Assay sensitivity and specificity

Dean et al. [93] used a high performance liquid chromatographic method for the simultaneous determination of primaquine and carboxyprimaquine in plasma with electrochemical detection. After the addition of the internal standard, plasma was deproteinized by the addition of acetonitrile. Nitrogen-dried supernatants, resuspended in mobile phase were analyzed on a C8 reversed-phase column. Limits of detection for primaquine and carboxyprimaquine were 2 and 5 ng/mL with quantitation limits of 5 and 20 ng/mL, respectively. The assay sensitivity and specificity are sufficient to permit quantitation of the drug in plasma for pharmacokinetics following low dose (30 mg, base) oral administration of primaquine, typically used in the treatment of malaria and P. carinii pneumonia. 

Although immunochemical assays can employ crude antisera, purification helps in improving assay sensitivity and specificity, reduces analysis time, and aids in standardization of the assay. Various degrees of antibody purification can be performed prior to incorporation of an antibody into the assay format. 

Measurement of Arginine Vasopressin Numerous immunoassays for measuring AVP in plasma or urine have been described. However, their routine clmical application has been hampered because of method complexity and lack of assay sensitivity and specificity. With most plasma assays, a prefiminary extraction procedure is required, not only to concentrate die minute amount of hormone that is present in the specimen, but also to remove nonspecific interfering substances. 

Antibody performance should be tested at all stages of lateral flow assay development, starting with the animal bleeds. ELISA of unfractionated antisera can help eliminate poorly performing antibodies and save time in assay development. The main goal of antibody testing is to evaluate potential assay sensitivity and specificity. 

In addition to enabling separate analysis of at least two types of sialic acid there are the additional advantages inherent in GLC assay sensitivity and specificity. As little as 1 fig of lipid-bound sialic acid could be conveniently analyzed and this could be reduced to 0.3/Ltg and perhaps less with suitable controls for background peaks. For routine assays OV-1 has been the column of choice, but the somewhat more polar OV-225 has proved quite useful in confirming peak identification where required. A typical analysis with both columns is shown in Figure 14 the sample was purified ganglioside mixture from ovine adrenal medulla (Price and Yu, 1976), which was found to contain NANA and NONA in a ratio of approximately 3 2, respectively. 

The microbial assay is based on the growth of l ctobacillus casei in the natural (72) or modified form. The lactic acid formed is titrated or, preferably, the turbidity measured photometrically. In a more sensitive assay, l euconostoc mesenteroides is employed as the assay organism (73). It is 50 times more sensitive than T. casei for assaying riboflavin and its analogues (0.1 ng/mL vs 20 ng/mL for T. casei). A very useful method for measuring total riboflavin in body fluids and tissues is based on the riboflavin requirement of the proto2oan cHate Tetrahjmenapyriformis which is sensitive and specific for riboflavin. 

Colepicolo, P., et al. (1990). A sensitive and specific assay for superoxide anion released by neutrophils or macrophages based on bioluminescence of polynoidin. Anal. Biochem. 184 369-374. 

In this chapter, the main aspects of mass spectrometry that are necessary for the application of LC-MS have been described. In particular, the use of selected-ion monitoring (SIM) for the development of sensitive and specific assays, and the use of MS-MS for generating structural information from species generated by soft ionization techniques, have been highlighted. Some important aspects of both qualitative and quantitative data analysis have been described and the power of using mass profiles to enhance selectivity and sensitivity has been demonstrated. 

Consistent with other analytical methods, immunoassays must be validated to ensure that assay results are accurate. Initial validation involves an evaluation of the sensitivity and specificity of the immunoassay, while later validation includes comparison with a reference method. Because a goal of immunoassays is to minimize sample preparation, validation also includes testing the effects of sample matrices and(or) sample cleanup methods on results. The final steps in validation involve testing a limited number of samples containing incurred residues to determine if the method provides reliable data. 

Differences in the relative proportion of f-PSA and PSA-ACT can affect the result obtained for t-PSA because of the differences in the nature of calibration and the molar response, sensitivity, and specificity of antibodies used in various immunoassays. The efficiency of these immunoassays has been evaluated by several investigators. Because the proportion of free and complexed PSA varies in benign and malignant diseases, these immunoassays measure one form or the other, giving rise to different results for different patient groups. It is very important that data from clinical studies support the proposed intended uses of these assays, since as many as 5 percent of men with a negative free PSA test (free PSA values >25 percent) will have cancer and not be recommended for biopsy. Therefore, a goal for standardization is to detect total and free PSA accurately in equimolar fractions. 

Factors such as assay variations, age, and prostate gland size are known to affect cutoff values. Also, free to total PSA cutoffs are influenced by the sensitivity and specificity values chosen, the reflex range for total PSA used, differences in free PSA assays, and differences in populations studied. Different PSA values are considered due to differences in cutoffs in different assays. Studies have shown that the comparison of a chemiluminescent free PSA showed a 25 percent difference in values. These types of variations suggest a need for standardization (9,29). 

Another approach has been to immobilize proteins within arrays of microfabricated polyacrylamide gel pads (Arenkov et al., 2000). Nanoliters of protein solutions are transferred to 100 x 100 x 20-pM gel pads and assayed with antibodies that are labeled with a fluorescent tag. Antigen imbedded in the gel pads can be detected with high sensitivity and specificity (Arenkov et al., 2000). Furthermore, enzymes such as alkaline phosphatase can be immobilized in the gel pads and enzymatic activity is readily detected upon the addition of an indicator substrate. The main advantage of the use of the threedimensional gel pad for fixation of proteins is the large capacity for immobilized molecules. In addition, the pads in the array are separated from one another by a hydrophobic surface. Thus, each pad behaves as a small test tube for assay of protein-protein interactions and enzymatic reactions (Arenkov et al., 2000). The disadvantage of the method is the need to microfabricate the array of gel pads in that microfabrication is... 

Fletcher et al. [123] used a sensitive and specific gas chromatography-mass spectrometry method for the assay of primaquine in plasma and urine for studying the plasma kinetics. Preliminary studies on the effects of single and multiple oral doses were carried out. In both cases, the drug was completely removed from plasma in 24 h. The concentration of primaquine in plasma usually reached a peak 1-2 h after oral administration. The plasma elimination half-life was about 4 h. 

Since the development of radioimmunoassay (RIA), many assays that rely on the specificity of the antigen-antibody binding reaction have been developed because of their inherent sensitivity and specificity. A typical competitive binding... 

The variety of assays reported here displays the great versatility of luminescent detection systems. As already described, in all luminescent systems the main advantages are the high sensitivity and specificity, which reduce to the minimum the sample treatment, and the ease of use of the reagents and the luminometer. Immobilized systems greatly reduce the cost per assay on the other hand, their preparation requires expertise, especially in the surface activation step on nylon tubes. 

The intensive research carried out to achieve good stability and easy handling of the BL reagents allowed development of many simple, rapid, highly sensitive and specific assays, many of which are available as commercial kits. More recently application of the newest techniques of molecular biology greatly accelerated the continuous improvement of BL assay performance. 

Lucigenin-Amplified CL as a Sensitive and Specific Assay of Superoxide Detection... 

The most commonly used screening method for HIV is an enzyme-linked immunosorbent assay, which detects antibodies against HIV-1 and is both highly sensitive and specific. False positives can occur in multiparous women in recent recipients of hepatitis B, HIV, influenza, or rabies vaccine following multiple blood transfusions and in those with liver disease or renal failure, or undergoing chronic hemodialysis. False negatives may occur if the patient is newly infected and the test is performed before antibody production is adequate. The minimum time to develop antibodies is 3 to 4 weeks from initial exposure. 

Given that, at least under some conditions, assays using these models tend to blow up (have high spontaneous tumor rates) once the animals are more than eight or nine months of age, how critical are age and other not currently apprehended factors to optimizing both sensitivity and specificity ... 

Develeeshouwer, M.J., Comil, M.F. and Dony, J. (1985). Studies on the sensitivity and specificity of the limulus amebocyte lysate test and rabbit pyrogen assays. Appl. Environ. Microbiol. 50 1509-1511. 

D3. Dhingra, K., Talpaz, M., Riggs, M. G., Eastman, P. S., Zipf, T., et al.. Hybridization protection assay A rapid, sensitive, and specific method for detection of Philadelphia chromosome-positive leukemias. Blood 77, 238-242 (1991). 

The final purity required depends on final use of the product e.g., vaccine with one immunization vs. hormone with chronic use. A detection range of 1 to 100 ppm of residual HCPs has been quoted as a regulatory (and analytical) benchmark for therapeutic proteins.11 Many biotech companies have limited the range to 1 to 10 ppm. The sensitivity and specificity of any unique HCP assay that is used to support such a target should be demonstrated accordingly.11... 

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