**4. Proper receptor for LFIA**

The basic requirements for antibodies used in LFIA are related to their affinity and selectivity. However, the topic of which characteristics of antibodies provide the most sensitive analysis requires additional clarification. Immune reactions during immunochromatography are carried out in the kinetic regime. Therefore, it is unimportant whether the detectable complexes will dissociate for hours or days. Their number is determined primarily by the kinetic constants of the association, which for receptors that are the same in structure and antigens that are similar in size vary within a limited range. In the case of competitive LFIA, the dependence of the number of complexes formed on the analyte concentration in the sample is determined primarily by the affinity of antibodies to the free analyte. Effective binding to a competitor modified by the analyte will interfere with the highly sensitive detection of the free analyte in the sample. In other words, the binding of antibodies to the analyte-protein conjugate should be somewhat worse than with the native analyte. The influence of the characteristics of immunoreagents on the sensitivity of analysis is considered in detail in works devoted to the mathematical modeling of LFIA [25–30].-

Given the above limitations, the affinity of antibodies is an important characteristic that- affects their analytical use. However, the possibility of natural production of antibodies- with more and higher binding to the analyte is limited. This is because an increase in the- half-life of an antigen complex with B-cellular receptors greater than the endocytosis time- of the complex is not supported by the selection of the corresponding B-cellular receptor- lines [31]. The cases of "infinite affinity" of antibodies are rare exceptions for the analytes- that form covalent bonds after the immune interaction [32]. An additional way to increase- affinity is the genetic modification (directed design) of the active center of antibodies. The- use of these methods in routine development is still very limited, despite confirmations of- their effectiveness [33].-

As far as specificity is concerned, an important problem is which series of structurally close compounds should be detected using this antibody to solve practical problems. Two kinds of situations are possible: (i) it is necessary to recognize a single compound possessing biological activity, in contrast to its analogs and metabolites and (ii) information is required on the total content in the sample of a significant number of homologous compounds. For the second, class-specific assay, it is desirable that affinity of antibodies to homologous compounds correlate with their biological activities, but this is not always possible. In some cases, regulatory documents establish maximum residue levels (MRLs) on the basis of the sum of concentrations of structurally similar toxicants, without correction factors, taking into account their biological activity. Therefore, class-specific analysis usually requires the detection of the maximum number of compounds of this class with at least 10–15% cross-reactivity with respect to the maximum [8].-

Additional practically important characteristics of antibodies are the values of their stability under storage and in the course of the assay. The stability may be effectively enhanced by chemical modification of antibodies as well as by addition of protective agents that are common for drying of different immunoreactants.-

The strategic tasks for improving receptors for immunoassays are summarized in **Figure 5**. However, in the final development of test systems, commercially available antibodies are usually used, and there is no possibility of directed production of new, improved antibodies. This is the reason for the interest in the use of receptor compounds of a different nature as a substitute for traditional immunoglobulins.-

**Figure 5.** Main research and development tasks to obtain proper receptors for LFIA.-

Thus, the single-domain antibodies produced by *Tylopoda* and sharks are characterized by significantly greater stability under different conditions [34]. Interest is caused by so-called protein scaffoldsof a non-antibody nature that also combine conservative basic structure with hypervariable segments providing receptor functions [35]. However, their analytic application is a matter of the future. The development of test systems based on aptamers—receptor oligonucleotides, selected from random libraries—is being actively pursued. Aptamers are significantly cheaper and more stable reagents in comparison with antibodies, and their properties are well reproducible. Limitations in affinity typical of many of the known aptamers are overcome by improving the selection procedures and subsequent directed design which is a much simpler process than for antibodies [35]. The possibilities of using aptamers in membrane test systems are shown in a number of works and summarized in recent reviews by Jauset-Rubio etal. [36], Chen etal. [37], and Dhiman etal. [38]. For developments on the use of nanobodies, see Tang etal. [39], who presented LFIA for the simultaneous determination of aflatoxin B1 and zearalenone.-

Requirements for proper receptors also include its effectiveness after immobilization on a membrane or on the surface of a marker nanoparticle label. Physical adsorption and random covalent coupling may be accompanied by significant loss of antibody reactivity. Therefore, systems are needed in which the oriented immobilization of antibodies is realized through the chemical conjugation of IgG in areas remote from the active center, or by their indirect binding with a preformed antibody-binding layer. In the role of this layer, staphylococcal protein A, streptococcal protein G, or (strept)avidin (reactive with biotinylated antibodies) can act. Approaches to the oriented immobilization of antibodies are systematized in a number of recent reviews [40–43].-

Filbrun etal. proposed a procedure for chemical modification of the lysine residues of antibodies before conjugation with gold nanoparticles and showed that it provided conjugates

**Figure 6.** Limitations in the use of common antibody-nanoparticle conjugates in competitive LFIA.-

 that were stable over a wide pH range [44]. Bauer etal. [45] developed a technique for the preparation and use of antibody conjugates modified with histidine-rich peptides (called "capture and release" antibody reagents). These preparations are affine to metal surfaces and magnetic particles and so may release immobilized antibodies when necessary. The benefits of oriented binding of antibodies to magnetic nanoparticles through modification of antibodies' carbohydrate components were shown by Puertas etal. using the example of LFIA for choriogonadotropin [46]. A comparison of methods of immobilization for receptors in bacteriophage-based LFIA is given in the works of Kim etal. [47, 48]. In particular, article [48] discussed the use of invivo-biotinylated peptide for oriented immobilization of receptor molecules on a test strip.-

The composition of conjugates of antibodies with nanoparticles also plays an important role. Although the increase in valencies for immune interaction is accompanied by an increase in conjugates' affinity [49], structural changes of antibodies or steric restriction of their availability to interact with antigens may occur in parallel. With adsorption immobilization of antibodies (i.e. the widespread approach for LFIA purposes), their excessive loading causes the formation of additional layers, the molecules in which can dissociate during the analysis, and preventing the formation of a detectable labeled complex. Additional complications are associated with the use of antibody-nanoparticle conjugates having high surface density in competitive LFIA (**Figure 6**). Such conjugates can form high-affine polyvalent complexes in the analytical zone, which impede competitive interaction with the monovalent analyte from the sample. Further, the resulting complexes contain a significant number of unreacted antibodies and can bind analyte molecules without weakening the detected signal [50]. Therefore, the composition of the conjugate should be selected in relation to the features of each analytical system as well as other variable parameters—see the list of tactical tasks in **Figure 5**. Describing the development of LFIA for aflatoxin M1 [51], Anfossi etal. found that the lowering the ratio between antibodies and gold nanoparticles caused improvement in the assay sensitivity. The proposed change was to decrease amount of antibodies used for immobilization twice as compared with saturating conditions and by that way to lower the limit of detection, too, almost twice with a minimal weakening of the staining.-
