Antibodies Applications and New Developments

This chapter deals with the discovery and application of the antibody-antigen reaction in the form of inoculation, variolation and vaccination to induce immunity - at first to smallpox, later to other diseases and common afflictions such as rabies, anthrax and cholera, caused by microorganisms.

The work of the pioneers in this field and their discoveries is briefly described here.

The in vitro applications of the antibody-antigen reaction - mainly in the form of radioimmunoassays - is covered and includes the early development of this technique both in assay design and data reduction, which allowed the rapid acceptance of radioimmunoassay in clinical diagnostic procedures, especially in the fields of endocrinology, microbiology and pharmacology.

The successors to radioimmunoassay which use non-radioisotopic labels have also been described in detail as well as listing the pioneers in immunoassay development.

The above topics have been considered in the context of antigen-antibody reactions and some of the persons who can be termed pioneers in this area of predominantly medically-related research.

The second chapter continues the history of immunoassay and mainly deals with the commercialization and automation of immunoassays, the impact of monoclonal antibodies and their potential role in standardization and the continued development towards microformats and multianalyte immunoassays.

This chapter also includes the terminology used in immunoassays and schematic representation of assay design in the form of diagrams, as well as a brief look into the possible future development of immunoassay techniques and their application and impact in clinical in vitro diagnostic procedures.

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.

While immunoassays are now used in a variety of fields including medical, food technology and environmental protection because of their high sensitivity, high specificity and ease of automation to provide highly cost effective analyses, it has been in medical testing that their standardization has been most intensely developed. The use of internationally agreed clinical protocols, common reference intervals or decision limits and even electronic health records across health-care institutions are all dependent on medical testing laboratories performing assays that are traceable to internationally recognized reference measurement systems. The application of metrological principles to achieve traceability and standardization for immunoassays is being pursued to this end. Comprehensive measurement systems are available for the total serum hapten assays currently measured in the clinical laboratory by immunoassay. Current research is investigating the usefulness of developing defined systems for the measurement of non-bound fractions or "free" hormone levels of these types of analytes. One strategy that has been successfully applied for the standardization of assays for large molecular weight polypeptide measurands has been to localise the biological activity to a small molecular weight moiety of the meausurand and to establish a reference measurement procedure for this moiety as a surrogate for the total molecule of clinical interest. The standardization or harmonization of assays for heterogeneous polypeptide hormones is also is another area of current research for standardization of clinical immunoassays. The accreditation of medical testing laboratories to ISO15189 standard requires that all testing methods including immunoassays are validated or verified to be fit for purpose including the accuracy of the assay and the estimation of measurement uncertainty as assessed by traceability to certified reference materials through a documented unbroken chain of calibrations.

Immunochemical methods, methods that employ antibodies as analytical reagents, have revolutionized laboratory medicine. Although the noncovalent bound between analyte in biological specimen and complementary antibody in reagent is specific, positive or negative interferences are possible. Interfering substances can be present in both specimen and reagents, respectively. Most interfering substances are inherent in patients’ blood under physiological and pathophysiological conditions, especially under diagnostic and therapeutic procedures. Some interferences are similar to those in chemical analyses and some are typical only for immunoassays (hook effect; cross-reactivity with structurally similar or identical epitopes; heterophile antibodies; anti-animal antibodies; autoantibodies; the matrix effect). The main characteristic of all immunoassays is that the reagent which discovers or quantifies the target analyte contains the specific antibody. Results higher than real values are recorded due to a low antibody specificity. Results can also be influenced by pre-analytical factors or assay formulations. One should suspect interferences a) after obtaining an unacceptable result; b) if there is non-linearity during dilution; c) if there is no agreement with other test results or clinical data; d) if different immunoassays in determination of the same analyte provide significantly different results. It is necessary to think about present predictable and always possible unpredictable and unrecognizable interferences. Unawareness and non-recognition of interferences could lead to diagnostic errors, unnecessary laboratory tests, inadequate and unnecessary treatment and therapy. There is no simple and practical way to identify interferences in specimens before analysis. Whenever interference is suspected, there are a few possibilities to solve them, e.g. serial dilution, pre-treatment with blocking reagent, use of more specific methods. At the same time a proper information upon pre-analytical diagnostic and therapeutic procedures is needed for accurate interpretation of the results. In this process, consistent communication and compliance between laboratory professionals and clinicians are required.

With new developments in agriculture and food production in the past two decades, bioanalytical detection of toxins and allergens in foods became a challenging task. In addition, efficient validation and labelling of food products demand reliable technical approaches. Complex, heterogeneous matrices and low amounts of specific analytes complicate detection and characterization of harmful substances in food. A highly relevant tool to achieve this goal is represented by several immunochemical approaches, based on antibody technology. Different types of antibodies have been developed, which are characterized by high affinity and specificity. Although, alternative methods have been introduced into food analysis (e.g., mass-spectrometry and PCR), antibodies still play a key role, especially in detection and characterization of allergens. In this chapter, I discuss basic immunochemical methods of food analysis based on antibody technologies. The increasing importance of establishing suitable detection schemes for several allergens (e.g., allergens from peanut and soy) in an immunoanalytical approach, will be particularly emphasized.

The food chain is threatened by various hazards and the presence of residues of antibiotics, used during cattle-breeding, is one of the serious risks for consumers. In food and feed analysis for antibiotics, screening technologies are powerful tools that provide a rapid screen of large numbers of samples when conventional analytical methods are too cumbersome. Due to their simplicity and/or high-throughput capacity, immunoassays are applicable for screening at critical control points in the food chain and in control laboratories. However, in general, they are very specific, and only suitable for the detection of one or two antibiotics, which seriously limits their application. New antibodies and new assay formats with multiplex capacity might give new possibilities for control agencies and food industries for increased and more efficient controls on antibiotics and other food contaminants. Multiplex applications and future perspectives of poly- and monoclonal as well as recombinant antibodies for the compound- or group-specific detection of antibiotics, using different immunoassay formats (e.g. ELISAs, dip sticks, biosensors and flow cytometers), are described and discussed.

The use of growth promoters to fatten cattle, once widespread, is now banned or strictly regulated worldwide, with a view to protecting consumer health. The European Union, in particular, has issued directives, regulations, and guidelines for controlling such substances. Immunoassays (radioimmunoassays, ELISAs, biosensor assays) provide quick, sensitive, low-cost, and highthroughput screening tools for detecting steroids, corticosteroids, and β-agonists in live animals and meat. Their development has led, for instance, to eradication of the use of the dangerous compound diethylstilbestrol. Current challenges include the growing number of growth-promoting “black market” substances and the emergence of “cocktails” containing low levels of several mutually potentiating compounds. New analytical approaches, such as immunochemical multiplexing or the development of receptor-based assays, may contribute to meet these challenges.

The application of antibodies for environmental analysis has a long history. In the first few years there have been developed several immunoassays, predominantly EIAs/ELISAs, for the detection of pesticides in water, which have been reviewed by many authors. Subsequently, the formats of the immunoassays were changed from enzyme immunoassays to fluorescent, (chemi/bio) luminescent, polarization fluorescent and other assays and from tube or microtiter plate to flow injection, lateral flow strips, dipstick, immune biosensor assays, etc. The goal was always to design faster and more sensitive assays. In addition, the range of target compounds was substantially extended. Initially, the more polar pesticides were chosen as target compounds for analysis because of their simple couplings chemistry for the synthesis of tracers and coating conjugates. However, more apolar pesticides followed as well as various industrial pollutants and even toxins and microorganisms. In the last few years, the attention was focused on other unwanted compounds present in the environment, such as Endocrine Disrupting compounds (EDC) and pharmaceuticals. Given the development in other fields of investigation, such as analytical chemistry, toxicology and bioassays, the list of emerging target compounds is increasing.

In this chapter, an overview is given of the development of immunoassays in various formats from the earlier days until now. In addition, attention will be given to various target compounds and organisms as well as the basis of selection.

Clean water, its secure delivery to consumers, and protection of resources are among the most important tasks for humans in future. To protect water resources and control water quality, it is necessary to develop fast, sensitive, cost-effective, and easy-to-use analytical systems capable of measuring a variety of small organic pollutants in aqueous samples.

Immunoassays use antibodies as a versatile analytical tool and are nowadays introduced into many different applications. The predominant testformats in the past have been the RIA (radioimmunoassay) and ELISA (enzyme linked immune sorbent assay). Together with the developments in the area of biosensors, the need for automated immunoassays carried out at the heterogeneous phase has lead to the development of new assay formats. Usually, these systems combine the approaches of various immunoassays with Flow Injection Analysis (FIA) systems and different fluorescence or label-free transduction technologies.

This chapter is dedicated to the application of antibodies in multi-analyte immunoassays for water monitoring with a focus on their application in fluorescence based optical biosensors. This includes practical aspects during assay development like affinity, cross-reactivity, and the validation process. In addition, a strategy to use antibodies with high cross reactivities (e.g. to the chemical group of estrogens) for the detection of sum concentrations will be explained as well as a strategy to reduce matrix effects caused by dissolved organic carbon.

Immunoaffinity Chromatography (IAC) is a type of liquid chromatography that uses the highly specific binding of antibodies for the retention of a target. This approach can be used in a myriad of techniques for the selective purification, concentration and/or analysis of target compounds. This chapter discusses the basic components of an IAC method, including the support, stationary phase, immobilization technique, and proper sample application and elution conditions. Several applications of IAC are also considered. These applications include immunoextraction, immunodepletion, chromatographic immunoassays, and post-column immunodetection. A wide range of targets that have been isolated or measured by these techniques are listed. Related methods based on antibody mimics are also discussed.

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.

Immunosensors are devices that comprise both a biomolecular recognition system, such as an antibody and its corresponding antigen, and a transducer to translate the high affinity and specific binding event into a physical signal.

Antibodies are produced by an immunological response to the presence of a foreign substance called an antigen. Antibodies may be immobilised onto a variety of platforms including bulk planar surfaces and nanoparticles by either covalent or adsorption strategies. Different interfaces between the biocomponents and the detector are available to monitor in ‘real-time’ the signal generated by biological interactions. The transducers detect, for example, the change in electron transfer, absorbance, fluorescence, refractive index, mass change or heat transfer as the antibody binds to the antigen of interest. Such analytical devices have allowed a wide range of analytes to be identified and quantified such as pathogens, toxins, environmental food contaminants and disease biomarkers.

The demand for sensitive, rapid, and ‘on-site’ techniques has taken advantage of the latest advances in microfluidics and nanotechnology. This chapter will highlight current trends in immobilisation, micro/nano-fluidics/and transducers utilised. A number of examples outlining the exploitation of these elements in immunosensors and their successful applications will be described.

Immunoassays are based on the molecular recognition occurring between an antigen and its antibody. They can be set up in a variety of formats (sandwich or competitive assays). The main differences between them are the immobilised species (antibody or target analyte), the number of experimental steps involved, and in which order the different reagents are exposed to the surface. The choice of the format depends on the molecular size of the analyte, the availability of reagents and the cost. Radioimmunoassays (RIA), fluorescence immunoassays (FIA) and enzyme immunoassays (EIA) are well established in clinical diagnostics. For the development of hand held devices which can be used for point of care measurements, electrochemical immunoassays are optimal alternative to existing immunochemical tests.

In this chapter, the current status of research in electrochemical immunoassays is considered. Primary attention was focused on label-free and enzyme-labelled immunosensors, and the analytical performance of these devices are discussed. Moreover, the use of magnetic beads as new materials for immunoassays coupled with electrochemical transduction is also described. Examples of such devices used for the detection of some biomarkers in clinical analysis are reported.

The advent of innovative recombinant DNA technology has made antibodies a new generation of pharmaceuticals for diagnosis and therapeutics. For the past few decades, minimal antigen-binding fragments, such as Fab (fragment antigen binding), Fv (fragment variable), and single variable domains (sVD), have emerged as credible alternatives to monoclonal antibodies (mAb) in many applications. These recombinant antibody fragments (rAbFs) retain the target specificity and affinity of whole mAbs and have the advantages of economical production, superior biodistribution, easy genetic manipulation and chemical modification to create reagents with required properties for a range of diagnostic and therapeutic applications. To enhance their application efficiency, these fragments have been engineered into multivalent, multispecific, or fusion molecules, or linked to therapeutic payloads (e.g. radionuclides, liposomes, toxins, cytokines, enzymes, and anti-sense RNA) that perform a specific function. The advances of selection technologies (e.g. phage-, ribosome-, and yeast-display) and the emergence of various production systems (e.g. bacteria, yeast, plants, and mammalian cells) have significantly facilitated rAbF development and diversified their applications. This chapter primarily focuses on the current advances in antibody engineering for improving the diagnostic or therapeutic applications of rAbFs.

With more than 25 molecules in clinical use, monoclonal antibodies have finally come of age as therapeutics, generating a market value of $11 billion in 2004, expected to reach $26 billion by 2010. While delivering interesting results in the treatment of several major diseases including autoimmune, cardiovascular and infectious diseases, cancer and inflammation, clinical trials and research are generating a wealth of useful information, for instance about associations of clinical responses with Fc receptor polymorphisms and the infiltration and recruitment of effector cells into targeted tissues. Some functional limitations of therapeutic antibodies have come to light such as inadequate pharmacokinetics and tissue accessibility as well as impaired interactions with the immune system. This review aims at giving an overview of the current state of the art and describes the most promising avenues that are followed for generating the next generation of antibody-based cancer therapeutics, with a special emphasis on bispecific antibodies.

Aptamers are small oligonucleotides (10 – 20 kD) that bind with high affinity and specificity to a large number of target molecules. K_d-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.