Antibody sandwich ELISA assays

Nowadays, antibodies are utilized in numerous immunoanalytical methods. Those widely used in practice, such as radioimmunoassays, fluoroimmunoassays and enzyme-linked immunosorbent assays (ELISA), require labelled reagents. Millions of ELISA tests for diagnostics of various diseases are daily performed in clinical laboratories. The detection of analytes by two-antibody "sandwich" ELISA, is schematically outlined in Figure 3. 

Given their high degree of sensitivity, which is 2-5 times more than assays in which the antigen is directly bound to the solid phase, antibody sandwich ELISA is arguably the most useful of the immunosorbent... 

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

Sandwich ELISA assays can be carried out in many different ways, but a typical procedure would be as follows. Antigen, at various dilutions, is added to individual chambers in a microtitre plate containing immobilised antibody. The plates are shaken gently for 2 h at room temperature and the solutions are then removed from the microtitre plate chambers. Non-specific binding is blocked by adding 200 pi... 

Figure 7. Standard curve of gel purified 60 kd protein endotoxin of Btk was generated using a double antibody sandwich ELISA. The arrows indicate dilutions of two Btk formulations absorbance values were used to determine the endotoxin concentrations of the formulations, based on the standard curve. The formulation dilutions gave curves that were virtually superimposable on the standard curve. Such similarity in shape and slope indicate that the antibody is likely binding to a specific determinant common to the purified Btk and the Btk in the formulation. In general immunoassays for biopolymers are much easier to develop than assays for small molecules. However, only recently has an interest in trace analysis of such materials begun to develop in the environmental field. Thus, sample cleanup and handling is not as sophisticated as with small molecules.

The Soy Protein Residue Assay is a double-antibody (sandwich) ELISA using specific anti-soy tripsyn inhibitor and other soy protein antibodies coated onto microwells. After addition of the sample, the enzyme conjugate, and the TMB substrate, a positive reaction (indicating the presence of soy protein), produces a blue color. Addition of the stop solution ends the assay and turns blue to yellow. The result may be read visually (in the qualitative method) or with an ELISA reader (in the qualitative or quantitative method). Quantification can be obtained by mnning positive control standards (2.5-5-10-25 ppm) together with the samples. A standard curve is then plotted using the optical density (OD) values of the control standards (OD vs. concentration). 

Conditioned medium was collected from drug- and untreated human B3 cells. AP(l-40) and AP(l-42) levels were measured using a standard sandwich ELISA assay where 2G3 and 21F12 antibodies captur Api-40 and AP M2, respectively. 266B antibody was used for detection. 

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. 

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

Table 6.2 Assay Sensitivity for Antibody-Antigen Microarrays and Standard Sandwich ELISA... 

In reality, these forays represent miniaturization of the standard sandwich ELISA to attain higher throughput assays by multiplexing a limited number (<50) of analytes, e.g., cytokine panels. Even at these low densities, quantification problems arise in part due to a lack of robustness in the printing process and also in the selection and stability of monoclonal antibodies that must be highly specific and of high binding affinity to be useful for microarrays. 

Test wells exhibiting growth for the presence of monoclonal antibodies when the hybridoma growth covers approx 25% of the base of the cell well. The assay system used should mimic the final test format required. Commonly, Triple Antibody Sandwich (TAS) (5) enzyme-linked immunosorbent assay (ELISA) is use for screening hybridomas for specific antibody. It is important when testing hybri-... 

It is not always possible to use exactly the same format for screening as will be used in the final assay but it is advisable to attempt to ensure that the position that the monoclonal antibodies (MAb) will occupy in the final format is the position that is used for screening. For example MAbs which will eventually be conjugated to enzymes and used in Double Antibody Sandwich (DAS) ELISA should be screened for using TAS ELISA. This ensures that the epitope-binding portion of the antibody is in the same position as it will be in the final test format with the analyte bound to the coating antibody. 

The dual-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA Fig. 1) is a sensitive and specific technique for quantifying molecules in solution. The technique is dependent upon the availability of two antibodies recognizing separate epitopes upon the antigen to be measured such that they are able to bind to the molecule simultaneously (1,2). The capture antibody specific to the substance to be measured is first coated onto a high-capacity... 

The ELISpot assay has been adapted for the detection of individual cells secreting specific cytokines or other antigens. ELISpot assays employ the quantitative sandwich ELISA technique. A monoclonal antibody specific for the cytokine is precoated onto a microplate. Cells are pipetted into the wells of the microplates. During the incubation period, the immobilized antibody (in the immediate vicinity... 

Instead of the sandwich ELISA-system, which was applied by Damle et al. (1998), Hildebrand et al. (1996) and Audebert et al. (1994), the competitive RIA-system was applied by Wring et al. (1996) and Basu et al. (1996) for toxicokinetic assays. The competitive RIA-system needs only one antibody but this may lead to a loss of selectivity. 

In addition to ELISA and Western blots for detecting host-cell protein impurities there is a third immunoassay now available to the bioanalyst for this purpose the immunoligand assay (ILA). Like the ELISA, it is configured as a double-antibody sandwich. The anti-host-cell protein antibodies are separately conjugated to biotin and to fluorescein. A tripartite immune complex is formed between host-cell protein impurities and these two anti-... 

These assays resemble a hybrid of an immunohistochemistry assay and ELISA. Whole cells are fixed, for example with 3.7% formaldehyde, to MTPs permeabilized by repetitive washing with 0.1% Triton X-100, blocked with a protein solution, probed with primary antibodies (phospho-spe-cific, and non-phospho-specific), washed, and subsequently the secondary antibodies labeled with infrared fluorescent tags are added. After washing, these assays are read in a reader (such as the Odyssey or Aerius) designed for high sensitivity detection of two colors. The two colors are useful because one color can be used to accommodate a stain assigned as a total protein or cell number control or as an antibody to total protein, which allows for normalization. These assays may only require a single antibody versus the dual antibody sandwich required for ELISA. 

A sandwich ELISA is used to search for a desired analyte in a test solution, as follows the solid phase is coated with analyte-specific capture antibodies to pull the analyte out of the test sample. After washing, the amount of analyte bound to the solid phase can be determined by adding an excess of enzyme-labelled analyte-specific antibody. The specificity of the method can be improved by using a sandwich-type, two-site assay, in which the capture and labelled antibodies have specificities for different parts of the analyte, as mentioned above. The performance of an immunoassay in a standard microtitre plate requires several hours. Such long incubation times are mostly linked to inefficient mass transport from the solution to the surface, whereas the immunocapture itself is a rapid process. 

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