Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water

Abstract

Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin-leucine-arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL(-1)) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 μg L(-1), respectively, and a recovery of 62-86 % with a coefficient of variation below 12.6 % in water samples.

A Mini-Review on Detection Methods of Microcystins

“…To elevate the sensitiveness of ELISA, a modified ELISA described as indirect competitive ELISA (IC-ELISA), which has high antibody specificity for MC arginine in position 4, was generated [ 56 , 57 ]. In subsequent studies, IC-ELISA based on anti-Adda monoclonal antibody (mAb2G5), bifunctional single chain variable fragment-alkaline phosphatase fusion protein (scFv−AP), anti-MC-LR scFv7-scFv (MscFv7-scFv), as well as anti-MC-LR polyclonal antibodies and scFv (PAbs and scFv) were constructed for high MC-LR specificity and sensitivity [ 79 , 80 , 95 , 98 ]. It is of interest that the novel fluorometry noncompetitive ELISA based on synthetic broad-specific anti-immunocomplex antibody SA51D1, Fluorescent ELISA (FELISA) based on silane-doped carbon dots and Norwegian ELISA successfully detected varying variants of MC below the WHO’s guideline value of 1 µg/L [ 87 , 97 , 108 ].…”

Development of Time-Resolved Fluoroimmunoassay for Detection of Cylindrospermopsin Using Its Novel Monoclonal Antibodies

“…Like other cyanotoxins, CYN is a low-molecular-weight nonimmunogenic toxin. To become immunogenic, these toxins need be conjugated to carrier proteins by means of different chemical approaches [ 28 , 37 ]. The Mannich reaction, a conjugation between one active hydrogen and primary amines in the presence of formaldehyde, has been used to link another alkaloid toxin-saxitoxin (STX) to BSA, while polyclonal antibody R895 was generated using the conjugate as an immunogen [ 38 ].…”

“…Suitable antibodies are essential to the establishment of immunoassays. In this regard, MCs have received a great deal of attention, and several types of antibodies including conventional polyclonal or monoclonal antibodies and novel recombinant antibodies have been developed [ 37 , 39 , 40 , 41 ]. Moreover, antibodies have been raised specifically against some MC variants [ 40 , 42 , 43 ].…”

“…However, the CYN ELISA kit from Abraxis LLC appeared to provide good quantification for the assessment of CYN and its variants [ 27 ]. The difference in performance may be attributed to antibody specificity, and some antibodies with broad specificity are capable of recognizing several cyanotoxins with similar structures [ 37 ].…”

Biotechnology for obtaining hybrid positive control samples for immunoassay for detecting antibodies against Chlamydia trachomatis

“…That is why testing for the presence of specific IgG and IgM antibodies against TORCH-infectious agents is an important diagnostic approach in the mother and child protection system. Based on literature data and our own experience (Galkin et al, 2013;Dovgan et al, 2016;Yang et al, 2016;Kalayu Yirga et al, 2017), we selected three principal methodological approaches for the synthesis of antibody-antibody conjugates that was evaluated for the synthesis of the appropriate PCs (non-specific IgM/IgA + anti-HRP McAbs): 1) NHS ether-maleimide mediated conjugation on the example of succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) as a cross linker; 2) glutaraldehyde method; 3) periodate conjugation method. The use of the above methodological techniques is possible through the following prerequisites.…”