A rapid and simple fluorescence enzyme-linked immunosorbent assay for tetrabromobisphenol A in soil samples based on a bifunctional fusion protein.

Tetrabromobisphenol A (TBBPA) is the largest brominated flame retardant which can be released to environment and cause long-term hazard. In this work, we developed a rapid and highly sensitive fluorescence enzyme-linked immunosorbent assay (FELISA) for monitoring of TBBPA in soil samples. TBBPA specific nanobody derived from camelid was fused with alkaline phosphatase to obtain the bi-functional fusion protein, which enable the specific binding of TBBPA and the generation of detection signal simultaneously. The assay showed an IC50 of 0.23 ng g-1, limit detection of 0.05 ng g-1 and linear range from 0.1 to 0.55 ng g-1 for TBBPA in soil samples. Due to the high resistance to organic solvents of the fusion protein, a simple pre-treatment by using 40% dimethyl sulfoxide (DMSO) as extract solvent can eliminate matrix effect and obtain good recoveries (ranging from 93.4% to 112.4%) for spiked soil samples. Good relationship between the results of the proposed FELISA and that of liquid chromatography tandem mass spectrometry (LC-MS/MS) was obtained, which indicated it could be a powerful analytical tool for determination of TBBPA to monitor human and environmental exposure.

Development of Cu(II)/Cu(I)-induced quantum dot-mediated fluorescence immunoassay for the sensitive determination of ethyl carbamate.

A series of haptens were rationally designed for producing monoclonal antibodies specific for EC and a simple fluorescence immunoassay platform was developed for the sensitive determination of EC based on alkaline phosphatase (ALP)-triggered Cu+ quenching of CdSe quantum dots (QDs). It was noted that Cd as a fluorescence substrate in CdSe QDs can be selectively substituted by Cu+ that resulted in a more significant fluorescence quenching in comparison with Cu2+. Meanwhile, because ALP catalyzed ascorbic acid phosphate and then assisted the transformation of Cu2+ to Cu+, the change in fluorescence intensity was found to be proportional to ALP concentration. After simple magnetic separation, the sensitivity and linear range of the established assay were improved approximately 53-fold and an order of magnitude, respectively, when compared with the conventional ELISA. The proposed platform was able to both amplify the signal and eliminate matrix interferences, making it a promising to determine EC as well as other contaminants in complex food matrix in a highly sensitive and simple manner. Graphical abstract.

Ciprofloxacin and Clinafloxacin Antibodies for an Immunoassay of Quinolones: Quantitative Structure⁻Activity Analysis of Cross-Reactivities.

A common problem in the immunodetection of structurally close compounds is understanding the regularities of immune recognition, and elucidating the basic structural elements that provide it. Correct identification of these elements would allow for select immunogens to obtain antibodies with either wide specificity to different representatives of a given chemical class (for class-specific immunoassays), or narrow specificity to a unique compound (mono-specific immunoassays). Fluoroquinolones (FQs; antibiotic contaminants of animal-derived foods) are of particular interest for such research. We studied the structural basis of immune recognition of FQs by antibodies against ciprofloxacin (CIP) and clinafloxacin (CLI) as the immunizing hapten. CIP and CLI possess the same cyclopropyl substituents at the N1 position, while their substituents at C7 and C8 are different. Anti-CIP antibodies were specific to 22 of 24 FQs, while anti-CLI antibodies were specific to 11 of 26 FQs. The molecular size was critical for the binding between the FQs and the anti-CIP antibody. The presence of the cyclopropyl ring at the N1 position was important for the recognition between fluoroquinolones and the anti-CLI antibody. The anti-CIP quantitative structure⁻activity relationship (QSAR) model was well-equipped to predict the test set (pred_R² = 0.944). The statistical parameters of the anti-CLI model were also high (R² = 0.885, q² = 0.864). Thus, the obtained QSAR models yielded sufficient correlation coefficients, internal stability, and predictive ability. This work broadens our knowledge of the molecular mechanisms of FQs' interaction with antibodies, and it will contribute to the further development of antibiotic immunoassays.

Production of Antigen-Binding Fragment against O,O-Diethyl Organophosphorus Pesticides and Molecular Dynamics Simulations of Antibody Recognition.

Immunoassay for pesticides is an emerging analytical method since it is rapid, efficient, sensitive, and inexpensive. In this study, a recombinant antigen-binding fragment (Fab) against a broad set of O,O-diethyl organophosphorus pesticides (DOPs) was produced and characterized. The κ chain and Fd fragment were amplified via PCR and inserted into the vector pComb3XSS and the soluble Fab on phagemid pComb3XSS was induced by isopropyl β-d-thiogalactoside in E. coli TOP 10F’. SDS-PAGE, Western blotting, and indirect competitive ELISA results indicated that Fab maintained the good characteristics of the parental mAb. To better understand antibody recognition, the three-dimensional (3D) model of Fab was built via homologous modeling and the interaction between Fab and DOPs was studied via molecular docking and dynamics simulations. The model clearly explained the interaction manner of Fab and DOPs, and showed that the Arg-L96 and Arg-H52 were mainly responsible for antibody binding. This work provided a foundation for further mutagenesis of Fab to improve its characteristics.

Preparative resolution of gatifloxacin enantiomers with pre-column esterification strategy and comparing their enantioselectivity to bacteria and antibody.

Gatifloxacin (GFX) is a kind of chiral fluoroquinolones compound due to the methyl group at the C-3 position of the piperazine ring[1]. Although the enantiomers of GFX show similar levels of antimicrobial activity and pharmacokinetics[2], the other biological activities (i.e., toxicity or enantioselective recognition to various receptors in vivo) of GFX enantiomers have not yet been studied. With this in mind, we developed a rapid and cost-effective high performance liquid chromatographic (HPLC) separation procedure for GFX enantiomers with a pre-column esterification strategy. With significant enhancement of drug solubility and optimization for chromatographic conditions, the proposed method was scaled up to preparative HPLC to obtain optical active S-(-)- and R-(+)-GFX. The antibacterial activities of GFX enantiomers after preparative separation were further verified by measuring the Minimum Inhibitory Concentration (MIC) values against Escherichia coli ATCC 25922. In addition, the binding selectivity of GFX enantiomers to protein receptor were evaluated by antibody using enzyme-linked immunosorbent assay (ELISA) for the first time.

Fluorescence immunosensor based on a specific monoclonal antibody for highly sensitive and rapid detection of gizzerosine in feed.

Gizzerosine is a biogenic amine produced in fish meal drying process and posted higher mortality due to gizzard erosion in poultry than histamine. However, it is difficult to obtain gizzerosine and achieve sensitive practical detection due to its simple structure. Herein, a monoclonal antibody (mAb) specific to gizzerosine was generated based on the new structural design and a fluorescence immunosensor for sensitive and on-site detection of gizzerosine in feed was first established. Molecular modeling of the three-dimensional (3D) structure and surface electrostatic potential of gizzerosine indicated that the carbonyl group of gizzerosine hapten might affect the important sites of antigen-antibody interactions. The proposed structure was used to obtain the sensitive and specific mAb with IC50 of 3.88 ng/mL in indirect competitive ELISA which was approximately 100-fold lower than that of direct competitive ELISA. Considering the practical application scenarios, a fluorescence immunosensor based on microporous dry method integrated with independent quality control line was established to improve detection stability. Under the optimum conditions, the proposed immunosensor showed a good linear relationship from 1.10 to 19.78 ng/mL and provided a low detection limit of 50 ng/g which was approximately 80-fold lower than the maximum recommended amount (0.4 mg/kg) of gizzerosine in feed. The recoveries of 6 kinds of feed ranged from 83.1 % to 114.3 %, which was in good consistence with that of UHPLC-MS/MS. Overall, this work provides a fast, cost-effective and reliable on-site tool for rapid screening of gizzerosine residues in feed samples.

Formation and Detection of Gizzerosine in Animal Feed Matrices: Progress and Perspectives.

Gizzerosine is responsible for gizzard erosion and black vomit, owing to excessive gastric acid secretion in poultry. It is a biogenic amine that forms during feed processing. Gizzerosine, a derivative of histamine, is a serious threat to animal feed safety and poultry production because it is more potent after ingestion and more harmful to poultry than histamine. The difficulty of obtaining gizzerosine and the lack of simple, rapid, and sensitive in vitro detection techniques have hindered studies on the effects of gizzerosine on gizzard health and poultry production. In this review, we evaluated the natural formation and the chemical synthesis methods of gizzerosine and introduced seven detection methods and their principles for analyzing gizzerosine. This review summarizes the issues of gizzerosine research and suggests methods for the future development of gizzerosine detection methods.

Controllable formation of polydopamine on carbon dots for ultrasensitive detection of alkaline phosphatase and ratiometric fluorescence immunoassay of benzocaine.

Sensing alkaline phosphatase (ALP) activity with high sensitivity and accuracy is critical for both ALP-related health and food safety supervision and the development of ALP-triggered immunoassay platforms. Herein, an ultrasensitive ratiometric fluorescence (RF) sensing system based on the controllable formation of luminescent polydopamine and efficient quenching of carbon dots was proposed for the ALP activity assay, achieving quantitative detection in the range of 0.01-100 mU/L. Furthermore, this RF sensing system was integrated with an ALP-based ELISA platform to construct an RF-ELISA for benzocaine, a potentially abused anesthetic in edible fish, and ultrasensitive assay at the level of fg/mL was realized. This ratiometric strategy-based platform effectively shields various interferences through the self-calibration effect, thus providing more accurate and reliable quantification results. This study not only offers an efficient method for ultratrace detection of ALP and benzocaine but also proposes a universal platform for ultrasensitive detection of diverse targets in food analysis by replacing the recognition unit.

Ratiometric Fluorescence Immunoassay Based on MnO2 Nanoflakes for Sensitive and Accurate Detection of Tricaine.

Tricaine is a common anesthetic used in the long-distance transport of live fish. Recently, its negative impact on human health has aroused extensive concern. Thus, rapid and reliable techniques for tricaine residue analysis are essential to ensuring the quality of aquatic products. Herein, a specific anti-tricaine monoclonal antibody (Mab) was prepared. Then, a sensitive and robust ratiometric fluorescence ELISA (RF-ELISA) was constructed for detecting tricaine based on two MnO2 nanoflake-mediated (MnO2 NFs) fluorogenic reactions. In the RF-ELISA protocol, MnO2 NFs with oxidase-like activity can trigger the formation of fluorescent 2,3-diaminophenazine (oxOPD) with an emissive peak at 570 nm from non-fluorescent o-phenylenediamine (OPD), while ascorbic acid (AA) can decompose MnO2 NFs to lose their oxidase-mimicking activity, which is accompanied by the oxidation of AA into dehydroascorbic acid (DHAA). The subsequent reaction between the generated DHAA and OPD will result in the production of 3-(1,2-dihydroxy ethyl)furo[3,4-b]quinoxalin-1(3H)-on (DFQ), which has a potent emission peak at 445 nm. By virtue of the alkaline phosphatase (ALP) labeled on the antibody, which can catalyze the production of AA from ascorbic acid 2-phosphate (AAP), the concentration of tricaine can be linked to the variation of the RF signal (F445/F570) via a competitive immunoreaction. After optimization, RF-ELISA displayed a detection limit (LOD) of 0.28 ng/mL toward tricaine (in buffer solution), which was 376-fold lower than that of the traditional colorimetric ELISA. For practical application, the LODs of RF-ELISA for tricaine detection in shrimp and tilapia samples were determined to be 2.8 and 5.6 ng/g, respectively. Recoveries for spiked shrimp and tilapia samples, as well as the validation data from LC-MS/MS, showed that RF-ELISA exhibited good accuracy, precision, and reliability. This RF-ELISA protocol opened up new ways for tricaine and other-target analyses in food safety detection.

Highly selective monoclonal antibody-based fluorescence immunochromatographic assay for the detection of fenpropathrin in vegetable and fruit samples.

Fenpropathrin (FPT) is a typical pyrethroid pesticide that can cause chronic toxicity to humans. Herein, an anti-FPT monoclonal antibody (mAb) was elicited via a novel hapten synthesized by introducing a carboxyl-containing spacer arm in the cyclopropane moiety of FPT. Characterized by enzyme-linked immunosorbent assay (ELISA), the mAb exhibited high affinity and selectivity to FPT with a half-maximal inhibitory concentration of 31.05 µg/L and negligible cross-reactivities with analogs of pyrethroids. Based on the mAb, a fluorescence immunochromatographic assay (FICA) for FPT detection was firstly developed. The detection limit of the FICA is 0.012 mg/kg which is much lower than the maximum residue limit of FPT for food samples. The average recoveries of FPT from spiked food samples by the FICA were 85.0-105.0%, and the obtained results were in good agreement with those of gas chromatography-tandem mass spectrometry. Overall, this work provided a reliable tool suitable for the detection of FPT residue for large-scale samples in a rapid and cost-effective manner.

Dual-modular immunosensor for bongkrekic acid detection using specific monoclonal antibody.

Bongkrekic acid (BA) is a mitochondrial toxin that causes high mortality but is often mistakenly categorized as other food poisonings. The immunoassay of BA is still challenging since the specific antibody is unavailable. In this work, a monoclonal antibody specific to BA was first generated and a dual-modular immunosensor for on-site and laboratory detection was established. The antibody showed good affinity (Kd=0.33 µM) and sensitivity (IC50 =17.9 ng/mL in ELISA) with negligible cross-reactivity with common mycotoxins. In dual-modular conditions, fluorescence assay (FA) was conducted based on the inner filter effect of carbon dots (CDs) and oxidized 3,3',5,5'-tetramethylbenzidine (TMB), while the colorimetric assay (CA) was conducted using TMB2+-mediated rapid surface etching of gold nanostars (Au NSs). The proposed immunosensor showed good sensitivity and reproducibility to BA in food samples, with a limit of detection lower than 10 ng/mL and recovery ranging from 80.0% to 103.6%, which was in good consistence with that of standard LC-MS/MS. Overall, the proposed immunosensor is an ideal tool for screening BA contaminants in food with good sensitivity and high effectivity.

A high-resolution colorimetric immunoassay for tyramine detection based on enzyme-enabled growth of gold nanostar coupled with smartphone readout.

In this work, a specific monoclonal antibody against tyramine was produced based on a new hapten design. Then, we developed a high-resolution multicolor colorimetric immunoassay for tyramine based on this antibody by integrating enzyme-induced multicolor generation with smartphone-assistant signal readout. The multicolor generation is due to the shift of the local surface plasmon resonance band of gold nanostructure controlled by alkaline phosphatase-induced the growth of gold nanostars. Quantitative detection of tyramine was achieved via analyzing the red/blue channel values of assay solution's image taken by a smartphone with the support of a color recognizer application. The limit of detection of this immunoassay for tyramine detection in beef, pork and yoghurt was 19.7 mg/kg or L. The average recoveries were between 83 % and 103 %., and the results were validated by high performance liquid chromatography to be reliable. Overall, this developed immunoassay provides a promising platform for on-site detection of tyramine.

Phosphate-triggered ratiometric fluoroimmunoassay based on nanobody-alkaline phosphatase fusion for sensitive detection of 1-naphthol for the exposure assessment of pesticide carbaryl.

The excessive use of carbaryl has resulted in the risk of its exposure. In this study, we isolated six nanobodies (Nbs) from a camelid phage display library against the biomarker of carbaryl, 1-naphthol (1-NAP). Owing to its characteristics of easy genetic modifications, we produced a nanobody-alkaline phosphatase (Nb-CC4-ALP) fusion protein with good stability. A dual-emission system based ratiometric fluoroimmunoassay (RFIA) for quick and highly sensitive determination of 1-NAP was developed. Silicon nanoparticles (SiNPs) was used as an internal reference and for aggregation-induced emission enhancement (AIEE) of gold nanoclusters (AuNCs), while AuNCs could be quenched by MnO2 via oxidation. In the presence of ALP, ascorbic acid phosphate (AAP) can be transformed into ascorbic acid (AA), the later can etch MnO2 to recover the fluorescence of the AuNCs. Based on optimal conditions, the proposed assay showed 220-fold sensitivity improvement in comparison with conventional monoclonal antibody-based ELISA. The recovery test of urine samples and the validation by standard HPLC-FLD demonstrated the proposed assay was an ideal tool for screening 1-NAP and provided technical support for the monitoring of carbaryl exposure.

Production and Characterization of Biotinylated Anti-fenitrothion Nanobodies and Development of Sensitive Fluoroimmunoassay.

A simple and sensitive fluoroimmunoassay (FIA) based on a heavy-chain antibody (VHH) for rapid detection of fenitrothion was developed. A VHH library was constructed from an immunized alpaca, and one clone recognizing fenitrothion (namely, VHHjd8) was achieved after careful biopanning. It was biotinylated by fusing with the Avi tag and biotin ligase to obtain a fusion protein (VHHjd8-BT), showing both binding capacity to fenitrothion and the streptavidin poly-horseradish peroxidase conjugate (SA-polyHRP). Based on a competitive assay format, the absorbance spectrum of oxidized 3,3',5,5'-tetramethylbenzidine generated by SA-polyHRP overlapped the emission spectrum of carbon dots, which resulted in quenching of signals due to the inner-filter effect. The developed FIA showed an IC50 value of 1.4 ng/mL and a limit of detection of 0.03 ng/mL, which exhibited 15-fold improvement compared with conventional enzyme-linked immunosorbent assay. The recovery test of FIA was validated by standard GC-MS/MS, and the results showed good consistency, indicating that the assay is an ideal tool for rapid screening of fenitrothion in bulk food samples.

Ki67 testing in the clinical management of patients with non-metastatic colorectal cancer: Detecting the optimal cut-off value based on the Restricted Cubic Spline model.

The proliferation of the biomarker Ki67 has been extensively studied in colorectal cancer (CRC). Although numerous Ki67 cut-off values have previously been reported, the optimal cut-off value remains unclear with previous studies providing contrasting results. The present retrospective cohort study aimed to determine the optimal cut-off value for CRC. Ki67 levels and the prognosis of patients with non-metastatic CRC were obtained from the Electronic Health Information System of a tertiary hospital in Kunming City. The Restricted Cubic Spline (RCS) model was used to analyze the non-linear association between Ki67 levels and the risk of patient death and metastasis. Moreover, the RCS model was used to determine the optimal cut-off value of Ki67. Cox proportional hazards models were used to verify the effects of the cut-off value. In total, 210 patients with CRC and a median age of 62.5 years (age range, 23.0-88.0 years) were studied. Results of the present study demonstrated a non-linear association between Ki67 levels and the risk of patient death based on the RCS model, and at Ki67 levels ≥60%, the hazard ratio (HR) of patient death gradually increased. Using multivariate-adjusted Cox proportional hazards models, the results of the present study demonstrated that Ki67 ≥60% indicated a high-risk ratio for both distant metastasis and death [HR, 2.640; 95% confidence interval (CI), 1.066-6.539], compared with Ki67 <60% (HR, 2.558; 95% CI, 1.079-6.064). Therefore, Ki67 ≥60% may be the optimal cut-off value for the prediction of death and metastasis in patients with CRC. Thus, Ki67 may act as a biomarker for predicting the prognosis of patients with CRC, and the optimal cut-off value for the prediction of both death and metastasis of patients with CRC is 60%.

Highly selective monoclonal antibody-based lateral flow immunoassay for visual and sensitive determination of conazole fungicides propiconazole in vegetables.

The conazole fungicide propiconazole is frequently found in vegetables although usage is not allowed. To overcome the high-cost and time-consuming labour requirements of instrumental methods, we developed a simple and visual lateral flow immunoassay for the sensitive determination of propiconazole. A hapten was carefully designed to raise a monoclonal antibody against propiconazole. Bal b/c mice were immunised with the hapten-carrier protein conjugate and a specific monoclonal antibody (mAb) was produced. Based on this mAb, a sensitive immunochromatographic strip assay (ICA) was established for rapid screening of propiconazole in vegetable samples. After optimisation of analytical parameters, the ICA strip showed a detection limit of 0.13 ng g-1 and a linear range from 0.5 to 80 ng g-1 using a strip reader. The assay also can be read by the naked eye with a visual limit of detection of 80 ng g-1. The recoveries for spiked vegetable samples by ICA ranged from 85.2% to 114.9%, with a coefficient of variation less than 11.7%. The assay time is within 45 min for a single sample including the sample pre-treatment. For spiked and blind samples, the detection capability of ICA was equivalent to liquid chromatography-mass spectrometry.

Nanobody-based fluorescent immunoassay using carbon dots anchored cobalt oxyhydroxide composite for the sensitive detection of fenitrothion.

Fenitrothion (FN) residue in food is a serious threat to public health. Consequently, a sensitive, cost-effective, and convenient immunoassay for FN urgently needs to be fabricated to safeguard human health. Herein, a nanobody-alkaline phosphatase fusion protein (Nb-ALP)-based fluorescent ELISA using red emissive carbon dots (r-CDs) anchored cobalt oxyhydroxide nanosheet (CoOOH NS) composite was developed for detecting FN. Briefly, a Nb-ALP was obtained by autoinduction expression and employed as a recognition, signal transduction, and amplification element. As the fluorescence signal source, r-CDs were assembled with CoOOH NS to yield the r-CDs@CoOOH NS composite, leading to the fluorescence quenching of r-CDs via Förster resonance energy transfer (FRET). After competitive immunoreaction, the Nb-ALP bounded to the immobilized antigen can mediate the production of ascorbic acid, which can reduce the CoOOH NS to Co2+, breaking the FRET between r-CDs and CoOOH NS, accompanied by the fluorescence recovery of r-CDs. This fluorescent ELISA is highly sensitive to FN with a detection limit of 0.14 ng mL-1, which is 25-fold lower than that of conventional colorimetric ELISAs. The recovery test of food samples and the validation by GC-MS/MS further demonstrated the proposed assay was an ideal tool for detecting FN.

The Simultaneous Determination of Chlorpyrifos-Ethyl and -Methyl with a New Format of Fluorescence-Based Immunochromatographic Assay.

The improper and excessive use in agriculture of chlorpyrifos-methyl (CPSM) and chlorpyrifos-ethyl (CPSE) may affect the health of human beings. Herein, a fluorescence-based immunochromatographic assay (FICA) was developed for the simultaneous determination of CPSM and CPSE. A monoclonal antibody (mAb) with equal recognition of CPSM and CPSE was generated by the careful designing of haptens and screening of hybridoma cells. Instead of labeling fluorescence with mAb, the probe was labeled with goat-anti-mouse IgG (GAM-IgG) and pre-incubated with mAb in the sample. The complex could compete with CPS by coating antigen in the test line. The new format of FICA used goat-anti-rabbit IgG (GAR-IgG) conjugated with rabbit IgG labeled with fluorescence microspheres as an independent quality control line (C line). The novel strategy significantly reduced nonspecific reactions and increased assay sensitivity. Under the optimal conditions, the proposed FICA showed a linear range of 0.015-64 mg/L and limit of detection (LOD) of 0.015 mg/L for both CPSE and CPSM. The average recoveries of CPS from spiked food samples by FICA were 82.0-110.0%. The accuracy was similar to the gas chromatography-tandem mass spectrometry (GC-MS/MS) results. The developed FICA was an ideal on-site tool for rapid screening of CPS residues in foods.

Conformational changes of hapten-protein conjugates resulting in improved broad-specificity and sensitivity of an ELISA for organophosphorus pesticides.

The type of hapten linkage to a carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon and a carboxamido spacer). These haptens were conjugated to bovine serum albumin (BSA) and used as immunogens to produce broad-specificity monoclonal antibodies (MAbs) to organophosphorus pesticides (OPs). Three-dimensional (3D) structures of hapten-lysine conjugates were optimized using molecular modeling (MM) to mimic conformations of hapten-BSA conjugates. The results from MM studies revealed a change of the 3D conformation and electrostatic potential of hapten 1 when the monocarboxylic acid linker was coupled to lysine. This result was consistent with the observed high-cross-reactivity of the corresponding MAb-H1 for the OPs. The competitive indirect enzyme-linked immunosorbent assay based on MAb-H1 is ideally suited to be used as a screening method for OP contaminants.

Development of a fluorescence polarization immunoassay for the detection of melamine in milk and milk powder.

A fluorescence polarization immunoassay (FPIA) based on a polyclonal antibody was developed for the determination of melamine in milk. To obtain an antibody with improved sensitivity and specificity, 6-hydrazinyl-1,3,5-triazine-2,4-diamine was coupled to bovine serum albumin and used as the immunogen for the rabbit immunization. Three fluorescein-labeled melamine tracers with different structures and spacer bridges were synthesized. The structural effect of the tracers on the assay characteristics was investigated. 6-(4,6-Diamino-1,3,5-triazin-2-ylamino)-N-(2-(3-(3',6'-dihydroxy-3-oxo-2,3-dihydrospiro[indene-1,9'-xanthene]-5-yl)thioureido)ethyl)hexanamide demonstrated better sensitivity than 5-(2-(4,6-diamino-1,3,5-triazin-2-yl)hydrazinecarbothioamido)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 3-(4,6-diamino-1,3,5-triazin-2-ylthio)-N-(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)thioureido)ethyl)propanamide. The limit of detection (10% inhibition) of the FPIA was 9.3 ng mL(-1) and the IC(50) (50% inhibition) value was 164.7 ng mL(-1). The antibody in the FPIA showed 21.2% cross-reactivity to the fly-killing insecticide cyromazine, but had no cross-reactivity to other natural structurally related compounds. Recoveries, measured in spiked milk and milk powder samples, ranged from 79.4 to 119.0%. Milk samples fortified with melamine were analyzed by this method and confirmed by high-performance liquid chromatography-mass spectrometry. Excellent recoveries and correlation with spiked levels were observed, suggesting that this immunoassay could be applied to the screening of melamine residues in milk and milk powder after a simple dilution procedure.