**3. Immunoassays for aflatoxins detection**

Among aflatoxin detection methods, there are those that base their operation on antibodyantigen reactions (Ab-Ag), known as immunoassays. Different kind of Aflatoxin molecules (AF) can be considered as antigens from the immunological point of view, so that it is possi‐ ble to develop antibodies against them.

Most of immunological the methods are based on enzyme-linked immunosorbent assay (ELISA), which require less expensive instruments, have good sensitivity, speed and sim‐ plicity. However, ELISA kits are expensive especially for third-world countries [16], so sev‐ eral studies have focused on developing less expensive methods, without losing the benefits they offer. Besides, other alternatives will have some advantages over ELISA, as the use of magnetic droplets together with RT-PCR (Reverse Transcription Polymerase Chain Reac‐ tion), which has sensitivity to 1000 times greater than ELISA [17].

#### **3.1. Antibody-Antigen reactions**

Immunoassays are based on antibody-antigen reactions, in which one of reactants is marked and the other is immobilized on a platform. There are several kinds of molecule mark‐ ers, which may be radioisotope, enzyme, fluorescent compound and colloidal Au. Be‐ cause of small size of AFs, they are bounded to proteins so they can be captured with antibodies (Abs).

There are three type of antibodies used in immunoassays: polyclonal, monoclonal and re‐ combinant. Polyclonal Abs (pAbs) is produced by rabbit, horse or goat blood immunized with protein-AF conjugate. This type of Abs is low-cost for preparation and easily produced. Monoclonal Abs (mAbs) is produced from positive hybridomas, which are usually pro‐ duced by fusing murine myeloma cells and spleen cells from immunized mice. In [16], the authors used an indirect-competitive ELISA to detect AFB1 by using a platform coated with monoclonal antibody. Recombinant Abs (rAbs) is produced by cloning the functional gene of some Abs and transmitted it into a prokaryotic or eukaryotic genetically-modified organ‐ ism to hybridoma or spleen cells with or without immunization.

#### **3.2. Competitive and non-competitive assay**

There are two types of immunoassays: competitive and non-competitive assays. Competi‐ tive assays in turn are divided into two types: indirect and direct assays. In an indirect com‐ petitive assay, aflatoxins are immobilized by a protein-aflatoxin conjugate (Ag). The set is exposed to a buffer with the tested sample. Antibodies are released into the buffer; some of them will bind to the immobilized conjugate, while the remainder will join the analytes in the buffer. After a while, it is released a second group of enzyme-labeled antibodies, or other fluorescent-core kind of signal material; these are joined to the first antibodies, which in turn are attached to the protein-conjugate immobilized aflatoxins.

The process of a direct competitive assay does not require a second labeled antibody. For this type of analysis, either aflatoxins (aflatoxin protein conjugate) or specific antibodies can be immobilized. The complementary component to that immobilized is marked and added to the sample. A competitive reaction occurs between the Ab-Ag, so that some marked components remain adhered to the immobilized one, while others adhere to those present in the sample.

For non-competitive assays, Abs are immobilized. When immobilized Abs make contact with the sample molecules and AF bind to them because Abs are attached to a sensitive sur‐ face, the amount of analyte bounded by Abs will result in an electrical or optical variation. However, sandwich format is preferred for this kind of assay, in which the sample is mixed previously with Bovine Serum Albumine (BSA) because AF molecules are small. Previously it was carried out a treating of the sample with Bovine Serum Albumine (BSA) (carrier pro‐ tein). BSA binds to the AFs and the conjugate is captured by immobilized Abs.

#### **3.3. Enzyme-Linked Immunoabsorbent Assays (ELISAs)**

ty in the selection of aflatoxin in samples contaminated with more compounds. So that, it is clear that the tendency of the technology is principally to improve the stage of pre-processes and derivatizations in order to achieve a more precise quantification. The more sophisticat‐ ed the technology of pre-process and derivatization the more high specificity and sensitivity

Among aflatoxin detection methods, there are those that base their operation on antibodyantigen reactions (Ab-Ag), known as immunoassays. Different kind of Aflatoxin molecules (AF) can be considered as antigens from the immunological point of view, so that it is possi‐

Most of immunological the methods are based on enzyme-linked immunosorbent assay (ELISA), which require less expensive instruments, have good sensitivity, speed and sim‐ plicity. However, ELISA kits are expensive especially for third-world countries [16], so sev‐ eral studies have focused on developing less expensive methods, without losing the benefits they offer. Besides, other alternatives will have some advantages over ELISA, as the use of magnetic droplets together with RT-PCR (Reverse Transcription Polymerase Chain Reac‐

Immunoassays are based on antibody-antigen reactions, in which one of reactants is marked and the other is immobilized on a platform. There are several kinds of molecule mark‐ ers, which may be radioisotope, enzyme, fluorescent compound and colloidal Au. Be‐ cause of small size of AFs, they are bounded to proteins so they can be captured with

There are three type of antibodies used in immunoassays: polyclonal, monoclonal and re‐ combinant. Polyclonal Abs (pAbs) is produced by rabbit, horse or goat blood immunized with protein-AF conjugate. This type of Abs is low-cost for preparation and easily produced. Monoclonal Abs (mAbs) is produced from positive hybridomas, which are usually pro‐ duced by fusing murine myeloma cells and spleen cells from immunized mice. In [16], the authors used an indirect-competitive ELISA to detect AFB1 by using a platform coated with monoclonal antibody. Recombinant Abs (rAbs) is produced by cloning the functional gene of some Abs and transmitted it into a prokaryotic or eukaryotic genetically-modified organ‐

There are two types of immunoassays: competitive and non-competitive assays. Competi‐ tive assays in turn are divided into two types: indirect and direct assays. In an indirect com‐ petitive assay, aflatoxins are immobilized by a protein-aflatoxin conjugate (Ag). The set is

the method gets.

**3. Immunoassays for aflatoxins detection**

tion), which has sensitivity to 1000 times greater than ELISA [17].

ism to hybridoma or spleen cells with or without immunization.

**3.2. Competitive and non-competitive assay**

ble to develop antibodies against them.

294 Aflatoxins - Recent Advances and Future Prospects

**3.1. Antibody-Antigen reactions**

antibodies (Abs).

Any type of assay involving Ab-Ag reaction, where one of the reactants is conjugated with an enzyme, is considered as an ELISA. Amplification and visualization of Ab-Ag interaction are achieved by this enzyme conjugation. ELISA is the most used immunoassays used in food-aflatoxin detection.

Antibodies or antigens are immobilized on a solid-phase matrix by linking them, either through adsorption or covalently. Reactants are usually adsorbed on to the wells of 96- or 384- microtitre plate of polystyrene, where adsorption is characterized by a strong hydro‐ phobic binding and slow dissociation rate. After this coating process, the residual proteinbinding capacity of solid matrix is blocked by exposing it to an excess of unrelated protein (e.g. gelatin or bovine serum albumin "BSA"). The next step is the addition of a test solution, which may be serum with an unknown concentration of antibodies against the immobilized antigen. After incubation and washing, binding of specific antibodies is visualized by the addition of antiimmunoglobulin-enzyme conjugate followed by a substrate, generating a colored product when hydrolysed. This change of color is proportional to the amount of an‐ tibodies bounded and may be recorded visually or spectrophotometrically. In case of an an‐ tigen measurement, the process is the same but may be done by using competitive- or sandwich-type assays. When using microarray format, ELISA may detect other toxins, such as AFs in a sample [18].

#### **3.4. Recent advances**

ELISA has been modified by using electrochemical techniques. Antibodies or antigens may be immobilized on an electrode with a free and enzyme-conjugate. So in a competitive as‐ say, some enzyme-conjugate will bind to the electrode, and enzyme density can be shown by current produced from the catalytic oxidation reaction of the enzyme with the substrates. In a non-competitive assay, the formation of an Ab-Ag complex generates a barrier of direct electrical communication between the immobilized enzyme and the electrode surface.

Every topic exposed in this chapter is so vast that could be by itself a single chapter. The aims of this chapter are to give a general overview of all the existing methods for the meas‐ urement and quantification of aflatoxins; to signalize their principles of operation; and to ex‐

Characteristics of Mycotoxin Analysis Tools for Tomorrow

http://dx.doi.org/10.5772/51683

297

Biosensors are multidisciplinary tools with an enormous potential in detection and quantifi‐ cation of aflatoxins. Thus, such devices have a huge impact in healthcare, food management,

The International Union of Pure and Applied Chemistry (IUPAC) define biosensor as:

"A device that uses specific biochemical reactions mediated by isolated enzymes, immuno‐ systems, tissues, organelles or whole cells to detect chemical compounds usually by electri‐

Dr. Leland C. Clark established the concept of a biosensor as a biological sensing element whose change its properties when reacting biochemically with a specific compound or ana‐ lyte [27]. Such reaction is converted into an electronic signal for its quantification. Dr. Clark

**a.** The enzyme metabolizes the analyte, thus the analyte can be determined through the

**b.** The analyte inhibits the enzyme, thus the decrease of the enzymatic product formation is correlated with the analyte concentration. This case is called "biosensor based on en‐

Biosensors are tools basically conformed of a substrate (silicon, glass or polymers). Common polymers are: polymethyl methacrylate, polydimethyl siloxane, etc. The substrate is often coated with a conductive layer like: polysilicon, silicon dioxide, silicon nitrite, gold, and metal oxides. The specific recognition elements include: antigens, antibodies, nucleic acids, whole cells, proteins, enzymes, DNA/RNA probes, and phage-derived biomolecular recog‐ nition probes. The changes in these elements are detected via optical, electrochemical, calori‐ metric, acoustic, piezoelectric (quartz crystal, potassium sodium tartrate, lithium niobate),

Optical sensors are analytical tools that satisfy requirements as accuracy, precision and spe‐ cificity in the selection of the analyte, allowing *in vivo* or *in vitro* investigations. Optical tech‐ niques provide a large realm of possibilities based on properties such as absorbance,

reflectance and luminescence of single elements or groups of analytes [29].

developed a glucose oxidase enzyme electrode for detecting glucose.

There are two different approaches which can be carried out by biosensors.

pose their tendencies.

**4.1. Biosensors**

agronomical economy and bio-defense [26].

measuring of the enzymatic product.

magnetic, and micromechanical transducers [28].

**4.3. Biosensor based on optical techniques**

cal, thermal or optical signals".

**4.2. Application of biosensors**

zyme inhibition".

Some authors have reported the use of electrochemical sensors. In [19] developed a sensor based on enzymatic silver deposition amplification to detect AFB1 in rice. A linear sweep voltage was done in order to read the sensor response. In [20] the authors proposed the use of electrical impedance spectroscopy and free-labeled molecules.

Optical ELISAs often uses surface plasmon resonance (SPR). They are similar to electro‐ chemical sensors, but in SPR, Ab or Ag is immobilized in an optical-sensitive surface. As AFB1 changes, the angle of Spectral Power Distribution (SPD) varies. A combination of com‐ petitive-direct ELISA and an immunochromatographic assay was done by [21], in order to increase its sensitivity.

In recent years, some articles have developed modifications on ELISAs (e.g. with the using of nano particles). In [22] the authors refined the ELISA process for aflatoxin detection by using anti-AFB1 single chain fragment variables, in order to detect only free AFB1 instead of an AFB1-protein conjugate. In the references [23] developed an ultra-sensitive ELISA by cou‐ pling a micro plate ELISA with sensitive magnetic particles. An important feature of this hy‐ brid system is its small column size, high capture efficiency and lower cost over other reported materials.

A combination of a competitive direct ELISA and gold nanoparticle immunochromatograph‐ ic strip was done by [21], with a detection limit of 1.0 ng/ml for AFM1 in milk. Immunochro‐ matographic assay (ICA) is rapid and simple, and can be carried out by untrained personnel without using electronic devices. However, this type of assay has low selectivity, so in [24], an improved ICA by using a new monoclonal antibody against AFB1 was developed.
