Microtitrator

Where in the scale of operations do the microtitration techniques discussed in Section 9B.8 belong ... 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

Avdeef, A., Nielsen, P. E., Tsinman, O. PAM PA - a drug absorption in vitro model. 11. Matching the in vivo unstirred water layer thickness by individual-well stirring in microtitre plates. Eur. J. 

Figure 7.9 Cross section of a pION 96-well microtitre plate PAMPA sandwich assembly.

Bevan, C. D. Lloyd, R. S., A high-throughput screening method for the determination of aqueous drug solubility using laser nephelometry in microtitre plates, Anal. Chem. 72, 1781-1787 (2000). 

H. Harma, T. Soukka, S.Lonnberg, J. Paukkunen, P. Tarkkinen, and T. Lovgren, Zeptomole detection sensitivity of prostate-specific antigen in a rapid microtitre plate assay using time-resolved fluorescence. Luminescence 15, 351-355 (2000). 

Lucas M, Mertens V, Corbisier A-M, Vanhulle S (2008) Synthetic dyes decolourisation by white-rot fungi development of original microtitre plate method and screening. Enzyme Microb Technol 42 97-106... 

One of the most popular bioassay for interferons is termed the cytopathic effect inhibition assay . This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye followed by spectrophotometric quantitation of the amount of dye assimilated can thus be used to quantitate percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of large numbers of samples with relative ease. 

EIA systems take advantage of the extreme specificity and affinity with which antibodies bind antigens which stimulated their initial production, coupled to the catalytic efficiency of enzymes, which facilitates signal amplification as well as straightforward detection and quantification. In most such systems, the antibody is immobilized on the internal walls of the wells in a multi-well microtitre plate, which therefore serves as collection of reaction mini-test tubes. 

Antibodies raised against the antigen of interest (i.e. the therapeutic protein) are first adsorbed onto the internal walls of microtitre plate wells. The sample to be assayed is then incubated in the wells. Antigen present will bind to the immobilized antibodies. After an appropriate time, which allows antibody-antigen binding to reach equilibrium, the wells are washed. 

For estimation of cloning efficiency and mutant induction, cells are plated out in 96-well microtiter plates. Flasks and microtitre plates are incubated at 37°C in a C02 incubator as in the cytotoxicity assays. 

For survival estimation, cells are placed into 96-well microtitre trays at a cell density of 1 cell per well as per the cytotoxicity assay. 

The Zymark robotic laboratory automation system Although detail procedures differ in each laboratory, the basic elements of binding and enzyme assays are similar. The generalized procedure shown in Table 1.10 highlights the common steps and indicates which Zymate laboratory systems are required. These procedures are performed using common laboratory glassware such as test tubes or in multiple tube devices such as microtitre plates. 

Hence, this assay is an extremely useful and selective assay to measure O2 secretion. Because of this selectivity and because it measures the initial product of O2 reduction, it is often used as the method of choice to detect NADPH oxidase activity. It is suitable for semi-automation because assays can be performed in 96-well microtitre plates (using ELISA plate readers with a suitable filter), or cytochrome c reduction can be detected using simple spectrophotometers. The assay, however, is not suitable for measuring O2 that may be generated intracellularly within activated neutrophils. 

In summary, chemiluminescence is a sensitive, non-invasive technique that can measure reactive oxidant production by small numbers of neutrophils indeed, neutrophil-derived chemiluminescence can be detected in as little as 5 fA of unfractionated human blood. The assay is suitable for automation using either multichannel luminometers or luminescence microtitre plate readers. Many researchers, however, have questioned the usefulness of this technique because of the uncertainty of the nature of the oxidant(s) that are detected. Nevertheless, in view of the recent developments made towards the identification of the oxidants measured and the assay s ability to detect intracellular oxidant production, it is has an important place in the phagocyte research laboratory. 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

Carrier protein Macromolecule to which a hapten is conjugated, thereby enabling the hapten to stimulate the immune response. catELISA Similar to an ELISA, except that the assay detects catalysis as opposed to simple binding between hapten and antibody. The substrate for a reaction is bound to the surface of the microtitre plate, and putative catalytic antibodies are applied. Any product molecules formed are then detected by the addition of anti-product antibodies, usually in the form of a polyclonal mixture raised in rabbits. The ELISA is then completed in the usual way, with an anti-rabbit second antibody conjugated to an enzyme, and the formation of coloured product upon addition of the substrate for this enzyme. The intensity of this colour is then indicative of the amount of product formed, and thus catalytic antibodies are selected directly. 

Pipette 5 (xl of standards and samples Into a clean, dry 96 well microtitre plate. 

Transfer 25 (jlL of the supernatant into a 96-well microtitre plate. 

Take 100 (xg of the cell lysates in 100 p,L of lysis buffer in a 91-well microtitre plate. 

Analysis was performed in a 96-well microtitre plate by ESI-MS on a Q-TOF mass spectrometer (Q-TOFmicro Micromass, Altrincham, UK) interfaced with a NanoMate M chip-based nanoESI source (Advion Biosciences, Ithaca, NY). Samples were infused at a flow rate 100 nL min . Calibration and sample acquisition were performed in positive ion mode in the range of m/z 500-5000. The ESI-MS screen identified noncovalent complexes of Bell and confirmed the preference of native Bell for certain... 

Resuspend the cells completely In 230 pil of Lysis Buffer supplemented with 1 mg/ml Lysozyme and either 3 units/ml of Benzonase (Merck, Germany purity grade I, >25 U/pil, Cat. No. 1.01694.0001) or 400 units/ml of DNAse Type I. This can be done by either repeated aspirate/dlspense with a suitable multichannel pipette or on an orbital microtitre plate shaker ( 1000 rpm for 30 min). Clear the lysate... 

Approximately 5 min before the end of the centrifugation run dispense 20 pel of the NI-NTA magnetic bead suspension (ensure full resuspension before you commence pipetting I) Into each well of a flat-bottomed microtitre plate (MTP). Not all microtitre plates are magnet compatible - check before you commence the assay. 

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