all negative. PT-PCR was found to be the most sensitive followed by EIA and LA test. Further development of LA test with other serotypes is necessary.