The Abs are glycoproteins called immunoglobulins (Ig), secreted by B-lymphocytes, components of the adaptive immune system, in response to an immunogen. There are many different isotypes or classes (IgG, IgA, IgM, IgE, and IgD), but the IgG isotype is often the major component of commercially available Abs and constitutes the most fractions of blood proteins. IgG is further divided into four subclasses (IgG1, IgG2, IgG3, and IgG4), with the numbers corresponding to the decreasing order in which they are found in the blood [30].

Abs production is simple, but there are several factors that affect the probability of an animal to produce Abs against the injected Ag (immunogen). The factors influencing immunogenicity are [31]:

• The molecular size of the injected Ags: the most active immunogens tend to have a high molecular mass (>14,000 Da). Indeed, small Ags (e.g., DOA) are known to be either non-antigenic or weakly antigenic.
• The foreignness: an antigen must be a foreign substance to the animal (not self) to elicit an immune response.
• The chemical complexity: the more complex the immunogen or substance is (chemically), the more immunogenic it will be. The DOA (BZD, heroin, amphetamine, morphine, etc.) are often of low molecular weight and, generally, for any very small Ag, the entire chemical structure is considered by the immune system as a single epitope to which an Ab binds.

Since they are unable to induce an immune response by themselves, they require a carrier molecule to act as a complete Ag. They are used as haptens or as a recognition site for the production of specific Abs by coupling them to a suitable carrier molecule (the immune response in the host animal can produce Abs against the entire immunogen and not just the drug molecule). Many proteins can be used as carriers, but the most commonly used ones are bovine serum albumin (BSA; 67 kDa) and keyhole limpet hemocyanin (KLH, 400 kDa), which are highly immunogenic because of their complexity (structure) and large sizes [32,33,34,35].

BSA is widely used as a blocking agent in development of immunoassays, such as ELISA and LFIA, because it is very accessible and available and has numerous useful groups to be linked to small molecules, including DOA, as a carrier molecule to induce the immune system. For this reason, it is recommended, for example, to use KLH as the carrier molecule (protein) to induce an immune response against the hapten and the BSA for Abs screening and purification, to assure the detection of the Ag (hapten) instead of the carrier Abs [32,33,35].

If morphine (MOP) is taken as a model for developing a DOA detection system based on LFIA, the carbon atoms in its positions 3, 6, 2, and group N, readily lend themselves to conjugation to the carrier protein (Figure 1).

The production of Abs directed against the MOP molecule using a carrier molecule in group N to produce a more specific assay for MOP detection is commonly used. This leaves positions 3 and 6 as antigenic determinants and, thus, allows the production of Abs more likely to be specific to MOP, without cross-reactivity to codeine (COD) or dihydrocodeine, for example. However, if the immunogen is produced via position 3, it is generally used to produce broad cross-reactive anti-opiate Abs (diacetylmorphine and MOP-3-glucuronide). Cross-reactivity to different opiates varies from one Ab to another. It is important that each Ab is fully characterized by the test developer. However, the production of MOP Abs in position 6 gives a better specificity to MOP relative to COD and MOP-3-glucuronide, and is expected to produce Abs with good cross-reactivity with 6-monoacetylmorphine and the active metabolite MOP-6-glucuronide [37].

When a mammal animal, such as a mouse, rabbit, sheep, goat, rat, or a horse (for large quantities of Ab) is immunized with an Ag immunogen, it will cause stimulation of all B-lymphocytes that produce Abs specific to that Ag. This stimulation will result in the clonal multiplication of these B-lymphocytes, which will turn into plasmacytes secreting the specific Ig. A clone produces the same Ig that has the same specificity for a given epitope. The Abs derived from a clone of plasmacytes are called monoclonal Abs (same Abs produced from a single clone).

The majority of the mAbs available in the market are IgG isotype because of their superior affinity and specificity compared to the other isotypes (IgM). However, in the natural situation, an Ag always produces a polyclonal serum [38]. Thus, the combination of Igs derived from different clones, but all recognizing different epitopes of the same Ag forms a polyclonal serum, called antiserum, specific for the given Ag.

The measure of binding strength between an Ag and an Ab is described by the affinity constant. This binding is non-covalent, reversible, and reaches equilibrium. In addition, high affinity Abs bind faster than low affinity ones and perform better in immunochemical methods [38].

In general, the commercial production of recombinant monoclonal antibodies (mAbs) follows principally similar workflow. The process begins with the generation of a mAb by immunizing an animal or by techniques using molecular biology methods involving the identification and optimization of the genetic coding sequence and the construction and identification of a stable high-producing clone.

Today, in the laboratory step, several techniques are well established and commonly used to obtain mAbs, namely: Epstein-Barr virus (EBV) lymphoblastoid transformation technique, hybridoma technique, and phage display technique (Scheme 1) [39].

Phage display technique is the most commonly applied technology to produce recombinant antibodies in the laboratory settings. This helps the isolation of proteins from diverse mutagenic libraries and investigates protein-protein, protein-peptide, and protein-DNA interactions, and consists, basically, in cloning Fab coding genes into bacteriophage plasmid vectors [39]. The advantages of this methodology are multiple: one library can generate a great number of new Abs, it is an in vitro process (so animal immunizations steps are not required), and, accordingly, even toxic antigens can be tested. Moreover, the Abs molecules can be rapidly obtained [39,40,41,42]. However, for LFIA development application, mAbs produced using this technique are still not widely used, and the mostly used ones are derived from mouse hybridoma (but exhibit a downside in human therapeutics).

In 1975, Georges Köhler and Cesar Milstein described the first technique developed for stable monoclonal antibody production. This technique consists of creating a hybridoma, a stable hybrid cell capable of producing a single type of antibody against a specific epitope present in an antigen (Scheme 2). It is also called the technique of hybridization cell and is a method for producing large numbers of mAbs. In LFIA development application (immunoassay diagnostic or screening tests in general), it is the mostly used technique to produce mAbs in all laboratories, but has a downside in human therapeutics. The hybridoma technique is currently performed following four main steps (Scheme 2):

• Step 1: fusing the secretory lymphocyte of an Ab to the Ag used in the animal’s immunization with the myeloma using polyethylene glycol.
• Step 2: identifying the Ab secretory hybridoma.
• Step 3: isolating one cell and maintaining it in culture to obtain a single clone or family of cells, all of which are identical and secretive of the same mAb. It is limit-dilution cloning, and several successive clones are sometimes necessary to obtain a genetically stable clone.
• Step 4: growing the cloned hybridoma in a bioreactor to obtain a mAb concentrate or in a roller system to obtain the less concentrated mAb as a culture supernatant. It can be injected into the abdomen of BALB/c mice (Bagg albino, laboratory-bred strain of the house mouse) to obtain ascites-concentrated mAb.

Overall, an immunization program usually involves injecting three to six animals with the same Ag. However, if appropriate Abs are not produced after multiple immunizations, it may be necessary to repeat the program with different animals and possibly a different immunogen [37].

This method was used by several authors for the development of mAbs against DOA. Indeed, Dehghannezhad et al. in 2012 [43] used it to produce a mAb and conjugated it to gold nanoparticles (GNPs) to develop a rapid competitive immunochromatographic strip test to detect MOP in urine samples. It was also used to develop Abs for the diagnosis and screening of different diseases and clinical cases, including arthritis, breast cancer, psoriasis, leukemia, transplant rejection, asthma, and toxicity [44,45,46,47,48,49,50,51,52].