Assay serial

This method is used for estimating analyte concentrations by immunoassay with maximum precision. Serial dilutions of a standard analyte solution are prepared and assayed serial dilutions of the unknown are also prepared and assayed. Responses from both dilution series are plotted against log10 of the dilution factor, as shown in Figure 16.6. 

Primary hits are usually cherry-picked for rescreening in triplicate. In order to prioritize compounds for which dry powders will be obtained for retesting, we also recommend assaying serial compound dilutions and discarding hits from further consideration that yield dose-response curves with Hill slopes far from I. In particular, compounds with very steep IC50 curves (Hill slope >4) should be avoided, because unspecific effects are likely at play. Once inhibitors have been confirmed from repurchased powders, compounds with reasonable IC50 values (e.g., <20 pM) should be tested in counterscreens against a panel of additional PTPs, in order to weed out... 

Compound Bioautography Agar Well Diffusion Assay Serial Broth Dilution Assay Alamar Blue Assay ... 

Digestibility assays Serial dilution assays Radioimmunoassay Endpoint methods... 

Figure 9.4 A direct ELISA linked ICP-MS assay of 3xFLAG-BAP (bacterial alkaline protein). In this assay, serial dilutions of 3xFLAG-BAP over a concentration range of 0.01-1000 ng/mL were incubated overnight at 4°C in the wells of a Maxisorp plate. Negative controls consisted of PBS without protein. The wells were then blocked for 1 h with 3% BSA/1 x PBS buffer at room temperature, washed, and exposed to anti-FLAG-Eu antibodies for 2 h in a shaken incubation. The wells were washed, dried, and the residue dissolved in 10% HCI spiked with 1 ppb Ho and 1 ppb Ir prior to sampling into the ICP-MS.

Methods to Detect and Quantitate Viral Agents in Fluids. In order to assess the effectiveness of membrane filtration the abihty to quantitate the amount of vims present pre- and post-filtration is critical. There are a number of techniques used. The method of choice for filter challenge studies is the plaque assay which utilizes the formation of plaques, localized areas in the cell monolayer where cell death caused by viral infection in the cell has occurred on the cell monolayer. Each plaque represents the presence of a single infectious vims. Vims quantity in a sample can be determined by serial dilution until the number of plaques can be accurately counted. The effectiveness of viral removal may be determined, as in the case of bacterial removal, by comparing the vims concentration in the input suspension to the concentration of vims in the effluent. 

The discovery and biological properties of lincomycin (1, R = OH, R = H) were described ia 1962 (1). This antibiotic is active in vitro and in vivo against most of the common gram-positive pathogens. Resistance by Staphylococci is developed slowly ia a stepwise manner, based on in vitro serial subculture experiments, and its activity is not iafluenced by body fluids up to concentrations of 50% ia the assay medium (2). 

The analytical sensitivities of the different quantitation methods have been compared using serial dilutions of patients specimens (Butterworth et al., 1996) and the Eurohep HBV DNA standards (Zaaijer et al., 1994). In both cases, bDNA was shown to be about 10-fold more sensitive than the liquid hybridization (Abbott) and the hybrid capture (Digene) assays. Using the Eurohep HB V standards, the detection limits were 2.5 X 106 genomes/ml for bDNA and 2.5 X 107 genomes/ml for both liquid hybridization (LH) and hybrid capture (HC) assays. 

The version 2.0 assay uses a different set of probes designed to hybridize to genotypes 1 to 6 with equal efficacy (Fig. 4). The new probe set not only enhanced the efficiency of binding to genotypic variants but also lowered the LOQ from 3.5 X 105 to 2 X 105 HCV RNA equivalents/ml (Detmer et al., 1996). The version 2.0 assay displayed almost a 600-fold dynamic range up to 1.2 X 108 RNA equivalents/ml. The LOQ was set at 2 X 105 to ensure a specificity of 95%. The assay was reproducible, with a mean CV of 14% for replicates of low-, middle-, and high-titer sera. Serial dilutions of quality level 1 RNA transcripts (Collins et al,... 

Figure 5.3 A convenient scheme for performing an inhibitor titration in 96-well format. Four compounds (1-4) are assessed in duplicate at each of 11 inhibitor concentrations. The inhibitor concentrations follow a threefold serial dilution from a maximum concentration of 1000 (molarity units nM, LlM, etc.). The right most column of wells is reserved for control samples. In this illustration four of the wells of column 12 are used for zero inhibitior positive controls, and the other four are used to establish the assay background as negative controls. Negative controls could represent any sample for which one knows that the enzymatic reaction has be abrogated. For example, the negative control wells could contain all of the reaction mixture components except the enzyme. See Chapter 4 for other potential forms of negative controls.

Human embryo lung fibroblasts infected with a reference laboratory strain of herpes simplex virus (HS V) type 2 were used to detect antibody to HSV type 2 in serum samples. After treatment of cells with serial dilutions of sera, HRP-labeled immunoglobulins to human IgG (class G immunoglobulins) were added and detected with CL substrate [36], In both cases a sharp detection of the specific antibodies was achieved with chemiluminescent assays, which proved more sensitive than the colorimetric immunoperoxidase assays. 

Comparison of inhibition curves generated by four compounds following serial dilution in assay buffer containing 1% (v/v) DMSO (circles) and in neat DMSO (triangles) in a protein tyrosine kinase assay. 

Level 2 of the screen uses, where possible, cells from the same patient s tumor and determines the potency of the compounds advanced from Level 1. Typically, six serial dilutions (of four- to five-fold each) for each sample are assayed in triplicate, and the 50% inhibitory concentration (IC50) is determined. Test samples with potency in the nanomolar range for pure compounds and at least the microgram per milliliter range for crude extracts are promoted to the third level of the screen. 

Level 3 of the screen is designed to determine the cytotoxic selectivity of samples for tumor cells vs. normal cells. Where possible, the same patient s tissues are used. As in Level 2, six serial dilutions (of four- to five-fold each) are assayed in triplicate for each sample. The diluted samples are then added to the tumor cell and normal cell cultures, and the IC50 is determined. A "selectivity index" (SI) is determined based on the IC50 for normal cells/IC5Q for tumor cells. Samples with an SI of three or more are advanced to Level 4 of the screen. Additionally, only purified and well-characterized compounds are promoted for further testing. 

If an approximate Km value for the enzyme-substrate combination of interest is known, a full-scale kinetic assay may be done immediately. However, often an approximate value is not known and it is necessary first to do a range finding or suck and see preliminary assay. For such an assay, a concentrated substrate solution is prepared and tenfold serial dilutions of the substrate are made so that a range of substrate concentrations is available within which the experimenter is confident the Km value lies. Initial velocities are determined at each substrate concentration, and data may he plotted either hyperholically (as V versus [S]) or with [S] values expressed as logio values. In the latter case, a sigmoidal curve is fitted to data with a three parameter logistic equation (O Eq. 4) ... 

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