Immunochromatographic techniques

The fundamentals of affinity chromatography can be taken from the literature [2-5]. Since affinity-based analytical methods are extremely dependent on the analytes and their respective binding molecules, it is not possible to go into much detail with regard to practical advice. It has to be mentioned that affinity chromatography is rarely a true chromatographic technique, and in most cases has to be considered to be a selective extraction method. Hence, the term affinity extraction or immunoaffinity extraction should be preferred where appropriate. 

In many affinity techniques, biomolecules such as DNA, oligonucleotides, proteins, peptides, cofactors, and other biochemical compounds are used as reagents. Mainly proteins and, in particular, antibodies are discussed herein, due to their broad applicability. However, many rules can be easily transferred to other binding molecules. 

HPLC Made to Measure A Practical Handbook for Optimization. Edited by Stavros Kromidas Copyright 2006 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim ISBN 3-527-31377-X 

From the serum of an animal, only polyclonal antibodies can be obtained, i.e. a mixture of different antibody species in different concentrations. Drawbacks of polyclonal antibodies are their critical long-term supply and a lack of reproducibility from batch to batch. The composition of a serum changes all the time and the sera of two individuals are completely different in terms of antibody concentration and their selectivity. Hence, polyclonal antibodies cannot be considered as defined reagents. On the other hand, the production of polyclonal antibodies is much cheaper than any other method and hence is often the economically most sensible way to obtain an immunoreagent. 

In contrast, monoclonal antibodies consist of only one protein species and are produced by cell culture. Hence, monoclonal antibodies can be considered to be pure chemical compounds (to a first approximation). The respective clones (cell lines) can be cultured indefinitely and can be stored for quite a long time by freezing in liquid nitrogen. Unfortunately, the generation and production of monoclonal antibodies (Mabs) is much more difficult and expensive than that of polyclonal antibodies (Pabs). There is an important misunderstanding in this respect, which should be mentioned here. It is definitely not true that polyclonal antibodies are less selective or show lower afSnity than monoclonal ones. On the contrary, polyclonal antibodies often show a better performance than monoclonal 

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Immunochromatographic techniques - a critical review Weller, M. G. Fresenius J. Anal. Chem. 2000, 366, 635-645. 

Quantify the amplification products by the sandwich immunoassay protocol of your choice or use the immunochromatographic technique detailed below. 

The nomenclature of immunochromatographic techniques is not consistent and may easily lead to misunderstandings. In addition, the respective hyphenations have their own designations. However, there are mainly three fimdamental setups that have to be taken into consideration (Fig. 1). 

Weller MG. Immunochromatographic techniques—a critical review. Fres J Anal Chem 2000 366 635-45. 

The first immunoassay performed in a capillary driven system was reported in 1978 [67]. Based on this technique, the commonly known over-the-counter pregnancy test was introduced into the market in the middle of the 80 s. Today, this microfluidic platform is commonly designated as a lateral flow test (LAT) [14]. Other terms are test strip , immunochromatographic strip , immunocapillary tests or sol particle immunoassay (SPIA) [68]. Astonishingly, hardly any publications from a microfluidic point of view or in terns of material classification exist, and apparently many company secrets are kept unpublished [69]. 

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