Schistosoma sp. circular anodic antigen (CAA) serum concentrations reflect actual worm burden in a patient and are a valuable tool for population screening and epidemiological research. However, for the diagnosis of individual imported schistosomiasis cases, the current enzyme-linked immunosorbent assay (ELISA) lacks sensitivity and robustness. Therefore, a lateral flow (LF) assay was developed to test CAA in serum for individual diagnosis of imported active schistosome infections. Application of fluorescent submicron-sized up-converting phosphor technology (UPT) reporter particles increased analytical sensitivity compared to that of the standard ELISA method. Evaluation of the UPT-LF test with a selection of 40 characterized epidemiologic samples indicated a good correlation between signal intensity and infection intensity. Subsequently, the UPT-LF assay was applied to 166 serum samples of Dutch residents (immigrants and travelers) suspected of schistosomiasis, a case in which group routine antibody detection frequently fails straightforward diagnosis. The UPT-LF assay identified 36 CAA-positive samples, compared to 15 detected by CAA-ELISA. In conclusion, the UPT-LF assay is a low-complexity test with higher sensitivity than the CAA-ELISA, well suited for laboratory diagnosis of individual active Schistosoma infections.

The development of up-converting phosphor reporter particles has added a powerful tool to modern detection technologies. Carefully constructed phosphor reporters have core-shell structures with surface functional groups suitable for standard bio-conjugations. These reporters are chemically stable, possess the unique property of infrared up-conversion, and are readily detected. In contrast to conventional fluorescent reporters, up-converting phosphor particles do not bleach and allow permanent excitation with simultaneous signal integration. A large anti-Stokes shift (up to 500 nm) separates discrete emission peaks from the infrared excitation source. Along with the unmatched contrast in biological specimens due to the absence of autofluorescence upon infrared excitation, up-converting phosphor technology (UPT) has unique properties for highly-sensitive particle-based assays. The production and characteristics of UPT reporter particles as well as their application in various bioassays is reviewed.

Background: A lateral-flow (LF) device using the new reporter up-converting phosphor technology (UPTTM) was applied to DNA (hybridization) assays for the detection of specific nucleic acid sequences, thereby aiming to perform the test outside well-equipped laboratories. The methodology reported here is sensitive and provides a rapid alternative for more elaborate gel electrophoresis and Southern blotting. In a preliminary study, it was applied to screen for the presence of human papillomavirus type 16 (HPV16) in a defined series of cervical carcinomas. Methods: A LF assay was used to capture haptenized DNA molecules and hybrids, which were immunolabeled (before LF) with 400-nm UPT particles. These particles emit visible light after excitation with infrared in a process called up-conversion. Because up-conversion occurs in only the phosphor lattice, autofluorescence of other assay components is virtually nonexistent. Results: The use of the UPT reporter in LF-DNA tests, as compared with colloidal gold, improved the detection limit at least 100-fold. UPT LF-DNA tests were successfully applied to detect (in a blind test) the presence of HPV16 in DNA extracts obtained from cervical carcinomas. Test results matched 100% with previous characterization of these carcinomas. Conclusions: The use of UPT in LF assays to detect specific nucleic acids provides low attamole-range sensitivity. Hybridization and consecutive detection of PCR-amplified HPV16 sequences were successful in a background of 10 μg of fish-sperm DNA. The sensitivity of UPT detection in these complex mixtures indicates that detection of viral infections without PCR or other amplification technique is achievable.

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A novel assay is described for multiplex detection of antibodies against different pathogens from a single sample. The assay employs a modified lateral flow format (consecutive flow, CF) together with a sensitive reporter particle technology (up-converting phosphor technology, UPT) that allows for fully instrumented assay analysis. Lateral flow (LF) strips developed for the detection of human antibodies against human immunodeficiency virus type-1 and -2 (HIV-1 and -2) with additional capture zones to detect antibodies against Myobacterium tuberculosis (TB) and hepatitis C Virus (HCV) provided the strips to test multiplexing. Data are presented that show the performance of the TB and HCV test, as well as two multiplex assays, TB with HIV and HCV with HIV. The TB/HCV assays demonstrate excellent detection capability, and HIV multiplexing does not affect the qualitative test result. The bench-top CF format was converted to a microfluidic platform and a first prototype semiautomated chip capable of performing CF is presented here.