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 IC₅₀ (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.      

Development of a lateral flow fluorescent microsphere immunoassay for the determination of sulfamethazine in milk

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL^?1 in buffer and 0.11 μg L^?1 in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6 % in the inter-batch assay with coefficient of variations of 6.0 to 14.3 %. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.     

Determination of chloramphenicol in milk by a fluorescence polarization immunoassay

A procedure for the determination of the drug chloramphenicol using a fluorescence polarization immunoassay (FPIA) was proposed. The optimum pairs of antibodies and antigens labeled with fluorescein were chosen, and the analytical characteristics of the procedure were determined. A rapid procedure for milk sample preparation with the use of a saturated solution of ammonium sulfate was optimized. The total time of sample preparation and determination of chloramphenicol in milk was no longer than 10 min. The detection limits of chloramphenicol in water and milk were 10 ng/mL and 20 μg/kg, respectively. The procedure developed for the determination of chloramphenicol was tested in the analysis of model and real milk samples. It was found that some milk samples contained chloramphenicol in concentrations of 38–41 μg/kg, which are several times higher than the maximum permissible concentration (MPC) (10 μg/kg).     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.     

Gold nanoparticle-based fluorescence immunoassay for malaria antigen detection

The development of rapid detection assays for malaria diagnostics is an area of intensive research, as the traditional microscopic analysis of blood smears is cumbersome and requires skilled personnel. Here, we describe a simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures. This homogeneous assay is based on the fluorescence quenching of cyanine 3B (Cy3B)-labeled recombinant Plasmodium falciparum heat shock protein 70 (PfHsp70) upon binding to gold nanoparticles (AuNPs) functionalized with an anti-Hsp70 monoclonal antibody. Upon competition with the free antigen, the Cy3B-labeled recombinant PfHsp70 is released to solution resulting in an increase of fluorescence intensity. Two types of AuNP-antibody conjugates were used as probes, one obtained by electrostatic adsorption of the antibody on AuNPs surface and the other by covalent bonding using protein cross-linking agents. In comparison with cross-linked antibodies, electrostatic adsorption of the antibodies to the AuNPs surfaces generated conjugates with increased activity and linearity of response, within a range of antigen concentration from 8.2 to 23.8 μg.mL(-1). The estimated LOD for the assay is 2.4 μg.mL(-1) and the LOQ is 7.3 μg.mL(-1). The fluorescence immunoassay was successfully applied to the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasite densities as low as 1,000 parasites.μL(-1).     

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.     

Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling

Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor’s surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein’s distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL⁻¹ apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1 h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient’s urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor.     

Quantum dot-based lateral flow immunoassay for detection of chloramphenicol in milk

A novel rapid (20 min) fluorescent lateral flow test for chloramphenicol (CAP) detection in milk was developed. The chosen format is a binding-inhibition assay. Water-soluble quantum dots with an emission peak at 625 nm were applied as a label. Milk samples were diluted by 20 % with phosphate buffer to eliminate the matrix effect. The result of the assay could be seen by eye under UV light excitation or registered by a portable power-dependent photometer. The limit of CAP detection by the second approach is 0.2 ng/mL, and the limit of quantitation is 0.3 ng/mL.

Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14 fM (1.4 pg mL⁻¹). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min.     

A one-step homogeneous immunoassay for cancer biomarker detection using gold nanoparticle probes coupled with dynamic light scattering

A one-step homogeneous immunoassay for the detection of a prostate cancer biomarker, free-PSA (prostate specific antigen), was developed using gold nanoparticle probes coupled with dynamic light scattering (DLS) measurements. A spherical gold nanoparticle with a core diameter around 37 nm and a gold nanorod with a dimension of 40 by 10 nm were first conjugated with two different primary anti-PSA antibodies and then used as optical probes for the immunoassay. In the presence of antigen f-PSA in solution, the nanoparticles and nanorods aggregate together into pairs and oligomers through the formation of a sandwich type antibody-antigen-antibody linkage. The relative ratio of nanoparticle-nanorod pairs and oligomers versus individual nanoparticles was quantitatively monitored by DLS measurement. A correlation can be established between this relative ratio and the amount of antigen in solution. The light scattering intensity of nanoparticles and nanoparticle oligomers is several orders of magnitude higher than proteins and other typical molecules, making it possible to detect nanoparticle probes in the low picomolar concentration range. f-PSA in the concentration range from 0.1 to 10 ng/mL was detected by this one-step and washing-free homogeneous immunoassay.     

Capillary electrophoretic competitive immunoassay with laser-induced fluorescence detection for methionine-enkephalin

Immunoassays are commonly used in bioresearch for the detection and quantification of small proteins and macromolecules in biological fluids and other complex matrices. In this report, a competitive immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence was developed for methionine-enkephalin (ME). The method is based on the competitive reaction between the ME and fluorescein conjugated ME (ME-F) with anti-ME antibody, capillary electrophoresis separation of the ME-antibody bound and free ME-F, followed by the laser-induced fluorescence detection of the fluorescent species. With the optimized separation conditions, it was possible to separate the antibody bound and free fluorescien conjugated ME by a capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis using an uncoated fused-silica capillaries. The results concluded that the assay specificity, selectivity and accuracy were excellent.     

Laser-induced fluorescence reader with a turbidimetric system for sandwich-type immunoassay using nanoparticles

A unique laser-induced fluorescence (LIF) reader equipped with a turbidimetric system was developed for a sandwich-type immunoassay using nanoparticles. The system was specifically designed to reduce experimental error caused by particle loss, aggregation and sinking, and to improve analytical performance through ratiometric measurement of the fluorescence with respect to the turbidimetric absorbance. For application to determine the concentration of salinomycin, magnetic nanoparticles (MNPs) and FITC-doped silica nanoparticles (colored balls) immobilized with antibody were synthesized for magnetic extraction and for tagging as a fluorescence probe, respectively. The detection limit of about 39 pg mL⁻¹ was obtained, which was an improvement of about 2-fold compared to that obtained without employment of the turbidimetric system. Calibration linearity and sensitivity were also improved, with increase from 0.8601 to 0.9905 in the R²-coefficient and by 1.92-fold for the curve slope, respectively. The developed LIF reader has the potential to be used for fluorescence measurements using various nanomaterials, such as quantum dots.     

Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation

The development of capillary electrophoresis (CE)-based competitive immunoassay for prion protein (PrP) using carboxymethyl beta-cyclodextrin (CM-beta-CD) as a buffer additive is described here. The assay was based on the competitive binding of PrP and a fluorescein-labeled peptide from the prion protein with a limiting amount of specific antibody. The amount of both free and fluorescein-labeled peptide bound to antibody (immunocomplex) were determined by CE with laser-induced fluorescence detection. In the presence of PrP, the peak height ratio of the immunocomplex and the free peptide was altered compared to the control. These changes were directly proportional to the amount of PrP present. The fluorescently labeled peptide spanning amino acid positions 140-158 of the PrP and its corresponding monoclonal antibody is reported here. The reaction times of the antibody with either the peptide or the recombinant PrP was less than 1 min and is a large improvement over the 16-18 h required to achieve equilibrium for polyclonal antibodies. CM-beta-CD was explored as a buffer additive to suppress analyte adsorption and enhance separation selectivity in the CE analysis. A fast (1.1 min), selective (resolution 4.7), and reproducible (relative standard deviations of migration time for free and bound fluorescein isothiocyanate (FITC)-peptide 0.56% and 0.64%, respectively) separation was obtained with 0.6% CM-beta-CD in 25 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) at pH 8.8. The concentration detection limit of the assay for recombinant PrP was determined to be 80 ng/mL (or mass detection limit 1 pg). When blood samples from scrapie-infected sheep and from normal sheep were tested, the results of the blood assay were consistent with scrapie status of the sheep as determined post mortem by Western blot analysis. Development of this assay will lead to a potentially robust, rapid, and specific preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) in animals and humans.     

Aflatoxin M1 determination in raw milk using a flow-injection immunoassay system

A flow-injection immunoassay (FI-IA) method with amperometric detection for aflatoxin M1 (AFM1) determination in milk has been developed. The first step consists in an incubation of the sample containing AFM1 (Ag) with fixed amounts of anti-AFM1 antibody (Ab) and of the tracer (Ag^*, AFM1 covalently coupled to HRP) until equilibrium is reached. In this mixture a competition occurs between Ag and Ag^* for the Ab. The mixture is then injected into a flow system where the separation of the free tracer (Ag^*) and the antibody-bound tracer (AbAg^*) is performed in a column with immobilized Protein G. The antigen–antibody complexes are retained in the column due to the high affinity of the Protein G for the antibody. The activity of the eluted enzyme label is then amperometrically detected.

The immunoassay was optimised relative to conditions for antibody–antigen incubation (pH, incubation time, ionic strength, temperature) and enzymatic label detection. This method showed a dynamic concentration range between 20 and 500 ppt AFM1, a low detection limit (11 ppt), good reproducibility (RSD < 8%) and a high throughput (six samples per hour in triplicate). Different milk samples were analysed and the results were in good agreement with those obtained by HPLC using the AOAC 2000.08 method.     

Single biosensor immunoassay for the detection of five aminoglycosides in reconstituted skimmed milk

The application of an optical biosensor (Biacore 3000), with four flow channels (Fcs), in combination with a mixture of four specific antibodies resulted in a competitive inhibition biosensor immunoassay (BIA) for the simultaneous detection of the five relevant aminoglycosides in reconstituted skimmed milk. Four aminoglycosides (gentamicin, neomycine, kanamycin and a streptomycin derivative) were immobilised onto the sensor surface of a biosensor chip (CM5) in the four Fcs of the biosensor system by amine coupling. In the Biacore, milk (reconstituted from skimmed milk powder) was 10 times diluted with a mixture of the four specific antibodies and injected through the four serially connected Fcs (1 min at a flow rate of 20 μl min⁻¹). The responses measured just prior to the injection (20 μl at a flow rate of 20 μl min⁻¹) of the regeneration solution (0.2 M NaOH + 20% acetonitril) were indicative for the presence or absence of the aminoglycosides in reconstituted milk. The limits of detection were between 15 and 60 ng ml⁻¹, which was far below the maximum residue limits (MRLs) (varying from 100 to 500 ng ml⁻¹) and the total run time between samples was 7 min.     

Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides

Specific polyclonal antibodies against s-triazine herbicides were obtained by preparing immunogens coupling home-synthesized haptens derivatives of simazine (6-chloro-N-ethyl-N′-ethyl-1,3,5-triazine-2,4-diamine) to lysine groups of hemocyanin from keyhole limpets and bovine serum albumin carrier proteins. Three highly sensitive rabbit antisera were obtained and evaluated with a battery of six enzyme tracers derived from triazine structures in an optimized ELISA format. The antiserum As8 and the HRP-2f tracer, which yield the best assay sensitivity for simazine (detection limit 0.11 ± 0.02 μg L⁻¹, IC₅₀ 0.88 ± 0.04 μg L⁻¹), were applied to the development of a sensitive flow-through immunoassay for the analysis of this herbicide. The automated assay was based on a direct competitive immunosorbent assay and fluorescence detection. The optimized method presents an IC₅₀ value of 0.35 ± 0.04 μg L⁻¹ with a detection limit of 1.3 ± 0.9 ng L⁻¹ and a dynamic range from 0.010 to 7.5 μg L⁻¹ simazine. The generic nature of the antiserum was shown by good relative cross-reactivities with other triazines such as atrazine (420%) or propazine (130%) and a lower response to terbutylazine (6.4%) and desethyl-atrazine (2.2%). No cross-reactivity was obtained for nonrelated pesticides such as 2,4-dichlorophenoxyacetic acid or linuron and the assay could be applied as a screening method for triazine herbicides. The total analysis time was 30 min per determination and the immunosensor could be reused for more than 150 cycles without significant loss of activity. The immunosensor has been successfully applied to the direct analysis of simazine in surface water samples at the nanogram per liter level. The results obtained by comparative analysis of the immunosensor with a chromatographic procedure for triazines showed a close correspondence.     

Potential of fluorescence polarization immunoassay for the detection of Aspergillus fumigatus galactomannan

Russian Chemical Bulletin - Galactomannan (GM) is a specific polysaccharide antigen of opportunistic mold fungi of the genus Aspergillus. The detection of GM in patients’ biological fluids is...     

Trace analysis of endectocides in milk by high performance liquid chromatography with fluorescence detection

An analytical method has been developed for the simultaneous determination of the following endectocide drugs in milk: ivermectin, abamectin, doramectin, moxidectin, eprinomectin, emamectin and nemadectin. Samples were extracted with acetonitrile, purified with solid-phase extraction on a reversed phase C₈, derivatised with N-methylimidazole, trifluoroacetic anhydride and acetic acid to a stable fluorescent derivative, and were further analysed by gradient high performance liquid chromatography (HPLC) on an endcapped reversed phase Supelcosil LC-8-DB. The derivatisation step was mathematically optimised and the method was validated according to the requirements of Commission Decision 2002/657/EC, using fortified raw bovine milk. Mean recovery was between 78 and 98%. The repeatability (CV_r) and within-laboratory reproducibility (CV_W) ranged from 4.6 to 13.4% and from 6.6 to 14.5%, respectively. Decision limits (CCα) for analytes with MRL values, namely eprinomectin and moxidectin, were determined to be 24.8 and 50.6 μg kg⁻¹, respectively. CCα values for unauthorised endectocides ranged from 0.1 to 0.2 μg kg⁻¹. Due to high acceptability regarding the required criteria and applicability to ovine and caprine milk, giving similar results, this multi-analyte method has been successfully implemented in pharmacokinetic research studies as well as statutory residue monitoring in Slovenia.