Immunoassay competitive format

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

To test our new signal reagent based on GZ-11 the detection system was applied to two competitive-format immunoassays. These two assays (for atrazine and clenbuterol) normally use chromogenic detection systems. While these colorimetric assays may be adequate for laboratory use, chemiluminescent detection offers potential advantages in sensitivity and on site screening applications [33],... 

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

Recently, a novel immunoassay has been developed for the quantitative determination of polybrominated biphenyls using indirect competitive format. The new method was optimized concenung the coating conjugate and antibody concentration, incubation time and temperature, the tolerance to organic solvents and so on. Under optimized conditions, PBB15 can be determined in the concentration range of 0.01-100 pg/L with a detection hmit of 0.02 pg/L. The cross-reactivities of the assays were below 8%. While water samples could be analyzed directly [94]. 

The utilization of lAC in analytical methods has received increasing retention in recent years [23,24], Of particular interest is the use of immobilized antibody columns in performing immunoassays, a technique known as a chromatographic immunoassay or flow-injection immunoassay. This approach has already been reported in a number of formats such as those involving simple analyte adsorption/desorption, sandwich immunoassays, competitive binding immunoassays, and multianalyte methods (see Figure 13,9) [23,24,73,74], Typical advantages of these methods include decreased analysis times and improved precision versus manual immunoassays. 

In heterogeneous assays the bound and free label forms must be separated by different means, such as precipitation of antibody or coupling of the antibody to a solid support. Heterogeneous immunoassays are more versatile as inclusion of a separation step eliminates most of the interferences before quantification. However, these assays are also more labor-intensive and time-consuming. Any of these methods can be performed in either competitive or non-competitive format. 

Like conventional immunoassays, CE-IA can be performed in either noncompetitive or competitive format. Sometimes different approaches for direct and indirect modes of analysis are also considered. 

Fig. 5 NRL Array Biosensor mixed format immunoassays (modified from [119]). Schematic of (a) the sandwich and (b) the competitive immunoassay fonnats used in the detection of the Campylobacter jejuni and aflatoxin Bi (AFBi), respectively, (a) Sandwich format antigen captured by the immobilized antibody then quantified by passing a second, fluorescently labeled, antibody over the surface, (b) Competitive format competition for binding sites on the fluorescently labeled antibody occurs between the unlabeled antigen in solution and the surface-bound antigen analog, (c) Final charge-coupled devices image taken with the NRL Array Biosensor of a waveguide exposed simultaneously to the C. jejuni (5 x 10" cfu/mL) sandwich assay (SAND) and the aflatoxin Bj (AFBi-1 ng/mL) competitive assay (COMP) in various combinations...

Enzyme immunoassay is widely used, both in competitive and non-competitive formats, for the bioanalysis of a broad range of low-molecular-weight compounds and macromolecules. Through the use of fluorogenic substrates and amplification systems such as avidin-biotin, the sensitivity of enzyme immunoassay has been developed to equal or exceed that of radioimmunoas-say.f ° The technique has found particularly wide applicability in the determination of new recombinant proteins, in demonstrating antibody responses to macromolecules, and in the measurement of biomarkers of disease, as well as in diagnostic medicine. 

Following principles similar to those of immunoassays, noncompetitive hybridization assays in general demonstrate better sensitivity than competitive formats of... 

Figure 5.4 Schematic representation of mode of operation of a direct competitive format lateral-flow immunoassay showing test results for a compliant (negative) sample (a) and a non-compliant (positive) sample (b) schematic representation of mode of operation of an indirect competitive format lateral-flow immunoassay showing test results for a compliant (negative) sample (c) and a non-compliant (positive) sample (d).

The time-resolved immunoassays can be performed by direct detection or in a competitive format. Direct detection is usually used for proteins which emtain mul-... 

Figure 4 Schematic representation of (a) heterogenous noncompetitive and (b) heterogenous competitive immunoassays. The noncompetitive format involves the formation of antibody-antigen-antibody sandwich . Signal is proportional to the sites occupied by analyte. The competitive format involves the analyte competing with probe (here, labelled antigen) for the available sites. Signal is proportional to sites not occupied by analyte.

In fact, most RIAs and many nonisotopic immunoassays use a competitive binding format (see Fig. 2). In this approach, the analyte in the sample to be measured competes with a known amount of added analyte that has been labeled with an indicator that binds to the immobilized antibody. After reaction, the free analyte—analyte-indicator solution is washed away from the soHd phase. The analyte-indicator on the soHd phase or remaining in the wash solution is then used to quantify the amount of analyte present in the sample as measured against a control assay using only an analyte-indicator. This is done by quantifying the analyte-indicator using the method appropriate for the assay, for example, enzyme activity, fluorescence, radioactivity, etc. 

Bioluminescence can also be used as the basis for immunoassay. For example, bacterial luciferase has been used in a co-immobilized system to detect and quantify progesterone using a competitive immunoassay format (34), and other luciferase-based immunoassays have been used to quantify insulin, digoxin, biotin, and other clinically important analytes (35). 

Most immunoassay kits and many commercial immunoassay analyzers are based on heterogenous EIA or FIA. These include an immunoassay system that uses FIA linked to radial partition chromatography of the antibody—antigen complex (39) a system that uses antibody-coated tubes for enzyme immunoassay of a variety of hormones and dmgs (40) and a system that uses either a sandwich or competitive FIA format to measure a variety of analytes (41). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Fig. 13. General protocol for heterogeneous enzyme immunoassay preparation of reagent cuvettes by coating Ab, competitive assay format, and sandwich assay format. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem, 1985, 57, 1321A. Copyright 1985, American Chemical Society)...

The microplate ELISA testis conducted in standard 96-well microplates. A microplate consists of a 12 X 8 grid of wells for test solutions. The three most widely used ELISA formats are immobilized antigen competitive immunoassay, immobilized antibody competitive immunoassay and sandwich immunoassay. " ... 

Another commonly used ELISA format is the immobilized antibody assay or direct competitive assay (Eigure 3). The primary anti-analyte antibody is immobilized on the solid phase and the analyte competes with a known amount of enzyme-labeled hapten for binding sites on the immobilized antibody. Eirst, the anti-analyte antibody is adsorbed on the microtiter plate wells. In the competition step, the analyte and enzyme-labeled hapten are added to microtiter plate wells and unbound materials are subsequently washed out. The enzyme substrate is then added for color production. Similarly to indirect competitive immunoassay, absorption is inversely proportional to the concentration of analyte. The direct competitive ELISA format is commonly used in commercial immunoassay test kits. 

Figure 11. Electrochemiluminescent PDMS-graphite biochip formats (a) nucleic acid-based biochip (b) immunochip (competitive immunoassay).

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

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