Development and validation of solvent free elution procedures for the isolation of mycotoxins by immunoaffinity

Table of Contents

1  Introduction and Scope of the work  4

2  Theoretical Part  7

2.1  Fungi moulds and mycotoxins  7

2.2  Major Groups of mycotoxins occuring in food and feed  8

2.3  Food Safety Aspects and Regulations  12

2.4  Mycotoxin-Analysis  13

2.5  Immunoaffinity Clean-Up  14

2.5.1  Immunosorbents and Antibodies  14

2.5.2  Supports for Immunosorbents  17

2.5.3  Bonding density  18

2.5.4  Selective Extractions and Cross-Reactivity  19

2.5.5  Capacity  19

2.5.6  Immuno-based applications for Mycotoxins  20

2.6  Determination of Mycotoxins  21

2.6.1  Principles of reversed-phase high performance liquid  

  chromatography (RP HPL)C  21

2.6.2  Individual Mycotoxins  23

2.6.3  Liquid chromatography mass spectrometry (LC-MS) as  

  universal detector for multi-toxin extracts  23

3  Results and Discussion  25

3.1  Exploratory Experiments with Deoxynivalenol  25

3.1.1  Background  25

3.1.2  The Surveillance procedure  27

3.1.3  pH solutions  27

3.1.4  Heated elution procedures  28

3.1.5  Statistical analysis  32

3.2  Zearalenone  36

3.2.1  Mixtures of water with organic solvents  37

3.2.2  Discussions  39

3.2.2.1Unspecific  bindings  39

3.2.2.2Recombination  of antibodies  41

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3.2.2.3Interfering  Peaks  41

3.2.3  Statistical analysis  44

3.3  Aflatoxins  46

3.3.1  Statistical Analysis  51

3.3.1.1Aflatoxin  B1  51

3.3.1.2Aflatoxin  B2  52

3.3.1.3Aflatoxin  G1  54

3.3.1.4Aflatoxin  G2  55

3.4  Ochratoxin A  56

3.4.1  Statistical analysis  57

3.5  Fumonisins  59

3.5.1  Statistical analysis  60

3.6  T-2 and HT-2 toxin  62

4  Technical  65

4.1  Alternative heating procedures  65

4.1.1  Self-built heating Block  65

4.1.2  Microwave  67

4.1.3  Soldering Rod  68

4.1.4  Oscillating circuit  69

4.1.5  Electrical operated heating  70

4.2  (Semi-) Automation  67

5  Summary  72

1 Introduction and Scope of the work

To ensure the health of humans and animals, the production of safe food and feed is indispensable. All over the world, food-borne diseases are among the most widespread health problems. These health problems are either of infectious origin, e.g. Salmonella, or they are associated with the consumption of toxic products, e.g. natural toxins such as mycotoxins or industrial chemical residues as for example polychlorinated biphenols (PCBs).

Especially for natural toxins the monitoring of possible contamination in food- and feedstuffs is an important and complex issue, causing a huge investment in time and effort by many regulatory and industrial laboratories. For more than 30 years, considerable research has also been devoted to develop methods for detecting and determining mycotoxins in foods, feeds and biological fluids.

But still, demands from consumers and regulators constantly grow to improve the quality and moreover the safety of food. To supply regulators, consumers and industry with long- term solutions to the complex problems associated with chemical contaminant monitoring, the European Commission has made several calls in its 4 th , 5 th , 6 th and the current 7 th Research Framework Program to improve methodologies for mycotoxin determinations. As a result of a recent call in the 6 th framework program, the so called BioCop – Project was launched. The aim of BioCop is to develop fast and cost-effective technologies for the screening of food contaminants. One approach within the project is to identify specific transcriptional "alarm" responses to phytoestrogens, organochlorine pesticides and also mycotoxins. 1 The Community Reference Laboratory (CRL) for Mycotoxins as a partner in BioCop focuses on novel determination techniques for mycotoxins.

Until today, various methods for the determination of mycotoxins exist, whereunder chromatographic methods are the most widespread used for final separation of matrix components and detection of the analyte of interest. High performance liquid chromatography (HPLC) methods have been developed for almost all major mycotoxins in cereals and other agricultural commodities, and are nowadays widely used because of their good performance and reliability. 2 Thin layer chromatography (TLC) is mostly the method of choice for rapid screening purposes and for situations where advanced HPLC equipment is not available, but can also be used for quantitative analyses with

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densitometric detection. The use of gas chromatography (GC) is restricted to a limited number of mycotoxins, especially the trichothecenes.

All these methods depend on a suitable extraction and isolation procedure prior to the measurement. This clean-up procedure is often equal for TLC, HPLC and GC, depending on the separation and the specificity of the detector.

Most methods are reliable and stable, so the main challenge today is to provide comparable results: Several projects of the European Commission deal with the production of calibrants and (certified) reference materials as well as the organisation of intercomparison studies between different laboratories, a prerequisite for the establishment and implementation of EU guidelines.

Inside the BioCop – Project a range of new technologies such as transcriptomics, proteomics and biosensors will be utilised. These new approaches are based on measuring effect rather than on measuring single target compound concentrations. For these new methods, rather pure and if possible organic solvent free extracts were aimed, as it was noticed that biosensors and proteomics can obtain more reliable results using aqueous solutions.

Nowadays, a key component for the sample extract purification in modern mycotoxin analysis is immunoaffinity column chromatography. Immunoaffinity columns (IACs) can be self made, but are also commercial available. Different suppliers provide different IACs using several immunosorbents containing different antibodies. Currently the only used and recommended procedure by manufacturers of IACs is to elute the purified mycotoxins from the column with neat organic solvents to break the antibody-antigen bond.

One limitation of immunoaffinity clean ups is, that the purified mycotoxins – eluted with pure organic solvents such as methanol or acetonitrile – cannot be directly injected in larger aliquots for chromatographic separation systems, as this would result in insufficient separation in commonly used reversed-phase systems. 3

Hence, only a rather small fraction of the eluate is injected after dilution with water or the IAC eluate is evaporated and re-dissolved in mobile phase, which is essential for rather polar analytes that require mobile phases with water contents higher than 85%. 4 In the first case, this means that the capacity of the IAC must be sufficient to allow the analysis of the fraction injected and that larger amounts of sample extracts must be applied. In the latter case, the evaporation limits the simple automation of the clean-up procedure.

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Therefore, this study aimed to investigate alternative, preferably solvent free procedures for the elution of mycotoxins from IACs. New elution procedures may contain the use of more dramatically conditions such as heat or protein denaturating agents such as glycin-HCl or urea. Goal should be a procedure which offers several advantages, such as automation, the need of less antibodies in an IAC and as result of that, better limits of detection.

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2 Theoretical Part

2.1 Fungi, Moulds and Mycotoxins

In terms of microbiology, moulds are summarized a systematic heterogeneous group of fungi, that are plant-like organisms composed of long filaments called hyphae. Mould hyphae grow over the surface and inside nearly all substances of plant or animal origin and under a wide range of climatic conditions on agricultural commodities (grains, spices, fruits, coffee, nuts, etc.) in the field and during storage. 5/6 Because of their filamentous construction and consistent lack of chlorophyll they are considered to be separate from the plant kingdom and members of the kingdom of fungi. They are related to the familiar mushrooms and toadstools, differing only in not having their filaments united into large fruiting structures.

Today there are about 500 7 identified filamentous fungi existing and not less of them play economically an important role – either positive as used in the food industry as Botrytis (precious-moulds) for example on cheese (Penicillium camemberti) or in the medical science as antibiotics ("Penicillin") as well as harmful as responsible for diseases and the production of mycotoxins. Potential diseases caused by fungi can emanate from two ways. Both, inhalation of their spores as well as direct affection of moulds often cause skin- or mucosa infections but can also trigger invasive infections concerning the viscera. The second and more important health risk emanating from moulds refers to their production of mycotoxins in harmful levels.

Mycotoxins are toxic secondary metabolites of low molecular weight, roughly in the range of 50 – 800g/mol produced by these filamentous fungi which enter the food chain when excreted by moulds growing on the agricultural commodities. The definition secondary metabolites refers to those compounds which are not essential for the growth and the survival of the fungi. Most of the mycotoxins that are considered to be important are produced primarily by three genera of fungi: Fusarium, Aspergillus and Penicillium species and have the following characteristics to be distinguished from primary metabolites 8 : (I.) The distribution in micro-organisms is restricted (II.) They are characteristic for an individual species, strain or gender (III.) They are formed along specialised pathways from only a few primary metabolites

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The name mycotoxin combines the Greek word for fungus "mykes" and the Latin word "toxicum" meaning poison. The evidence of mycotoxin poisonings can be traced back to the middle age. The gangrenous poisoning "Ergotism", known as St. Anthony's fire, was caused by the fungi Claviceps Purpurea or Claviceps paspali. Although of this, mycotoxins were first discovered in the 1960s, when the cause of the so called Turkey-X-Disease was identified. The disease resulted in the death of more than 100.000 turkeys from an acute necrosis of the liver after they were fed with peanut meal. The main responsible toxic metabolites were identified as the aflatoxins. 5/6 In the 1960s it was first possible to isolate other mycotoxins, such as for example zearalenone in 1962 by Stob. Others, like the fumonisins were first discovered in the 1980s. However, even today the diagnosis of mycotoxicoses is difficult because the effects observed are not necessarily unique to a single mycotoxin.

Today, there are more than 300 mycotoxins known, however only about 20 of these can be detected in food and feed at levels that are considered as a risk for the health of humans and animals. 9 Some toxins are lethal, some cause identifiable diseases or health problems, some weaken the immune system without other observable symptoms, some act as allergens or irritants, and some have no known effect on humans. The most relevant mycotoxins in food and feed are aflatoxins, fumonisins, ochratoxin A, the trichothecenes (e.g. deoxynivalenol, T-2 and HT-2 toxin), zearalenone and patulin. Minor relevant as food contaminants are for example cyclopiazonic acid (CPA), gliotoxin, citreovidrin or sterigmatocystin. 11

2.2 Major Groups of Mycotoxins occurring in Food and Feed

Aflatoxins (Aspergillus flavus toxins) are a group of approximately 20 related fungal metabolites, although only the types B1, B2, G1 and G2 (B=blue and G=green, according to their fluorescence colour under UV-light, while the subscript number designates relative chromatogaphic mobility) are normally found in foods. They are widely associated with commodities produced in the tropics and sub-tropics, such as groundnuts, figs, spices and maize. Aflatoxin B1 (AfB1), as the most toxic aflatoxin, is recognized as potent carcinogen and related to cause liver cancer as most animal species exposed to aflatoxins show signs of liver disease ranging from acute to chronic. 9 Also immunosuppression is an important consideration in aflatoxin-exposed animals. In humans aflatoxins also are under suspicion to cause hepatitis B, according to an often co-occurrence of hepatitis B in the high-risk areas for aflatoxin contaminations. In 2004 in Kenya 125 people died and nearly

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200 others were treated after eating aflatoxin contaminated maize. The deaths were mainly associated with homegrown maize that had not been treated with fungicides or properly dried before storage. Due to food shortages at the time, farmers may have been harvesting maize earlier than normal to prevent thefts from their fields, so that the grain had not fully matured and was more susceptible to infection.

Ochratoxin A (OTA) is produced by some species of Aspergillus, such as Aspergillus ochraceus, mainly in tropical regions and by Penicillium verrucosum, a common storage fungus in temperate areas such as Canada, eastern and north western Europe and parts of South America. Aspergillus ochraceus as ochratoxin producing contaminant is found on a wide range of commodities including cereals and their products, fruit and a wide range of beverages and spices. It causes kidney damage in humans and is a potential carcinogen. 9 The nephrotoxic effects of OTA in swine are a major disease in certain countries such as Denmark. Presently, OTA is also the most probable mycotoxin, involved in an endemic nephropathy in the Balkan countries.

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Figure 2-5: Ochratoxin A, chemical structure

empirical formula: C 20 H 18 Cl NO 6 molecular weight: 403.8 g/mol

The trichothecenes are a family of nearly 150 structurally related compounds produced by several fungual genera as Fusarium species and others and are acutely toxic to humans, causing sickness and diarrhoea and potentially death. Fusarium toxins are a range of mycotoxins including the fumonisins and the trichothecenes, including deoxynivalenol, T-2 toxin and HT-2 toxin. T-2 toxin is presumably associated with a disease observed in Russia during war-time known as alimental toxic aleukia. 10

Deoxynivalenol (DON) also belongs to the group of the trichothecenes and is sometimes termed as vomitoxin. Because DON is toxic and often found in foodstuffs, sometimes in high concentrations, it has recently been of concern to international organizations and government food agencies. 10 The effects on animals exposed to DON range from feed refusal and vomiting to immunosuppression and loss of productivity. Swine are considerably more sensitive to DON than poultry are, and cattle are quite intensive. Fehler! Textmarke nicht definiert.

10

Figure 2-8: Deoxynivalenol, chemical structure

molecular weight: 296.3 g/mol

The fumonisins are a group of at least 15 related toxins produced by Fusarium verticillioides that occur in maize frequently. The largest widespread is fumonisin B1 (FB1) which seems to be the most likely cause of human oesophageal cancer, but this has not been conclusively demonstrated. 11

Figure 2-9: Fumonisin B1, chemical structure

empirical formula: C 34 H 59 NO 15 molecular weight: 721.8 g/mol

The most common Fusarium species besides the trichothecenes in agricultural commodities is zearalenone (ZON). 10 ZON is detected in wheat, barley, rice, maize, and other cereals and tend to develop particularly during cool, wet growing and harvest seasons. ZON is an endocrine disruptor, interferring with estrogen receptors. It is known to cause hormonal effects in animals and may be an important etiological agent of intoxication in young children or fetuses, which results in premature thelarche, pubarche and breast enlargement.

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Figure 2-10: Zearalenone, chemical structure

molecular weight: 318.4 g/mol

Summarized, the diverse chemical properties of the mycotoxins result in various toxic

effects to humans and animals and therefore mycotoxins are also classified into different

groups corresponding to their toxicity [Table 2-1].

2.3 Food Safety Aspects and Regulations

Avoiding mycotoxin occurrence in the food chain in harmful levels is a highly important

issue, which is still getting bigger as nowadays consumers are removed more and more

from the source of the foods bought, either by time or distance. Each year about 10 million

tonnes of foodstuffs enter into the European Union through the port of Rotterdam alone. 12

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Therefore extensive studies of food-based mycotoxins have been accomplished worldwide throughout the 20th century. In Europe, statutory levels of a range of mycotoxins permitted in food and animal feed are set by a range of European directives and Commission regulations. As a result, the exposure of humans and animals to mycotoxins was mainly limited through chemical monitoring/screening programmes of the suspected commodities. 13 Due to the potential risk of mycotoxins to humans and animals, in at least 77 countries worldwide, legal limits in food and feed have been established. In the European Commission several regulation papers have been established in recent years and maximum levels for mycotoxins in several foodstuffs have been set down in the Council Regulations (EEC) no. 1881/2006 and no. 401/2006. According to these regulations, the legal limits for OTA range from 0,5μg/kg for dietary food to 10μg/kg for instant coffee. Aflatoxins are limited from 0,1μg/kg AfB1 and 0,5μg/kg total aflatoxins in dietary food up to 2μg/kg for AfB1 and 10μg/kg for total aflatoxins in spices and peanuts. The levels for ZON and DON are higher and vary between 20μg/kg ZON in dietary food and 100μg/kg in untreated grains, DON is restricted to 200μg/kg in nursling food and a highest of 1750μg/kg in untreated durum and oat. Legal limits for the trichothecenes T-2 and HT-2 toxin are not set by the EC yet. The legal limits for FB1 are set down by the commission but are first in force at the latest from October 1 st , 2007.

Furthermore, there are also national laws and regulations in the member states covering either foodstuff not regulated by European law yet or concerning other mycotoxins.

To ensure the abidance of the legal limits, requirements for analytical methods determining mycotoxins have also been established at a national as well as at an international level. Hence, relevant requirements of the methods have been prescribed by international organizations such as the "Association of Official Analytical Communities International (AOAC International)" or the "European Committee for Standardization (CEN)". An analytical method should be well characterised and validated in-house, but it should also be validated through an interlaboratory trial according to ISO 5725, 1994 (Accuracy - trueness and precision - of measurement methods and results) or following the Harmonised IUPAC protocol. 14

2.4 Mycotoxin-Analysis

Both in- and outside the activities of BioCop, all existing analytical methods for the determination of mycotoxins contain clean-up procedures such as solid phase extraction (SPE) or immunoaffinity chromatography, which are essential for the purification of

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various types of analytes. SPE has rapidly developed in the last years with sorbents such as n-alkylsilicas or highly cross-linked copolymers whereby retention is based on hydrophobic interactions. These interactions often only allow poor selectivity for trace analysis in complex matrices. Co-extraction of analytes and matrix interferences generally occur, and this can become a major problem when analytes are at trace levels and interferences at higher concentrations. 15 Summarized the disadvantages, additional clean-up procedures are necessary, but then the sample pre-treatment involves several steps and consequently the risk of loss or contamination increases. Therefore the need for one step sample treatment, which can be coupled directly to the separation technique, was given.

Today, especially in the field of food contaminant analysis clean-up procedures based on immunoaffinity are widely used for the determination of a variety of relevant mycotoxins. The final determination of the mycotoxins is possible, depending on the particular toxin by HPLC-fluorescence (FL) detection, HPLC-UV detection, (HP)-Thin Layer Chromatography (TLC), HPLC-mass spectrometry (LC-MS) or GC-electron capture detection (GC-ECD).

2.5 Immunoaffinity Clean-up

2.5.1 Immunosorbents and Antibodies

Antibodies (also called as immunoglobulins) are proteins, which are produced by mammalians as a reaction to the penetration of certain contaminants, so called antigens. Each antibody consists of four polypeptides – two heavy chains and two light chains are joined together to form a "Y" shaped molecule. The amino acid sequence in the tips of the "Y" varies greatly among different antibodies. This variable region, composed of 110-130 amino acids, give the antibody its specificity to tie the antigen. The variable region includes the ends of the light and heavy chains. The design of the antibody is the key parameter that defines the potential of the immunosorbent for future complete bonding of antigens.

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Figure 2-11: Scheme of an antibody

from http://www.biology.arizona.edu/IMMUNOLOGY/tutorials/antibody/graphics/antibody.gif

By definition, an immunosorbent (IS) is an insoluble support for antigens or antibodies used to absorb homologous antibodies or antigens, respectively, from a mixture; the antibodies or antigens so removed may then be eluted in pure form. 16 In the medical and biological fields, immunosorbents have long been established using techniques such as ELISA (enzyme linked immunosorbent assay) or EIA (enzyme immuno assay). Compared to that, their application for environmental analysis is relatively recent, because of difficulties in synthesizing selective antibodies for small molecules, such as mycotoxins. Today numerous immunosorbents for the field of environmental analysis have been developed. 15 The most important step in making an immunosorbent is to develop antibodies with the ability to recognize either one or a group of analytes. Low molecular compounds are unable to cause an immune response from the sorbent, so they usually first have to be modified by binding to a larger carrier molecule (e.g. proteins like bovine serum albumin). To make the coupling between the small molecule and the protein possible, it is often necessary to derivate the selected small molecule while introducing a functional group. This coupling intermediate is called hapten.

Antibodies are attained while firstly producing antigens or protein-complexes including the attended haptens. These are squirted to animals such as mice or rats whose immune

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systems are producing antibodies against them (immunization). After immunization, either polyclonal (PAbs) or monoclonal (MAbs) antibodies can be obtained by different procedures. When antiserum is directly taken from the animals, polyclonal antibodies are obtained [Figure 2-12(a)]. This serum contains a mixture of different antibodies produced by different cells. Monoclonal antibodies are produced by removing mammalian splendic antibody-producing cells. Each one of these cells is fused with an immortal line of myeloma (tumor) cells in culture [Figure 2-12(b)]. The resulting hybrid cells are screened in order to select one cell that will produce a desired antibody indefinitely. The resulting anti-

Figure 2-12: Production of (a) polyclonal and (b) monoclonal antibodies 15

bodies are referred as monoclonal when homogenous. Both, PAbs and MAbs have been selected for immobilization with an increase in the use of MAbs in recent years, although the production is more costly. 15/17 The degree of purification of the antibody solution is very important. Mycotoxin specific antibodies are purified by precipitation with ammonium sulfate, by ion exchange chromatography, by gel filtration, or by affinity chromatography. 2

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2.5.2 Supports for Immunosorbents

For the use in immunoaffinity clean-up procedures the antibodies are linked (immobilized) onto a solid matrix, like agarose-gel (sepharose) or synthetic polymers, which is called support [Figure 2-13 and 2-14]. The solid immunosorbent is than brought into a plastic cartridge and welled within a buffer-solution, usually aqueous PBS (phosphate-buffered saline), pH 7.4.

The support selected for the immobilization of the antibodies is the second critical parameter besides the antibody itself in designing an immunosorbent. A good solid support is distinguished through various parameters:

(I.) chemical and biochemical inertness (II.) mechanical stability (III.) uniformity in particle size (IV.) large pore sizes (V.) refusal of non-specific interactions (VI.) easy to activate to allow antibody attachment To enable the immobilization of the antibodies, the supports first have to be activated by an activating agent like carbonyldiimidazol, glutaraldehyde or cyanogen bromide. Most important in this whole activating process is that the bio-specific activity of the antibody has to be retained. 18 The coupling between the antibodies to the activated supports is considered as random immobilization, because it involves the association via lysine ε-amino groups which are encountered throughout antibody molecules, allowing thus several orientations of the antibody. The amino groups are usually reacting with epoxide or aldehyde groups provided by the support. Orientated antibodies can be achieved by using hydrazide activated supports involving covalent bonds via carbohydrate components and were primarily designed to provide greater column binding capacity. 17 Another way to achieve a better orientation and thus to increase the binding surface area is to employ antibody fragments. These fragments will increase the number of integral binding sites without causing steric hindrance. 19 Nevertheless, immunosorbents based on sepharose are still the most widespread supports used for IACs due to a greater flow-rate and thus faster clean-up time. Sepharose based IACs can in most cases only be used for offline clean-up due to weak pressure stability. However, development of various types of supports containing different materials or non-covalent binding mechanisms are continuously done by for example B. Spitzer et al. 17

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(a) (b)

Figure 2-13: Agarose: (a) molecular structure and (b) microscopic structure: Agarose consist mainly of the two carbohydrates - D-galactose and 3→6 L-anhydro-galactose

Figure 2-14: Scheme of immobilized antibodies on an agarose surface

2.5.3 Bonding Density

The number of antibodies linked to the surface of the sorbent is known as bonding density (IgG) and usually expressed in mg/g of sorbent. Thus, the bonding density is an important parameter, because it determines the future antigen-binding capacity and can be measured experimentally. It depends on the specific surface area of the support, which is receptive for the immobilization of the antibodies. Small pore sizes lead to high surface area, but they have low accessibility for the large antibody molecules. On the other hand supports with large pore sizes have a small surface area but a good accessibility for the antibodies. For immunoaffinity sample preparation usually a compromise is necessary to reach sufficient numbers of bound antibodies.

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2.5.4 Selective Extractions and Cross-Reactivity

Methods based on the interactions between antigen and antibody (molecular recognition) allow, compared to "classical" SPE, selective extractions. These selective extractions are the primary objective of using antibodies. The reversible association between antibodies and their corresponding antigens is called as immunological reaction. The binding forces involve hydrogen bonds and hydrophobic interactions as well as weak molecular bonds like Coulomb- and Van der Waals- forces. Therefore, an antibody can also bind one or more analytes with a structure similar to the desired analyte. This effect is the so-called cross-reactivity of antibodies. Cross-reactivity is not generally considered as a negative feature, in the biological field, it is often of interest to determine a group of related drugs and their metabolites. Even in the field of mycotoxin analysis, cross-reactivity allows the simultaneous determination of e.g. T-2 as well as HT-2 by only one MAb as a result of their structural similarity.

As a result of that, group-specific immunosorbents have been developed and established as for example for the determination of natural food contaminants like the mycotoxins, veterinary drugs or drugs of abuse such as LSD and its metabolites. Also enantioselective immunoextraction has been developed, which employ an immobilized antibody to specifically isolate fragments with the objective of structural analysis of drug-protein adducts. 20

Due to this, the only way to achieve reproducible ISs is to use MAbs. Generally, a larger cross-reactivity exists, while using PAbs, because of their inhomogeneity. But when the target molecule is small, the polyclonal mixture cannot contain large number of different specific antibodies for the different parts of the small molecules and the probability is high, that PAbs and MAbs have similar properties. 17

2.5.5 Capacity

The total number of antibodies associating to the immunosorbent is affected by their capacity. So the capacity is defined as the maximum amount of analytes, which can be bound on the IAC. If this amount is exceeded a so-called breakthrough of analytes can occur, due to the overloading of the capacity. The capacity of an immunosorbent corresponds to the total number of accessible specific immobilized antibodies. The capacity is conditioned by random orientation and steric hindrance and can therefore not

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