Sandwich ELISA protein

Sandwich ELISAs (Eigure 4) are the most common type of immunoassay used for the detection of proteins. A capture antibody is immobilized on the wells of a microplate. The solution containing the analyte is introduced and antibody-analyte... 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

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],... 

Fig. 2. Biochemical classification of the nia mutants, mutated domains and intragenic complementation. A collection of Nicotiana plumbaginifolia nia mutants (defective for NR apoenzyme) was tested for NR protein and activities (Cherel et al., 1990) and checked for in vivo and in vitro intragenic complementation (Pelsy Gonneau, 1991). NR protein amounts were measured by a sandwich ELISA test using as first antibody the anti-corn NR monoclonal antibody 96.9.25 (Ch6rel eta/., 1985) shown to be directed against the haem domain (M. Kavanagh, personal communication Meyer etal., 1991). The result is shown here as + or when a positive or negative ELISA test, respectively, was observed. Many class 4 mutants, most probably frameshift and deletion mutants, complement class 3 but not class 2 mutants.

Holzhauser, T., O. Stephan, and S. Vieths. 2002. Detection of potentially allergenic hazelnut (Corylus avellana) residues in food A comparative study with DNA PCR-ELISA and protein sandwich-ELISA. JAgric Food Chem 50 (21) 5808—5815. 

Corporation, Lansing, MI) is a sandwich ELISA used for the quantitative analysis of minimally processed soy flour protein in food products such as cookies, crackers, chocolate bars, and cereals in the range of 2.5-25mg/kg. 

In the case of too high protein concentrations or high amounts of strongly cationic proteins, solid-phase ELISA will give low extinctions or false negatively no results Either use less volume of fraction, which allows all proteins present to stick to the surface of the ELISA plate, or use sandwich ELISA. 

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.

In fishes possessing two or more Vg proteins, differential responses of the liver to estrogen with regard to production of each distinct Vg are just beginning to be elucidated. For example, Takemura and Kim191 developed sandwich ELISAs for two distinct Vgs (VTG210 and VTG140) in a tilapia species (O. mossambicus). 

The principle of a double sandwich assay is also used for the sandwich ELISA, which avoids the use of radioactivity. The second antibody contains an enzyme (e.g., horseradish peroxidase), which serves as catalyst for a color reaction of a suitable substrate. Quantification is performed using UV-spectrophotometry. This type of ELISA may be used for trace impurity analyses. Immunization for such a multiantigen assay requires the representative preparation of all host cell proteins, but this must be completely free from the product protein. 

Martin, R., Azcona, J.I., Casas, C., Hamandez, P.E., and Sanz, B. 1988. Sandwich ELISA for detection of pig meat in raw beef using antisera to muscle soluble proteins. J. Food Prot. 51,190-194. 

Heimig C, Rink L, Fagin U, Jabs WJ, Kirchner H. The influence of naturally occurring heterophiUk anti-immunoglobuhn antibodies on direct measurement of serum proteins using sandwich ELISAs. J Immunol Methods 2000 235 71-70. 

O Callaghan, J. P. 1991. Quantification of glial fibrillary acidic protein eomparison of slot-immunobinding assays with a novel sandwich ELISA. Neurotoxicology and Teratology 13 275-281. 

Most blood proteins do not show up in the urine, but hCG does. And it is produced very soon after the egg is fertilized, and then in increasing amounts as the pregnancy progresses. Sandwich ELISA (see Figure 4.35 in the text) is the ideal method for complex biological fluids, and it is relatively easy to produce two different monoclonal antibodies to epitopes on opposite sides of the protein. All home pregnancy test kits are based on variations of this method. 

Another technique, the enzyme-linked immunosorbent assay (ELISA), combines the specificity of antibodies with the sensitivity of an enzyme assay. The ELISA can be performed in a variety of combinations that involve either a specific antibody or the total cellular protein immobilized on a solid support, such as the wells of a plastic microplate. In one version of the method, the sandwich ELISA, the primary antibody is bound to the wells. When a mixture of proteins is added, the protein of interest binds to the antibody, and other proteins are washed away. A second labeled antibody, specific to a different epitope on the protein, is added, and the amoruit of signal is proportional to the amoimt of the particular protein in the sample. The method can be modified to detect specific antibodies in a mixture by using their antigen as the immobilized bait. ELlSAs also have the advantage of being able to be performed in 96-weU plates so many samples can be analyzed in one experiment. 

Stephan O, Weisz N, Vieths S, Weiser T, Rahe B, Vatterott W (2004). Protein quantification, sandwich ELISA, and real-time PCR used to monitor industrial cleaning procedures for contamination with peanut and celery allergens. J. AOAC Int., 87(6) 1448-1457. 

Two additional false-negative examples are described below. The first type, the prozone/high-dose hook effect, may occur in lateral-flow assay and one-step sandwich ELISA, which happens when the target protein is present in a sample at a very high concentration. Both capture and labeled antibodies are fully occupied by existing target protein and fail to form the sandwich complex (i.e., labeled-antibody/target... 

Double-antibody (sandwich) ELISA using specific anti-soy trypsin inhibitor and other soy protein antibodies coated onto microwells... 

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). 

Morinaga Institute Sandwich ELISA, Wheat Protein quantitative ELISA Kit... 

FIGURE 22.10 (a) Immunoblot using polyclonal anti-lupin antibody (1, molecular-weight marker 2, L. aftMj-derived extract 3, L. angustifolius flour extract) (b) Representative linear six-point calibration curve based on the standard curve obtained using the lupin protein standard in the sandwich ELISA. (From Holden etal., 2005.) (Reprinted with permission from Reference Citation. Copyright 2005 American Chemical Society.)... 

Furthermore, a novel polyclonal/monoclonal-based sandwich ELISA for the detection of lupin proteins in foods was described (Holden et al., 2007). The monoclonal antibody reacts specifically with a-conglutin (Dooper et al., 2007). The assay has a detection limit of Img protein/kg food and is sensitive to both native and processed proteins from L. angustifolius and L. albus. A selection of 112 food samples, both with and without lupin declaration, was analyzed showing that the majority of products were labeled correctly. 

ELISA Systems Mustard Seed Protein Residue kit was released in June 2007. A polyclonal rabbit antiserum was raised and used to develop a quantitative sandwich ELISA that has been demonstrated to detect mustard seed protein from aU three species of mustard plants S. alba, B. nigra, and B. juncea [5]. The detection limit of the kit has been shown to be less than 0.5 ppm (mg/kg) of soluble mustard protein, which corresponds to mustard seed concentrations below 3.4ppm S. alba, below 4.9 ppm B. nigra, and below 5.5 ppm B. juncea. An example of a calibration curve is presented in Figure 23.1. Cross-reactivity studies were conducted on full-strength extracts from 50 plants and other common foods, and cross-reactivity was observed only with rapeseed (Canola), Brassica napus. This cross-reactivity was approximately 50%, but purified canola oil did not cross react. 

FIGURE 23.1 Example of a mustard seed protein calibration curve for a sandwich ELISA. (Courtesy of EUsa Systems.)... 

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