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ISA assay for retroviruses
Helicobacter pylori
Quantitative measurement of HIV RNA is biomarkers essential for monitoring the progression It is estimated that half of the world's of HIV in infected patients and evaluating population is infected with Helicobacter the efficacy of antiretroviral drug therapylori, ,he bacteria known to cause pies. Many analytical methods have been gastrointestinal diseases, such as pepreported for quantifying HIV RNA, and tic ulcers and stomach cancer. In several commercial assays are now availmany cases, H. pyllri bacteria aan nxable. Such assays, however, require the change genes freely with one another, extraction of viral RNA from blood or resulting in strains that are genetically plasma samples, which is a time-consumheterogeneous. Changes in the bacteing step that often introduces error into ria's genotype over time cause subsethe analysis. M. J. Stapleton and K. Wei of quent changes in protein expression Gene Tec Corp. report on a new, faster, and, therefore, changes in their ability and more accurate method for the quantito cause disease. Carol L. Nilsson of tative determination of retroviruses that Goteborg University (Sweden) has does not require the isolation of viral nuturned to MALDI time-of-flight (TOF) cleic acids. MS to establish biomarkers for moniThe assay, referred to as immobilized toring changes in the genetic material sample amplification (ISA), involves colof H pylori ia tth ephnotypii level. lecting a small amount of blood or plasma A well-defined protocol is essential onto a nylon or polyester matrix, where it is for monitoring biomarkers in intact dried quickly to immobilize, inactivate, and bacteria and bacterial extracts by preserve the viral RNA Immobilized samMALDI-TOFMS because a wide range ples are rehydrated, and the retroviral RNA of factors, including pH, hydrophobicis directly amplified, eliminating the need ity of the extracting solvent, type of for extraction. The assay is particularly well matrix, and sample preparation procesuited for the analysis of small sample voldures, all affect the end result. Nilsson umes, from which RNA is difficult to exexamined lysates and extracts from six tract. Plasma or whole blood samples as small as 5-10 uL were successfully immobilized onto the matrix. The analysis of whole blood samples RNA. On the other hand, with plasma by ISA results in the amplification of retsamples, only retroviral RNA from the roviral DNA in lymphocytes and retroviral free virus is amplified. The researchers
different strains of H. pylori using three different MALDI matrixes— a-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid, and ferulic acid. In addition, the effects of adding the detergent »-octylglucoside were studied to determine whether the sensitivity of larger protein biomarkers in H. pylori could db improved. Ferulic acid was found to be superior to CHCA and sinapinic acid for the analysis of lysates, which Nilsson attributes to ferulic acid's higher tolerance of the high analyte/matrix ratios used in the experiments. In contrast, sinapinic acid was superior for the analysis of bacterial extracts. The greatest sensitivity was achieved, however, for all samples that were dried and reconstituted in 20 mM «-octylglucoside with a matrix of ferulic acid in formic acid/2-propanol/water. Some of the larger virulence proteins could not be observed using any of the methods Work is currently under way in Nilsson's laboratory to further characterize the virulence proteins expressed in H pylorr .Rapid Commun Mass Spectrom 1999 13 1067-71)
believe that by using ISA it may be possible to determine the ratio of free virus to infected lymphocytes, simply by analyzing equal volumes of whole blood and plasma from an individual patient. For a 10-uL viral sample, the ISA assay generated results equal to or better than those of reverse transcriptase-polymerase chain reaction (RT-PCR) for an equivalent amount of RNA. Because extraction of RNA is nearly impossible from a 10-uL sample, RNA was extracted (for comparison) from a larger sample volume to obtain an equivalent amount of RNA for RT-PCR amplification. The ISA assay is capable of detecting
