Enzyme-linked immunosorbent protocol

As an example of microchip-based electrochemical immunoassays, we describe here the protocol established for the analysis of interleukin IB by enzyme linked immunosorbent assay (ELISA) with amperometric detection at the sub-pM level in DiagnoSwiss microfluidic chip called Immuchip . 

A standard double-sandwich enzyme-linked immunosorbent assay (ELISA) or Western blot analysis is used. As the concentration of factor is very low in normal plasma (approximately 0.1 mg/1 for factor VIII), it is necessary to subject plasma samples to cryoprecipitation in order to concentrate the sample, prior to Western blot analysis. The cryoprecipitation protocol described by Bi et al. is as follows (Bi et al., 1996 Sarkar et al., 2000 Mah et al., 2003). Plasma samples are collected as described above for the Coatest assay. Plasma is then frozen at -80 °C overnight. Frozen samples are then subject to centrifugation at 7000 x g for 20 min at 4°C. The precipitate is washed with... 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

This chapter describes the procedures that can be used to determine compounds that have antiviral activity against HIV. These include maintenance of lymphoblastoid cell lines, preparation of peripheral blood mononuclear cells (PMMCs), and determination of the infectivity of the HIV stock-supernatant and antiviral assays. The assays described use both acutely and chronically infected cells. Toxicity of compounds is assessed by measuring 14C uptake. These protocols are used for the evaluation of compounds that can be carried out by a single individual in a Category 3 containment laboratory. The number of compounds analyzed would be about 10, which is a convenient number to fill a single 96-well p24 enzyme-linked immunosorbent assay (ELISA) plate. 

Antibodies can be used for a variety of applications in the molecular characterization of receptors and receptor-hgand interactions. Antibodies can be used for the detection of receptors in tissue shces. Western blot experiments [48], or ELISAs (enzyme-linked immunosorbent assays) [49]. They can also be used in competition experiments to map the binding epitope of a hgand [50]. Even though the use of antibodies is routine, fhere is no general protocol for fheir generation. 

Many types of assay are available to be used in HTS protocols to identify inhibitors of PPIs, but a competition assay, in which inhibition of complex formation is measured, is most common. Fluorescence polarization (FPA), fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assays (ELISA), and other assay formats have been used. The interacting proteins can be used in their full-length forms though, more frequently only the interacting domains are employed, and if possible the excised interacting peptide is usually preferred. 

A further alternative is to implement these approaches with the use of redox and/or nanoparticle labels. The redox and nanoparticle labels replace the familiar enzyme labels in enzyme-linked immunosorbent assays (ELISAs) and offer the advantages of simplified protocol, wider linear dynamic range, higher stability compared to enzymes, and higher separation efficiency. 

The most widely used immunological approaches are those based on enzyme-conjugated antibodies, offered as kits that allow immunochemical detection of allergens and toxic components in foods. These products are popular in that they offer ease of automation, the possibility of analyzing a large number of samples in a single approach, besides relying on safe chemicals and simple protocols. Either direct or competitive enzyme-linked immunosorbent assay... 

A ricin detection method has been developed that couples LC-MS/MS with an enzyme-linked immunosorbent assay (ELISA).The analyte target for MS detection is adenine, which is released from ricin during the assay. Adenine detection limits were achieved at 0.1 ng/mL or 1.56 pM in a 500-pL sample volume with this method. This LC-MS method may be chosen over other techniques because milk and drinking water are the intended real-world application for this protocol [77]. Another MS/MS technique detects tryptic peptide fragments from ricin produced by an immunoassay technique. This technique utilizes the inherent speed of MS analysis to confirm peptide identity. This technique is applied to the detection of ricin in food and body fluids such as blood serum and saliva [78]. A similar LC-MS/MS method uses an organic solvent-assisted tryptic digest to prepare samples of crude ricin extracts. This sample preparation... 

Hornbeck, P. Assays for antibody production. Enzyme-Linked Immunosorbent Assays. Current Protocols in Immunology Immunology 2.1.1-2.1.22, 1991. Antibodies—Enzyme-Linked Immunosorbent Assays. 

The dot blot will provide a qualitative estimate of antibody titer (Protocol 21.3). The same test can be done quantitatively by ELISA (enzyme-linked immunosorbent assay), but the technically simpler dot blot gives adequate information, uses less protein, and does not require an ELISA plate reader. If the same protein has been expressed in two different fusion-protein vectors, the test should be performed with the protein not used for immunization. If there is only one fusion protein, e.g., a GST-fusion, then a second dot blot can be done in parallel with just GST alone to compare the titers. If the antiserum recognizes the protein, there should be a lower titer of antibodies against GST alone as compared to against the entire fusion protein. A preimmune control is always advisable. Figure 21.1 shows a sample dot blot from a monoclonal antibody screen. 

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