Qualitative Screening Assays

This kind of interference is called cross-reactivity, or specific interference, and is usually quantitated by assaying for the interferent in the absence of analyte. Validation procedures involve screening of a large number of potential interferents at concentrations higher than their expected levels in real samples. Cross-reactivity methods for immunoassays have been described in Chapter 6. 

True positive Both methods yield positive results. 

True negative Both methods yield negative results. 

False positive New assay positive, accepted assay negative. 

False negative New assay negative, accepted assay positive. 

---

Assessment and definition of sensitivity are often described for quantitative analysis but are of equal importance for qualitative devices of the dip-stick type that are very popular for farm- or field-based screening assays. Because of the somewhat subjective nature of visually assessed assays, the assay s sensitivity must be validated using a number of observers to determine at what level a test is deemed positive. The number of false positives and false negatives must be carefully determined in order to balance consumer safety and potential economic loss to animal producers. 

The test based on motility inhibition of the bacterium Spirillum volutants is a very simple and rapid test for the qualitative screening of wastewater samples or extracts [48]. The organisms are observed under the microscope immediately after the addition of the test solution. The maintenance of a bacterial culture is necessary as in the previous type of assay. 

Intense efforts are being undertaken both to overcome detection limits and to increase the number of determinable compounds, also for the purpose of developing alternative analytical methods that are simpler, cheaper, and require less solvent. Researchers are currently experimenting new methods for purifying samples and new extraction systems specially tailored to the bee s body that rely on techniques such as gel permeation [68] and solid phase extraction. A particular focus is placed on the study of enzymatic and immunoenzymatic assays for classes of compounds these methods may allow samples to be passed through a fast prehminary qualitative screening and thus drastically reduce the number of analyses to be performed. 

Arginine prototrophs were screened for Ars-activity by using the qualitatively X-SO4 assay. After growing cells on plate during anaerobic (A) or aerobic (B) adaptation, X-SO4 was dropped on colonies of strain MR16 generated by transformation with plasmid pMR16. 

An entire antibiotic screen can be carried out using 4 samples with the assay time being 15 min to 1 h depending upon whether the qualitative or quantitative mode is desired. 

Therefore, directed evolution was applied to solve these problems. To screen for enantioselectivity, the Miilheim MS-based high-throughput ee assay (92,93) (Section III.C) was applied (46). In this case, the necessary isotope labeling focused on the use of in the pseudo-meso compound N-(J )-17 (see Section III.C for a detailed discussion). An (5)-selective nitrilase leads preferentially to N-(5)-18, whereas an 7 -selective variant results in the picw o-enantiomer (J )-18. They differ by one mass unit and can therefore be distinguished by MS, both qualitatively and quantitatively (by integration of the relevant peaks). 

This experiment impressively demonstrated how an improved protein assay accessible by IPCR has significantly enhanced the opportunity for the early detection of potentially dangerous false-negative samples and emphasized the value of IPCR as a screening tool for low-level infections. Although IPCR sensitivity facilitates an early therapy, actual therapy monitoring and adaptation is supported by the broad dynamic quantification range. As the actual increase/decrease of the virus load is detectable in contrast to the simple qualitative presence/absence of the virus, detailed studies of therapy and medicament influences are possible. 

The authors describe an ultrasensitive method that measures RT activity. The assay adopts polymerase chain reaction (PCR) amplification for detecting the cDNA product of the reaction, and therefore was named Amp-RT (3,4). Amp-RT measures the ability of a sample to produce a DNA copy of a known heteropolymeric RNA template by extending a complementary DNA oligoprimer. The RNA template used in Amp-RT is a sequence from the genome of the encephalomyocarditis virus. This chapter describes in detail the Amp-RT method and its use as (1) a qualitative assay for the generic detection of retroviruses, (2) a quantitative method to measure virus loads of the human immunodeficiency virus type 1 (HIV-1), and (3), a screening method for susceptibility of HIV-1 to RT inhibitors. 

---