Capture ELISA

Figure 15.9 The results of capture ELISA on native RNase A and formalin-treated RNase A. Right panel, native RNase A (curve 1) and unfractionated formalin-treated RNase A (curve 2). Left panel, individual fractions of formalin-treated RNase A monomer (curve 3), dimmer (curve 4), trimer (curve 5), tetramer (curve 6), and a mixture of oligomers with >5 cross-linked proteins (curve 7). The ELISA plate wells were coated with monoclonal antibody against bovine pancreatic RNase A (1 pg/mL) overnight at 4°C and then blocked with bovine serum albumin. The wells were incubated for lh at 37°C in the presence of various concentrations of antigen in lOOpL of PBS. After washing, each plate well received a 1 4000 dilution of horseradish peroxidase conjugated rabbit polyclonal anti-RNase A antibody followed by incubation at ambient temperature for lh. After washing, detection was achieved using a mixture of 2,2,-azino-di-(3-ethylbenzthiazoline-6-sulphonate) and hydrogen peroxide. Absorbance was monitored at 405 nm. See Rait etal.11 for details.

Capture ELISA on selected oligomeric fractions of formalin-treated RNase A (see curves 3-7 in Fig. 15.9) also reveal that the plateau values increase with an increase in the number of cross-linked molecules in the fractions. This is due to an increasing proportion of bound epitopes per binding site or, in other words, epitope density on the surface. Thus, the nearly identical plateau values for the titration of native RNase A and formalin-treated unfractionated RNase A (curves 1 and 2 in Fig. 15.9) are fortuitous, being caused by the particular composition of oligomers present in the formalin-treated RNase A preparations that was analyzed. 

Capture ELISA was used to follow the restoration of immunoreactivity in formalin-treated RNase A preparations after incubation at elevated temperatures in TAE buffer at pH 4.11 Two different formulations of formalin-treated... 

Kumarasamy, V. Wahab, A. H. A. Chua, S. K. Hassan, Z. Chem, Y. K Mohamad, M. Chua, K. B., Evaluation of a commercial dengue NS1 antigen capture ELISA for laboratory diagnosis of acute dengue virus infection, J. Virolog. Methods 2007, 140, 75 79... 

Sehr P, Zumbach K, Pawlita M. (2001) A generic capture ELISA for recombinant proteins fused to glutathione S-transferase validation for HPV serology. J Immunol Methods 253, 153-62. 

The final application of the antibody must be borne in mind when deciding the extent of characterization. Initially, the antibody must be tested to establish whether binding occurs with the immunogen, with and without any carrier molecules used in the immunization. This test should be carried out with reference to the intended application to control for bridge binding. For instance, a reagent intended for use in a capture ELISA should be tested when coated onto the assay solid phase. If the antibody is intended for in vivo immunoneutralization, it should be tested initially in a liquid phase assay. If the final use is to be immunocytochemical, then the testing should be conducted on tissue sections. 

ELISA is also of limited application when the antigen is extremely diluted, such that other molecules interfere with the performance of the technique. One approach in this case is the so-called substrate-capture ELISA . Here, a substrate for a protease, for example, is bound to the plastic plate in order to absorb and concentrate the specific enzyme for detection by immune reaction. The first modification of a standard capture assay technique in which a metalloprotease substrate is used to capture the enzyme of interest was described by Wacher et al. in 1990, and is summarized in generalized form below. The substrate-capture ELISA greatly simplifies the mixture in which the enzyme is detected and removes potentially interfering substances, thus avoiding some of the difficulties... 

Vaughn DW, Nisalak A, Solomon T, Kalayanarooj S, Nguyen MD, Kneen R, Cuzzubbo A, Devine PL (1999) Rapid serologic diagnosis of dengue virus infection using a commercial capture ELISA that distinguishes primary and secondary infections. Am J Trop Med Hyg 60(4) 693-698... 

Jones RD, Offutt DM, Longmoor BA. Capture ELISA and flow cytometry methods for toxicologic assessment following immunization and cyclophosphamide challenges in beagles. Toxicol Lett 2000 115 33 4. 

Visentin GP, Wolfmeyer K, Newman PJ, Aster RH (1990) Detection of drag-dependent, platelet-reactive antibodies by antigen-capture ELISA and flow cytometry. Transfusion 30 694—700 von dem Borne AE, Pegels JG, van der Stadt RJ, van der Plas-van Dalen CM, Hehnerhorst FM (1986) Thrombocytopenia associated with gold therapy a drag-induced autoimmune disease ... 

Antigen-capture enzyme-linked immunosorbent assay (ELISA) testing, IgM-capture ELISA, polymerase chain reaction (PCR), and virus isolation can be used to confirm a case of Marburg hemorrhagic fever within a few days of the onset of symptoms. The IgG-capture ELISA is appropriate for testing persons later in the course of disease or after recovery. The disease is readily diagnosed by immunohistochemistry, virus isolation, or PCR of blood or tissue specimens from deceased patients. 

Use of Capture ELISA to Detect and Titrate Antigen Learning Principles... 

Since you are now familiar with the indirect assay, the steps in the optimization of the capture ELISA should be straightforward. The first essential step is to determine the amount of capture antibody to be attached to the wells. We have two situations in the laboratory depending on the availability of specific reagents. We can use capture antibody as an IgG preparation, or if sufficiently... 

Essentially, the same parameters have to be standardized as for capture ELISA for antigen detection. However, the test is used to measure antibodies against a fixed amount of antigen captured on the plate. Thus, we must optimize the system to have the correct amount of capture antibody and antigen necessary to bind any test or control antisera. The test offers the ability to specifically capture antigen using a solid-phase antibody. Thus, relatively crude... 

Kashiwase, H., Ishimura, M., Ishikawa, Y., Nishigald, T. (1997) Characterization of One Monocloneil Antibody Against FeUne Immunodeficiency Vitus p24 and Its Application to Antigen Capture ELISA. 

Typically, capture ELISAs are used for screening potential transformed plants, followed by Western blotting to confirm the correct relative molecular mass of the recombinant product The techniques are identical to those in routine standard use, however due to the ubiquitous nature of plant produas in aU diets and the consequent presence of plant protein reactive antibodies, it is important to use affinity purified antibodies at all stages. Similarly, if monoclonal antibodies are used it is often necessary to remove any contaminating antibodies that might be present, for example in fetal calf serum. 

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