Development of colloidal gold-based immumochromatographic assay for the rapid detection of medroxyprogesterone acetate residues in biological materials

A competitive colloidal gold-based immunoassay in lateral-flow format for the rapid detection of medroxyprogesterone acetate (MPA) in biological materials was developed. A nitro-cellulose membrane strip was separately coated with goat anti-rabbit IgG (control line) and MPA hapten-OVA conjugate (test line). Anti-MPA polyclonal antibody labelled with colloidal gold particles was first incubated with MPA. The limit of detection for lateral flow was 5?ng?g^?1 for detecting an MPA standard solution, and the limit of detection was 10?ng?mL^?1 for detecting the MPA spiked in pig urine and 10?ng?g^?1 for spiked in pig liver. The results were confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS) and indicated that there was a good agreement between both methods (R ²?=?0.976). The assay time for the test was less than 5?min, suitable for rapid testing on site.     

Highly Sensitive Chemiluminescent Analysis of Residual Bovine Serum Albumin (BSA) Based on a Pair of Specific Monoclonal Antibodies and Peroxyoxalate–glyoxaline–PHPPA Dimer Chemiluminescent System in Vaccines

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence analysis have been combined to develop a highly sensitive, simple, and rapid method for analysis of bovine serum albumin (BSA) based on a pair of specific monoclonal antibodies in vaccines. A typical ??sandwich type?? immunoassay was used. Reaction of 3-(4-hydroxyphenyl propionate) (PHPPA) with hydrogen peroxide-urea, catalyzed by HRP, produced fluorescence of 3-(4-hydroxyphenyl propionate) dimer, which was detected by chemiluminescence analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)?CH₂O₂?Cglyoxaline?CPHPPA dimer chemiluminescent system. This method exhibited high performance with a linear correlation between response and amount of bovine serum albumin (BSA) in the range 0.1 to 100.0?ng?mL^?1 (r?=?0.9988), and the detection limit was 0.03?ng?mL^?1 (S/N?=?3). Intra- and interassay coefficient variations were all lower than 9.0% at three concentrations (1.0, 20.0, and 80.0?ng?mL^?1). The proposed method has been used for successful analysis of the amount of residual BSA in vaccines. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA.     

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I₅₀ of the assay ranged from 0.64 to 20.98 μg mL⁻¹ for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I₅₀ and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL⁻¹, was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.     

Immunoassay for the detection of lead ions in environmental water samples

A rapid, simple, and reliable competitive immunoassay was developed for measurement of lead ions Pb(II) in environmental samples. Avian antibodies were produced against Pb(II). Since lead ions are too small to elicit an immune response, the metal was coupled to protein carrier Bovine serum albumin (BSA) using a bifunctional chelator 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N′,N′-tetra acetic acid (ITCBE). Poultry birds (layers) were immunised with this Pb(II)–ITCBE–BSA immunoconjugate and the avian antibodies (IgY) isolated from egg yolk recognised Pb(II)-ITCBE complexes as capture reagent and a Pb(II)–ITCBE conjugate of Alkaline phosphatase as an enzyme label. Antibody reaction was optimised for different concentrations of antigen and antibody dilutions. Cross reactivity with other metals were below 1% in competitive ELISA. The IC₅₀ value of this avian antibody was 0.19?µg?mL^?1. The detection range and the detection limit were 0.02–1000?µg?mL^?1and 0.2?µg?mL^?1, respectively.     

Monoclonal antibody-based enzyme-linked immunosorbent assay for detection of total malachite green and crystal violet residues in fishery products

A rapid easy-to-use trace level direct competitive enzyme-linked immunosorbent assay (dc-ELISA) detection of total residual malachite green (MG), crystal violet (CV) and their corresponding primary metabolites leucomalachite green (LMG) and leucocrystal violet (LCV) in fishery products in a single assay was developed. The monoclonal antibodies, anti-MG and anti-CV mAbs, were prepared using carboxyl-malachite green (CMG) and cationized bovine serum albumin (cBSA) conjugates as immunogen. The linear range for the quantitative detection of total MG, CV and their primary metabolites LMG and LCV was between 0.15 to 4.5?ng?mL^?1 with a half maximal inhibitory concentration (IC₅₀) at 0.56?±?0.04?ng?mL^?1 (n?=?5). The anti-MG mAbs exhibited 98% cross-reactivity to CV, less than 0.1% cross-reactivity with LMG and LCV, and no cross-reactivity with chloramphenicol, enrofloxacin, sulfadiazine, and tetracycline. Application of the dc-ELISA in fish tissue samples gave a limit of detection (LOD) of 0.37?ng?g^?1. The improved total detection lead to a recovery of 74.60?±?8.38% at 0.5?ng?g^?1 and 87.47?±?12.83% at 2.0?ng?g^?1 that was better than existing techniques. The dc-ELISA showed total MG in 7 out of 44 field fish samples that were confirmed with LC-MS/MS. The easy-to-use, inexpensive, and rapid dc-ELISA for the detection of total MG, CV and their corresponding primary metabolites holds promise for field applications.     

Chemiluminescent Detection of Gatifloxacin Residue in Milk

Abstract

An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for detection of gatifloxacin residue in milk was developed in this study. Compared with conventional colorimetric ELISA using the same antibody, the developed CL immunoassay shows a significant improvement in sensitivity and detectability with an IC₅₀ of 0.4 ng mL^?1 and a detection limit of 0.001 ng mL^?1 and thus is suitable to be used as a highly sensitive screening method to detect and regulate illegal use of gatifloxacin in food and food products. The test kit was applied to detect milk samples spiked by gatifloxacin, and satisfactory results were obtained.     

Characterization of Anti-Chloramphenicol Antibodies by Enzyme-Linked Immunosorbent Assay

Polyclonal antibodies against conjugates of chloramphenicol succinate and chloramphenicol base with proteins were obtained and characterized in direct ELISA. Antiserum against a conjugate of chloramphenicol (CAP) base with BSA (direct coupling) was very specific and showed cross-reactivity only with CAP succinate (11.3%) and CAP base (4.6%); whereas, antisera against a conjugate of CAP succinate with a protein recognized CAP succinate strongly as an initial compound. In direct ELISA, antisera against a conjugate of CAP succinate with KLH (homologous assay) and CAP base with BSA (heterologous assay) showed similar sensitivity: IC₅₀ were 1.3 and 1.5 ng mL^?1, respectively. Applicability of the immunoreagents obtained was shown in the analysis of CAP residues in milk (3.5% fat content). Detection limit of 0.3 ng mL^?1 was obtained for milk diluted 5 times.     

An Indirect Competitive Enzyme-linked Immunosorbent Assay for Simultaneous Determination of Florfenicol and Thiamphenicol in Animal Meat and Urine

A high-affinity polyclonal antibody was prepared by immunizing animals with haptens FFD and FFM. Under the optimal combination of coating antigen and antibody, an indirect competitive enzyme-linked immunosorbent assay (icELISA) for simultaneous detection of florfenicol and thiamphenicol residues in animal meat and urine samples was developed. The icELISA showed an IC₅₀ value of 1.32 ng mL^?1 for florfenicol and 2.13 ng mL^?1 for thiamphenicol, respectively. The linear ranges were from 0.31 to 5.61 ng mL^?1 with a limit of detection of 0.12 ng mL^?1 for florfenicol, and 0.41 to 11.2 ng mL^?1 with a limit of detection of 0.15 ng mL^?1 for thiamphenicol, respectively. The average recoveries of florfenicol and thiamphenicol in spiked samples ranged from 77.2% to 116.0% with a relative standard deviation of less than 15%. Therefore, this proposed icELISA provided a valid detection method for florfenicol and thiamphenicol residues in animal tissue and urine samples.     

Synthesis of derivatives and production of antiserum for class specific detection of pyrethroids by indirect ELISA

Two series of haptens including 3-phenoxybenzoic acid (PBA) and 3-(2-chloro-3, 3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclo-propanecarboxylic acid (CF₃MPA) were used to prepare immunogens through attachment of 4-C or 6-C handles. Class selective antibodies were produced by immunising rabbits. Ab502 showed the highest reactivity towards tau-fluvalinate (IC₅₀ 1.3 ng mL^?1), λ-cyhalothrin (IC₅₀ 2.3 ng mL^?1), cyfluthrin (IC₅₀ 2.2 ng mL^?1) and fenpropathrin (IC₅₀ 18.5 ng mL^?1) among the antibodies in a competitive ELISA. The effects of methanol, pH and salt concentration were optimised for maximum efficiency of the ELISA (Enzyme-Linked ImmunoSorbent Assay). Ab502 (1:80000)/2-OVA-1(0.2 µg mL^?1) was chosen for ELISA optimisation. Finally, 0.05 M phosphate buffered saline (PBS) at pH 6.5 containing 30% methanol (v/v) was used to dilute the standards. Target analytes in honey samples were extracted with ethyl acetate by sonication. The samples were spiked with three different concentrations of each compound (tau-fluvalinate, 0.5 ng g^?1, 3 ng g^?1, 12 ng g^?1; λ-cyhalothrin and cyfluthrin 1 ng g^?1, 5 ng g^?1, 65 ng g^?1). The recoveries were 36–59% at the lowest spiking concentration and 61–81% at the higher concentration. This assay might be useful to screen pyrethroid residues in honey or other matrix.     

Mixed immunoassay design for multiple chemical residues detection

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23?～?68 ng?mL^?1 for DONs and 4.1?～?49 ng?mL^?1 for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45–1.3 ng?mL^?1 for DONs and 0.59–6.9 ng?mL^?1 for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng?mL^?1 ranged from 91.3 to 102.2 % for DONs and 88.7–98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic assay for several (fluoro)quinolones in milk

We have developed a heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) and a visual colloidal gold-based immunochromatographic assay (CGIA) for simultaneous determination of ofloxacin, marbofloxacin, and fleroxacin residues in milk using polyclonal antibodies. The half-maximum inhibition concentrations (IC₅₀) of ofloxacin, marbofloxacin, fleroxacin, and limits of detection (LODs; calculated as IC₁₅ values) are between 0.20 and 0.53?ng mL^?1, and between 0.02 and 0.05?ng mL^?1, respectively. The average recoveries range from of 78% to 113%, and the coefficients of variation of intra- and inter-assays are between 2 and 11%, and 3 to 19%, respectively. The LODs for ofloxacin, marbofloxacin, fleroxacin in milk are between 3.5 and 8.9?ng mL^?1. The visual minimum detection limit of the optimized CGIA is 2?ng mL^?1 for milk samples. The detection process can be completed within 10?min. The strips can be stored at 4?°C for 8?weeks without significant loss of activity. The results of the analysis of spiked samples showed that the CGIA can be applied to preliminary, fast, and on-site screening of milk samples. The ELISA and CGIA allow for a rapid, sensitive, and low-cost determination of (fluoro)quinolones residues in milk samples.

Comparative surface plasmon resonance and enzyme-linked immunosorbent assay characterisation of a monoclonal antibody with N-acyl homoserine lactones

This study shows the detection of (N-acyl) homoserine lactones (AHLs or HSL) with monoclonal antibodies via a surface plasmon resonance (SPR)-based immunosensor in comparison to conventional microtiter plate-based enzyme-linked immunosorbent assay (ELISA). An HSL derivative, named HSL2 (Table 1), was attached to bovine serum albumin (BSA) and the conjugate (HSL2-BSA-r2) was either covalently immobilised on the SPR sensor chip surface via free amino groups or via adsorption on the ELISA polystyrene plate surface. With a newly developed rat monoclonal antibody (mAb HSL1/2 2C10), AHLs were detected sensitively in a competitive format with SPR and ELISA. Well comparable experiments between SPR and ELISA could be obtained in buffers. Moreover, the SPR sensor surface with the immobilised conjugate HSL2-BSA-r2 could be regenerated at least 340 times (regeneration cycles) without loss of activity. The measurement time per cycle was approximately 15 min. The competitive detection format for SPR and ELISA allowed the detection in the μg L⁻¹ range.     

Simultaneous determination of fluoroquinolone and tetracycline antibacterials in sewage sludge using ultrasonic-assisted extraction and HPLC-MS/MS

A reliable method was proposed for the simultaneous determination of five fluoroquinolones (FQs) and two tetracyclines (TCs) in sewage sludge using ultrasonic-assisted extraction (USE) followed by SPE cleanup and high-performance liquid chromatography-mass spectrometry (HPLC-MS)/MS analysis with electrospray ionisation (ESI) in a positive mode. The USE conditions (e.g. extraction solvent, pH, and extraction cycles) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) parameters were optimised. Quantification was performed by internal standard calibration in multiple reaction monitoring mode. Recoveries of the antibacterials ranged from 41 to 123%, with relative standard deviations within 17%. The sample-based limits of quantification were 10–63?ng?g^?1 dry weight (dw) for FQs (ciprofloxacin, enrofloxacin, lomefloxacin, norfloxacin, and ofloxacin) and 250–500?ng?g^?1 dw for TCs (tetracycline and oxytetracycline). The method was applied to determine the antibacterials in sewage sludge and sediment samples were collected from the Pearl River Delta, China. Ciprofloxacin, norfloxacin, and ofloxacin were frequently detected, ranging from 1052 to 17740?ng?g^?1 dw in dewatered sludge samples, 585–3545?ng?g^?1 dw in untreated solids, and 98–258?ng?g^?1 dw in an urban stream sediment sample, respectively. Lomefloxacin and enrofloxacin were also occasionally detected.     

A simplified method for simultaneous quantitation of alkylphenols and alkylphenol ethoxylates in meat and fish using high-performance liquid chromatography with fluorescence detection

Nonylphenol (NP), octylphenol (OP), nonylphenol monoethoxylate (NP₁EO) and nonylphenol diethoxylate (NP₂EO) are products of the biodegradation of alkylphenol polyethoxylates (AP _n EO) which are used worldwide as detergents and surfactants. NP and OP are categorized as definitely endocrine disruptors. 2,4-Tert-butylphenol (BP) is extensively used for anti-oxidant of rubber and plastics. This work proposed a simple and stable method for simultaneously determining the concentration of NP, OP, BP, n-NP₁EO and n-NP₂EO in meat and fish, without requiring the complex pretreatments of current methods. This study used liquid extraction with acetonitrile and hexane and solid extraction using Florisil, in that order to pretreat samples. The sample solutions were analyzed to identify NP, OP, BP, n-NP₁EO and n-NP₂EO by HPLC with fluorescence detection. The mean recoveries were 85.3?±?3.32% for OP, 87.5?±?6.01% for BP, 90.9?±?4.72% for NP, 86.4?±?4.81% for n-NP₂EO and 90.9?±?4.84% for n-NP₁EO. The average coefficients of variation were about 6%. The method's detection limits were 5.4?ng?g^?1 for OP, 5.2?ng?g^?1 for BP, 8.9?ng?g^?1 for NP, 8.7?ng?g^?1 for n-NP₂EO and 8.1?ng?g^?1 for n-NP₁EO. This work analyzed 5 kinds of usual foodstuffs of meat and fish that are frequently consumed by residents of Taiwan. All of these samples contained NP, but not detectable levels n-NP₁EO. Only salmon was contaminated with n-NP₂EO. The NP level was highest in cod (198.41?±?129.34?ng?g^?1, wet weight). The fried chicken had the highest BP level (48.0?±?41.3?ng?g^?1, wet weight), and the uncooked chicken had the highest OP level (66.6?±?53.0?ng?g^?1, wet weight).     

Study of a toxin–alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4–15 ng mL⁻¹ with a limit of detection (LOD) of 2 ng mL⁻¹ (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2–50 ng mL⁻¹ and the LOD was1 ng mL⁻¹.     

Enzyme-linked immunosorbent assay for the determination of T-2 toxin in cereals and feedstuff

A reliable enzyme-linked immunosorbent assay method was developed for the assay of T-2 toxin in cereals and feedstuff. A hapten of the T-2 toxin was synthesized, and a polyclonal antibody with high affinity and specificity was obtained after immunization of rabbits. Compared to the other ELISA methods, the assay is simple, rapid and affordable. The concentration of T-2 causing 15% inhibition is 0.01?±?0.001 ng mL^?1, which makes the method more sensitive than others. The cross-reactivity against other mycotoxins is low, except for the HT-2 toxin. Sample extraction was achieved within 3 min. The recoveries from samples including barley, wheat, corn, oat, rice and feedstuff were between 75% and 102%, and the detection limit for T-2 toxin was lower than 4 ng g^?1. The method was validated by high-performance liquid chromatography with tandem mass spectrometry detection.     

Immunoassay based on a polyclonal antibody for sex steroid hormones produced by a heterogeneous hapten-conjugated immunogen: Estimation of its potentiality and antibody characteristics

A method in which antibodies are produced by using an immunogen heterogeneously conjugated with two or more kinds of haptens having unlike chemical structures against a same carrier protein was offered as an efficient approach for development of antibody to low molecular compounds. To appreciate the potentiality of the approach, 17β-estradiol (E2) and testosterone were selected as model compounds. The I₅₀ values of antiserum developed were 6 and 8 μg L⁻¹ with the detection limits of 0.02 and 0.15 μg L⁻¹ for E2 and testosterone, respectively. Antiserum owned an interesting characteristic that it was possible to independently analyze E2 and testosterone without mutual interference by making proper use of coating antigens. When using β-estradiol 17-hemisuccuinate (EH) conjugated with bovine serum albumin (BSA) as a coating antigen, the enzyme-linked immunosorbent assay (ELISA) was very selective to E2 and some estrogen analogues. Therefore, if testosterone coexisted in the ELISA for E2 detection, it showed no interference with it. From these findings, it was suggested that the verified method was an efficient and rational approach in development of polyclonal antibody to low molecular compounds.     

Determination of synthetic musks in the sediment of the Taihu lake by using accelerated solvent extraction (ASE) and GC/MS

Synthetic musks, substitutes for natural musks, are widely distributed in environment. They have been detected in water, sludge, fish, shrimp, mussels and other aquatic animals, and even in human's adipose tissue, blood and breast milk. In this study, a new extraction procedure, based on the accelerated solvent extraction (ASE) and in cell clean-up technique was developed and successfully coupled with gas chromatography-mass spectrometry (GC/MS) for the analysis of musks in sediment samples. With this method, the limits of detection as low as 0.03–0.05?ng?g^?1 and the recovery rate of 86.0%–104% are achieved. When compared with soxhlet extraction (SE) and ultrasonic extraction (USE), ASE not only has the best extraction efficiency but also has advantage in extraction time and solvent consumption. Eight synthetic musks, including six polycyclic musks (Tonalide (AHTN), Galaxolide (HHCB), Phantolide (AHDI), Traseolide (ATII), Cashmeran (DPMI) and Celestolide (ADBI)) and two nitro musks (musk xylene (MX) and musk ketone (MK)), were evaluated in sediment samples collected from 15 selected locations of the Taihu lake, one of the largest freshwater lakes in China. The contents of synthetic musks in sediment samples range from 0.336 to 3.10?ng?g^?1 for HHCB, 0.184 to 1.21?ng?g^?1 for AHTN, below detection limit (BDL) to 0.349?ng?g^?1 for MX, and BDL to 0.0786?ng?g^?1 for MK. The contents of DPMI, ADBI, AHMI and ATII are below detection limit in all samples. The results reflect current status of fragrance compound pollution in this area, and provide basic data for environmental policy making.     

Preparation and characterization of ultrasensitive and specific polyclonal antiserum against ciprofloxacin based on cationized bovine serum albumin

An ultrasensitive polyclonal antiserum against ciprofloxacin (CPFX) was developed by introducing cationized bovine serum albumin (BSA) as the carrier. Conjugates used as immunogens were synthesized using CPFX coupled to BSA cationized with ethylenediamine and hexamethylenediamine, namely CPFX-eBSA and CPFX-hBSA. The results showed that the antisera immunized with CPFX-hBSA exhibit an improved sensitivity and specificity compared with those immunized with CPFX-eBSA. Under the conditions of optimal heterologous enzyme-linked immunosorbent assay, the optimal antiserum yielded an IC₅₀ of 0.097 ng mL^?1, which proves it to be more sensitive than any CPFX antibody. Cross-reactivities with norfloxacin, enrofloxacin, and ofloxacin were < 3.0 %. No cross-reactivities with penicillin and gentamicin were found. The limit of detection was 0.01 ng mL^?1, which is far below the maximum residue level established by the EU regulation, suggesting the great potential of the presented antiserum for quantitative assays of CPFX in samples.     

Direct competitive ELISA based on a monoclonal antibody for detection of aflatoxin B1. Stabilization of ELISA kit components and application to grain samples

A direct competitive enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody has been developed and optimized for detection of aflatoxin B₁ (AFB₁), and an ELISA kit has been designed. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for aflatoxin monitoring. AFB₁ concentrations determinable by ELISA ranged from 0.1 to 10 μg L⁻¹. The IC₅₀ value was 0.62 μg L⁻¹. Recovery from spiked rice samples averaged between 94 and 113%. The effect of different reagents on the stability of HRP–AFB₁ conjugate solution was studied. The performance of a stabilized enzyme tracer in ELISA was determined and compared with that of a freshly prepared control solution of HRP–AFB₁ conjugate. The results showed that stabilizing media containing 0.02% BSA, 0.1% Kathon CG, and 0.05 mol L⁻¹ calcium chloride in 0.05 mol L⁻¹ Tris-HCl buffer (pH 7.2) maintained the activity of HRP–AFB₁ at a dilution of 1:1000 for a period of at least 12 months at room temperature whereas the reference conjugate solution without the additives lost its activity within a few days. Several additives were tested for their stabilizing effect on a monoclonal antibody (MAb) immobilized on the surface of polystyrene microtitre plates. It was shown that immobilized MAb, treated with post-coating solutions containing PVA, BSA, and combinations of these substances with trehalose, retained its activity for at least 4 months at 4°C, whereas the untreated MAb-coated plate lost its activity within 2 days.