Assay control, negative

Assay Controls. Negative Control is re-calcified negative human plasma. HCV core Ag Positive Control (AgPC) was diluted from a unit of HCV RNA positive / HCV Ab negative human plasma. 

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.

Additionally, we tested cell extracts in the colorimetric assay both negative control strains E. coli pET28a containing the empty vector and E. coli BFDL476Q (Table 2.2.1.2) did not show any enzyme activity in the TTC assay, whereas E. coli BAL (Table 2.2.1.2) led to the formation of an intense red color (data not shown). 

The unit is equipped with devices such as remote switches to alleviate problems such as missing the pickup of a test tube or pipette tip. In a sequence of events, the robot would pick up a test tube and move to a location that has a remote switch. It would then press down the switch which would send a response to the controller. The Zymate program and the response from the switch location would indicate whether the robot had picked up the test tube. If the response was positive it would continue with the assay. If negative, it would take other actions that might be programmed into the system. These actions might include another attempt at picking up the test tube or a system shutdown. These actions thus allow safety checks on system operation or status. 

Assay Control The assay control involves omission of the primary antibody (test article or negative control antibody) during the staining reaction and permits determination of staining by the secondary or tertiary antibodies or other components of the reaction process. This control slide may or may not be included based on the immunohistochemical method chosen. 

Easy to use Ability of trained users to correctly read instructions and execute assay with negative/positive controls Report showing controls used properly = 3 sites/3 naive operators n = 10 devices at each control... 

If assessment by eye is being used, control negative sera giving OD levels at the upper limit of negativity (around two times the mean) might be used. Color development in such assays should then be allowed until color is just detectable in the negative controls. The test should then be stopped. Therefore, any wells showing color more intense than the control wells would be positive for antibody. 

The amount of a signal produced in an assay or screen in the absence of a test substance (2) the signal detected from an assay in the absence of TARGET activity often equivalent to negative control. 

In a micronucleus assay using male B6C3Fj mice dosed with 0, 250, 500, or 1,000 mg/kg diisopropyl methylphosphonate, a small but significant increase in micronuclei were observed at mid- and high-dose levels (DOD 1991a). However, the maximum response was found to be within the laboratory historical control limits. The assay was repeated and the increase in micronuclei was not observed, therefore, it is believed that diisopropyl methylphosphonate did not cause micronuclei induction in this experiment. Diisopropyl methylphosphonate was also negative for the induction of micronuclei in the rat bone marrow after administration of up to 800 mg/kg (DOD 1991b). 

Light emission from the chemiluminescent substrate is directly proportional to the amount of the target nucleic acid in the sample, and the results are recorded as relative luminescence units (RLUs). All samples, standards, and controls are run in duplicate, and the mean RLU is used in data analysis. The percent coefficient of variation (%CV) for duplicate RLU for controls and samples must be within the recommended limit for that assay for the results to be valid. For example, negative samples must have a CV of <30% and positive samples <20% in the HCV assay. 

Comments There are several suggested controls for this assay, including use of yeast total RNA as a negative control (check for probe species specificity) and a no RNAse control to determine probe stability. In Fig. 6.3A, the positive control marker lane was produced by addition of R-luc-4 sites or F-luc mRNA only to the assay. Also, optimal times for RNAse digestion will vary from probe to probe. In addition, for maximum sensitivity a probe with high specific activity is preferable (yet still in molar excess to the mRNA). 

The immobilized immunoprecipitates are washed twice with lysis buffer containing 0.5 MNaCl and twice with buffer A. The beads are resuspended in 20 /il of kinase buffer also containing the appropriate concentration of the specific peptide. Reactions should also be set up without peptide as a negative control for nonspecific or self-incorporation of radiolabel. To start the reactions, 5 /il of ATP is added (final concentration 0.1 mM unlabeled ATP, 1 /iCi [7 -32P]ATP (per assay) in kinase buffer). The assays are allowed to proceed for 15 to 30 min at 30° with constant shaking at 900 rpm, and stopped by spotting 20 /il of the sample (slurry) onto a square (1.5 X 1.5 cm) of phosphocellulose (P81) paper. The P81 papers are immediately immersed in 500 ml of 1% (v/v) orthophosphoric acid, and then washed 3 times with the same solution (to remove the excess ATP). The washes therefore contain almost all of the radiolabel and must be handled carefully and disposed of appropriately. The papers are briefly rinsed in ethanol and air-dried. The incorporation of 32P-label is measured by Cerenkov counting. 

---