Antibody assay

Figure 3. Antigen-Antibody assay. Lane 1 moleeular mass standard, lanes 2 and 4 glucose culture medium, lanes 3 and 5 apple pectin culture medium. Lanes 2 and 3 to isolate T2, lanes 4 and 5 to isolate rij.

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 8 Structure of immunogen haptens for pyrethroids with spacer arm attachment at the a-position of the alcohol moiety. Since the whole pyrethroid molecule is available for recognition by the antibody, assays resulting from these immunogens were selective for the parent pyrethroids...

Many issues surrounding neutralizing antibodies remain such as standardization of the neutralizing antibody assay, testing recommendations, and treatment recommendations for positive tests 41 Neutralizing antibodies may disappear even with continued treatment. Neutralizing antibodies exhibit cross-reactivity with the other beta interferons.41... 

T.-S. Zhong and G. Liu, Silica sol-gel amperometric immunosensor for Schistosoma japonicum antibody assay. Anal. Sci. 20, 537-541 (2004). 

F.C. Gong, Z.J. Zhou, G.L. Shen, and R.Q. Yu, Schistosoma japonicum antibody assay by immunosens-ing with fluorescence detection using 3,3, 5,5 -tetramethylbenzidine as substrate. Talanta 58, 611-618 (2002). 

Rolling circle amplification (RCA) is an alternative method to polymerase chain reaction it is also a generic amplification technique that can be used in antibody assays. Using a replication process similar to that used by viruses, RCA allows the recognition,... 

Quenching can actually be used to create analytical methods. An example is that of fluorescent antibody assay. Here, a fluor is bound to an antibody but the binding constant is less than that for the antibody-antigen pair for which the antibody was generated. Binding quenches the fluorescence of the fluor. When a solution containing the native antigen is mixed with the fluor complex, the fluor is released and can fluoresce normally. There are numerous examples of this approach. 

Antibody assays are the most widely used methods for HIV testing because they are better suited than other methods for routine use in blood banks and screening programs. 

Some patients will have repeatedly nonspecific reactivity in an ELISA assay, since the HIV-1 antigen used for the antibody assays is produced in cultured human T cells. It is not unexpected that occasional false positive assays occur in human sera from individuals with autoimmune diseases a history of multiple pregnancies or multiple transfusions or antibodies to certain class II histocompatibility antigens (especially HLA-DR4). Block reagents have been added to specimen diluents to minimize cross-reactions in these sera. This necessitates the use of confirmatory tests, especially the Western blot. With the use of both ELISA and Western blots, false positives decrease to less than 1 per 100,000. 

The RIPA is especially sensitive for antibodies to the higher-molecular-weight major env glycoproteins gp 160 and gp 120, which are missed by some WB techniques. Sera from blood donors with probable false-positive WB patterns are often negative by RIPA. Thus, the technique may be useful in resolving conflicting results from other HIV antibody assays. 

However, not all antibody detected by ELISA corresponds to the antibody molecule that blocks the biological activity or active sites of the therapeutic protein. These neutralizing (blocking) antibodies are only a small fraction of the binding antibody population. Different cell- and receptor-based neutralizing antibody assays are developed for different protein drugs. 

Lane, D. P. and Lane, E. B. (1981) A rapid antibody assay system for screening hybridoma cultures. J. Immunol. Methods 47, 303—307. 

Analyte Antibody Assay Concentration Detected Referen- ences... 

Antibody Assays Monoclonal antibodies can be analyzed by the solid-phase assay techniques such as enzyme linked immunosorbent assays (ELISA) or radioimmuno assays (RIA). The typical assay procedures are as follows (Figure 5.9)... 

Fig. 5.9 Schematic diagram for the ELISA antibody assay. The enzyme will be replaced by a radioactive label in the case of RIA assay.

Wherever an antibody assay is used now, there will be the prospect of producing an immunosensor for the same analyte. Therefore, the development of immunosensors is directed to obtain sensors with fast response, low detection limits, little preparation efforts, low price availability and high specificity. Thanks to the possibility of fabricating fast portable sensors, immunosensors now constitute a potential alternative to centralized and sophisticated bioanalytical systems. 

Competitive binding immunoassay (e.g., RIA) Enzyme-linked immunosorbent assay (ELISA) Immunoradiometric assay (IRMA) — dual monoclonal antibody assay Receptor binding assay Cell binding assay... 

Ahlstedt, S., Holmquist, I., Koner, A. et al. 2002. Accuracy of specific IgE antibody assay for diagnosis of cow s milk allergy. Ann Allergy Asthma Immunol 89 21-25. 

Kleine-Tebbe, J., M. Eickholt, M. Gatjen, T. Brunnee, A. O Connor, and G. Kunkel. 1992. Comparison between MAGIC LITE- and CAP-system Two automated specific IgE antibody assays. Clin Exp Allergy 22 (4) 475 184. 

In CD, autoantibody assays have generally replaced gliadin antibody assays. Measurement of gliadin antibodies is, however, advantageous in small children. Autoantibodies are often absent in CD children under 2 years of age [148, 149]. In another study, antibodies against tTG were found to develop not before an age of 1.3 years [150], Increased concentration of gliadin antibodies may be helpful in the investigation of other... 

The TRH radio-immunoassay was developed as a double antibody assay, with modifications mainly depending on the antiserum used, and on the conditions of incubation. Addition of an enzyme inhibitor to the assay tubes is essential to avoid degradation of TRH in samples and of radioiodinated TRH. 

G9. Gomez, B., Ardakani, J. J., Jenkins, D., Cerelli, M. J., Daniloff, G. Y., and Kung, V. T., Monoclonal antibody assay for measuring bone specific alkaline phosphatase in serum. Clin. Chem. 41,1560-1566 (1995). 

Van der Zee JS, de Groot H, van Swieten P, Jansen HM, Aalberse RC Discrepancies between the skin test and IgE antibody assays Study of histamine release, complement activation in vitro, and occurrence of allergen specific IgE. J Allergy Clin Immunol 1988 82 270-281. 

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