Aptamers are small oligonucleotides (10 – 20 kD) that bind with high affinity and specificity to a large number of target molecules. Kd-values for the aptamer-target interactions vary from a few picomolar (pM) to a few nanomolar (nM), which is comparable to the binding of antibodies. They are selected from combinatorial libraries consisting of about 1015 different oligonucleotides in an entirely in vitro system. As no animal is used, the target molecule does not have to be immunogenic and is allowed to be (highly) toxic. After characterization, using standard molecular biological techniques, aptamers are chemically synthesized in small quantities or on large scale.
These “chemical antibodies” can easily be modified in a side directed way for different purposes, such as immobilization on a surface, addition of labels, or changing their pharmacodynamic and pharmacokinetic profiles. Over the last 20 years, aptamers have been widely used in diagnostic (sensors), biotechnology and therapeutic applications. To date, there is one marketed aptamer-based drug for the treatment of wet macula degeneration, and several aptamers are currently in the clinical pipeline.
A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most cases, coloured colloidal nanoparticles serve as a label. The method is very user-friendly, as only the liquid sample has to be added. Results are available within 5-15 minutes and after evaluation of the signal by visual inspection, a desktop scanner with image analysis software, or a dedicated reader, the used strips can be discarded. With respect to the specificity, sensitivity and efficiency the technology is heavily dependent on the recognition of the analyte by the corresponding antibody. Lateral flow assays are mainly used for qualitative or semi-quantitative detection of (un)wanted substances in the biomatrix or the environment. The technology requires a minimum of resources and skills of the operator. Many applications already reached the market. We will address here a bit of history and the general principle of the technique, and critical parameters influencing the performance of the assay. Amongst those are the material of the membrane, the sample pad, the conjugate pad and the absorbent pad, properties of currently used labels, formats of the tests and properties of good recognition elements. Processing of the results will be discussed as well.
Fundamental immunological principles and factors influencing production strategies for antibodies used as reagents in analytical and diagnostic methods are highlighted. A role of the peptideprotein design for an antibody formation, covering particularly epitope selection, peptide and protein chemistry, carrier-protein conjugation including multiple antigen protein approach are described. Special attention is paid to hapten design and production of antibodies against molecules having lowmolecular character. Critical stages in polyclonal and monoclonal antibody development as well as practical hints for their production are discussed.