Generation of Dual functional Nanobody-Nanoluciferase Fusion and its potential in Bioluminescence Enzyme Immunoassay for trace Glypican-3 in Serum.

Glypican-3 (GPC3) is a serological biomarker for the diagnosis of Hepatocellular carcinoma (HCC), but it is a challenging task to develop a bioassay for determination of the trace GPC3 in serum. In this study, Bioluminescense immunoassay based on bifunctional nanobody-nanoluciferase fusion was developed with the ultra-sensitive feature to achieve this goal. First, nanobodies special against GPC-3 binder as biological recognition element were generated by immunization and phage display technology. Second, The best clone GPN2 was fused with nanoluciferase as a dual-functional immunoreagent to establish an ultra-sensitive bioluminescence enzyme immunoassay (BLEIA), which is 30 and 5 times more sensitive than the traditional colorimetric assay and fluorescent assay, respectively. The cross-reactivity analysis of BLEIA showed that there was no cross-reactivity with HCC related tumor markers AFP, CEA, CA19-9 and GPC1/GPC2. The limit of detection (LOD) of developed BLEIA was 1.5 ng/mL, which assured its application in the diagnosis of GPC3 in 94 serum samples. This study indicates that BLEIA based on nanobody-nanoluciferase fusion could be used as a useful tool for the diagnosis of HCC patients.

An Ultrasensitive Bioluminescent Enzyme Immunoassay Based on Nanobody/Nanoluciferase Heptamer Fusion for the Detection of Tetrabromobisphenol A in Sediment.

Tetrabromobisphenol A (TBBPA) is a flame retardant and has become a widely concerning environmental pollutant. An ultrasensitive nanobody-based immunoassay was developed to monitor the exposure of TBBPA in sediment. First, the anti-TBBPA nanobody was fused with nanoluciferase, and then a one-step bioluminescent enzyme immunoassay (BLEIA) was developed with high sensitivity for TBBPA, with a maximum half inhibition concentration (IC50) at 187 pg/mL. Although approximately 10-fold higher sensitivity can be achieved by this developed BLEIA than by the classical two-step ELISA (IC50 at 1778 pg/mL), it is still a challenge to detect trace TBBPA in sediment samples reliably due to the relatively high matrix effect. To further improve the performance of this one-step BLEIA, a C4b-binding protein (C4BP) was inserted as a self-assembling linker between the nanobody and nanoluciferase. Therefore, a heptamer fusion containing seven binders and seven tracers was generated. This reagent improved the binding capacity and signal amplification. The one-step heptamer plus BLEIA based on this immune-reagent shows an additional 7-fold improvement of sensitivity, with the IC50 of 28.9 pg/mL and the limit of detection as low as 2.5 pg/mL. The proposed assay was further applied to determine the trace TBBPA in sediment, and the recovery was within 92-103%. Taking advantage of this heptamer fusion, one-step BLEIA can serve as a powerful tool for fast detection of trace TBBPA in the sediment samples.

Isolation of Bactrian Camel Single Domain Antibody for Parathion and Development of One-Step dc-FEIA Method Using VHH-Alkaline Phosphatase Fusion Protein.

A heavy chain variable fragment of heavy chain only antibodies derived from camelids termed VHH shows beneficial characteristics for immunoassay in terms of high sensitivity, outstanding stability and ease in expression. In the present study, we isolated six VHHs from phage display library against parathion, which is a widely used organophosphorus pesticide with high toxicity and persistence. One of six selected VHHs named VHH9, showed highest specificity and superior thermo-stability. A VHH9-alkaline phosphatase (AP) fusion was constructed and used to establish a one-step direct competitive fluorescence enzyme immunoassay (dc-FEIA) with a half maximal inhibitory concentration (IC50) of 1.6 ng/mL and a limit of detection of 0.2 ng/mL which was 4-fold or 3-fold higher sensitivity than direct competitive enzyme-linked immunoassay (dc-ELISA) and indirect competitive enzyme-linked immunoassay (ic-ELISA) for parathion. Furthermore, our assay indicated a 50% reduction on operation time compared with the ic-ELISA method. The presented immunoassay was validated with spiked Chinese cabbage, cucumber, and lettuce samples, and confirmed by UPLC-MS/MS. The results indicated that the VHH-AP-based dc-FEIA is a reproducible detection assay for parathion residues in vegetable samples.

VHH antibodies: emerging reagents for the analysis of environmental chemicals.

A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Discovered nearly 25 years ago, they have been investigated for their use in clinical therapeutics and immunodiagnostics, and more recently for environmental monitoring applications. A new and valuable immunoreagent for the analysis of small molecular weight environmental chemicals, VHH will overcome many pitfalls encountered with conventional reagents. In the work so far, VHH antibodies often perform comparably to conventional antibodies for small molecule analysis, are amenable to numerous genetic engineering techniques, and show ease of adaption to other immunodiagnostic platforms for use in environmental monitoring. Recent reviews cover the structure and production of VHH antibodies as well as their use in clinical settings. However, no report focuses on the use of these VHH antibodies to detect small environmental chemicals (MW < 1500 Da). This review article summarizes the efforts made to produce VHHs to various environmental targets, compares the VHH-based assays with conventional antibody assays, and discusses the advantages and limitations in developing these new antibody reagents particularly to small molecule targets. Graphical Abstract Overview of the production of VHHs to small environmental chemicals and highlights of the utility of these new emerging reagents.

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase.

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris.

The expression efficiency was improved for the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse and expressed in the methylotrophic yeast Pichia pastoris GS115, by redesigning and synthesizing the DNA sequence encoding for CBL-scFv based on the codon bias of P. pastoris. The codons encoding 124 amino acids were optimized, in which a total of 156 nucleotides were changed, and the G+C ratio was simultaneously decreased from 53 to 47.2 %. Under the optimized expression conditions, the yield of the recombinant CBL-scFv (41 kDa) antibodies was 0.223 g L⁻¹ in shake culture. Compared to the non-optimized control, the expression level of the optimized recombinant CBL-scFv based on preferred codons in P. pastoris demonstrated a 2.35-fold higher yield. Furthermore, the recombinant CBL-scFv was purified by Ni-NTA column chromatography, and the purity was 95 %. The purified CBL-scFv showed good CBL recognition by a competitive indirect enzyme-linked immunoassay. The average concentration required for 50 % inhibition of binding and the limit of detection for the assay were 5.82 and 0.77 ng mL⁻¹, respectively.

Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli.

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)(1). Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC(50) value of 1.46±0.01 ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.

Highly specific nanobody against herbicide 2,4-dichlorophenoxyacetic acid for monitoring of its contamination in environmental water.

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a small organic chemical pollutant in the environment. To develop a nanobody-based immunoassay for monitoring trace levels of 2,4-D, a step-wise strategy for the generation of nanobodies highly specific against this small chemical was employed. Firstly, we synthesized three novel haptens mimicking 2,4-D and assessed their influence on the sensitivity and specificity of the existing antibody-based assay. Polyclonal antibodies (pAb) from rabbits showed good sensitivity and moderate specificity for 2,4-D, pAb from llama based on selected haptens showed similar performance when compared to those from rabbits. Secondly, nanobodies derived from llama were generated for 2,4-D by an effective procedure, including serum monitoring and one-step library construction. One nanobody, NB3-9, exhibited good sensitivity against 2,4-D (IC50 = 29.2 ng/mL) had better specificity than the rabbit pAb#1518, with no cross-reactivities against the 2,4-D analogs tested. Thirdly, one-step fluorescent enzyme immunoassay (FLEIA) for 2,4-D based on a nanobody-alkaline phosphatase (AP) fusion was developed with IC50 of 1.9 ng/mL and a linear range of 0.4-8.6 ng/mL. Environmental water samples were analyzed by FLEIA and LC-MS/MS for comparison, and the results were consistent between both methods. Therefore, the proposed step-wise strategy from hapten design to nanobody-AP fusion production was successfully conducted, and the resulting nanobody based FLEIA was demonstrated as a convenient tool to monitor 2,4-D residuals in the environment.

Generation of functional single-chain fragment variable from hybridoma and development of chemiluminescence enzyme immunoassay for determination of total malachite green in tilapia fish.

To determine malachite green (MG) and its major metabolite, leucomalachite green (LMG) residual levels in tilapia fish, chemiluminescent enzyme immunoassay (CLEIA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. At first, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against LMG, and then thoroughly by database-assisted sequence analysis. Finally, the productive VH and VL were assembled to an intact scFv sequence and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step CLEIA against LMG was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 1.3 and 0.04 ng/mL, respectively. The validation results of this novel competitive CLEIA was in line with those obtained by classical HPLC method for determination of total MG in spiked and field incurred samples.

Broad-specificity immunoassay for O,O-diethyl organophosphorus pesticides: application of molecular modeling to improve assay sensitivity and study antibody recognition.

A monoclonal antibody (mAb) against 4-(diethoxyphosphorothioyloxy)benzoic acid (hapten 1) was raised and used to develop a broad-specificity competitive indirect enzyme-linked immunosorbent assay (ciELISA) for 14 O,O-diethyl organophosphorus pesticides (OPs). Computer-assisted molecular modeling was used to model two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationships (QSARs) to study antibody recognition. On the basis of insights obtained from the QSAR models, two heterologous coating haptens, 4-(diethoxyphosphorothioylamino)butanoic acid (hapten 2) and 4-(diethoxyphosphorothioyloxy)-2-methylbenzoic acid (hapten 3) were designed, synthesized, and used to develop heterologous ciELISAs with significantly improved sensitivity. The heterologous ciELISA using hapten 2 as the coating hapten showed good sensitivity in a broad-specific manner for eight O,O-diethyl OPs and may be used as a screening method for the determination of these OPs. Our studies demonstrated that molecular modeling can provide insights into the spatial and electronic effects of molecular structures that are important for antibody activity, which can then be used to improve immunoassay sensitivity.

Expression and purification of an anti-clenbuterol single chain Fv antibody in Escherichia coli.

Recombinant antibodies with desirable characteristics that can replace polyclonal or monoclonal antibodies are important for enzyme-linked immunosorbent assay (ELISA) of residues of clenbuterol (CBL), an illicit veterinary drug. Here, we report our work on expression and purification of a mouse-derived anti-CBL single chain Fv (scFv) antibody in Escherichia coli (E. coli). An expression plasmid pBV220-CBL was constructed and transformed into E. coli BL21 (DH3) strain cells. After induction by temperature, the 6x His-tagged anti-CBL scFv antibodies were expressed with the yield of 31%. The solubilized inclusion bodies were extracted, denatured and then purified by Ni-NTA column chromatography. The purified recombinant target protein was analyzed by high performance liquid chromatography, SDS-PAGE and Western blotting, respectively. The results showed the prepared anti-CBL scFv antibodies posed HRP-anti-His-tag antibody-recognized activity and their purity was up to 96%. Moreover, an indirect competitive ELISA based on the anti-CBL scFv antibodies revealed that the limit of detection for CBL was 0.5 ng/ml and the linear range was 1.5-10.6 ng/ml. Taken together, these findings suggest that the prepared recombinant antibody can be used for future immunoassay detection for CBL.

Development, Screening, and Validation of Camelid-Derived Nanobodies for Neuroscience Research.

Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well-established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First-strand cDNA synthesis; VH H and VH repertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small-scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb-pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. coli bacterial strains.

Development of a Simple Pretreatment Immunoassay Based on an Organic Solvent-Tolerant Nanobody for the Detection of Carbofuran in Vegetable and Fruit Samples.

Nanobodies are one-tenth the size of conventional antibodies and are naturally obtained from the atypical heavy-chain-only antibodies present in camelids. Their small size, high solubility, high stability, and strong resilience to organic solvents facilitate their use as novel analytical reagents in immunochemistry. In this study, specific nanobodies against pesticide carbofuran were isolated and characterized from an immunized library via phage display platform. We further established an indirect competitive enzyme-linked immunosorbent assay (ELISA) using nanobody Nb316 to detect carbofuran in vegetable and fruit samples. The results showed a half-maximal inhibitory concentration (IC50) of 7.27 ng/mL and a detection limit of 0.65 ng/mL. A simplified sample pretreatment procedure omitting the evaporation of organic solvent was used. The averaged recovery rate of spiked samples ranged between 82.3% and 103.9%, which correlated with that of standard UPLC-MS/MS method. In conclusion, a nanobody with high specificity for carbofuran was characterized, and a nanobody-based sensitive immunoassay for simple and rapid detection of carbofuran in real samples was validated.

Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine.

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules.

A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

A Nanobody Against Cytotoxic T-Lymphocyte Associated Antigen-4 Increases the Anti-Tumor Effects of Specific CD8+ T Cells.

Adoptive cell-based immunotherapy typically utilizes cytotoxic T lymphocytes (CTLs), expanding these cells ex vivo. Such expansion is traditionally accomplished through the use of autologous APCs that are capable of interactions with T cells. However, incidental inhibitory program such as CTLA-4 pathway can impair T cell proliferation. We therefore designed a nanobody which is specific for CTLA-4 (CTLA-4 Nb 16), and we then used this molecule to assess its ability to disrupt CTLA-4 signaling and thereby overcome negative costimulation of T cells. With CTLA-4 Nb16 stimulation, dendritic cell/hepatocellular carcinoma fusion cells (DC/HepG2-FCs) enhanced autologous CD8+ T cell proliferation and production of IFN-γ in vitro, thereby leading to enhanced killing of tumor cells. Using this approach in the context of adoptive CD8+ immunotherapy led to a marked suppression of tumor growth in murine NOD/SCID hepatocarcinoma or breast cancer xenograft models. We also observed significantly increased tumor cell apoptosis, and corresponding increases in murine survival. These findings thus demonstrate that in response to nanobody stimulation, DC/tumor cells-FC-induced specific CTLs exhibit superior anti-tumor efficacy, making this a potentially valuable means of achieving better adoptive immunotherapy outcomes in cancer patients.

Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif).

Lead identification and optimization are essential steps in the development of a new drug. It requires cost-effective, selective and sensitive chemical tools. Here, we report a novel method using nanobodies that allows the efficient screening for potent ligands. The method is illustrated with the cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), a virulence factor secreted by the opportunistic pathogen Pseudomonas aeruginosa. 18 nanobodies selective to Cif were isolated by bio-panning from nanobody-phage library constructed from immunized llama. 8 out of 18 nanobodies were identified as potent inhibitors of Cif enzymatic activity with IC50s in the range of 0.3-6.4 µM. A nanobody VHH219 showed high affinity (KD = 0.08 nM) to Cif and the highest inhibitory potency, IC50 = 0.3 µM. A displacement sandwich ELISA (dsELISA) with VHH219 was then developed for classification of synthetic small molecule inhibitors according their inhibitory potency. The developed assay allowed identification of new inhibitor with highest potency reported so far (0.16 ±â€¯0.02 µM). The results from dsELISA assay correlates strongly with a conventional fluorogenic assay (R = 0.9998) in predicting the inhibitory potency of the tested compounds. However, the novel dsELISA is an order of magnitude more sensitive and allows the identification and ranking of potent inhibitors missed by the classic fluorogenic assay method. These data were supported with Octet biolayer interferometry measurements. The novel method described herein relies solely on the binding properties of the specific neutralizing nanobody, and thus is applicable to any pharmacological target for which such a nanobody can be found, independent of any requirement for catalytic activity.

Bispecific Monoclonal Antibody-Based Multianalyte ELISA for Furaltadone Metabolite, Malachite Green, and Leucomalachite Green in Aquatic Products.

A new multianalyte immunoassay was designed to screen furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and leucomalachite green (LMG) in aquatic products using a bispecific monoclonal antibody (BsMAb). Gradient drug mutagenesis methods were separately used to prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient. BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid-hybridomas of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3 ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA, 3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced from MG by potassium borohydride was recognized by BsMAb. Recoveries of AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with HPLC analysis. Thus, the multianalyte ic-ELISA is suitable for rapid quantification of AMOZ, MG, and LMG in aquatic products.

Enhanced sensitive immunoassay: noncompetitive phage anti-immune complex assay for the determination of malachite green and leucomalachite green.

To develop a more sensitive immunoassay for malachite green (MG) and leucomalachite green (LMG), we identified the immunocomplex binding phage-borne peptides for use in the noncompetitive phage anti-immunocomplex assay (PHAIA). An anti-LMG monoclonal antibody (mAb) was used to select immunocomplex binding peptides from a circular random eight-amino-acid phage-displayed library. After three rounds of panning-elution, five peptides that bound the LMG-mAb immunocomplex were obtained. One of the phage-borne peptide clones that resulted in an assay with the highest sensitivity was chosen for further research. The concentration of LMG producing 50% of the saturated signal and the limit of detection of the assay were 7.02 and 0.55 ng/mL, respectively, with a linear range of 1.35 to 21.56 ng/mL. The PHAIA based on the same antibody was 16 times more sensitive compared to the competitive immunoassay. PHAIA was used to analyze LMG, MG, and two mixtures of spiked fish samples, with validation by high-performance liquid chromatography (HPLC) with fluorescence detector. Results showed a good correlation (R(2)LMG = 0.9841; R(2)MG = 0.993; R(2)Mixture = 0.9903) between the data of PHAIA and HPLC, thus the assay was an efficient method for monitoring food safety.