Specific Monoclonal Antibody-Based Enzyme Immunoassay for Sensitive and Reliable Detection of <Emphasis Type="Italic">Alternaria</Emphasis> Mycotoxin Iso-Tenuazonic Acid in Food Products

In this paper, we report the development of a sensitive and specific monoclonal antibody-based immunodiagnostic method for the detection of iso-tenuazonic acid (ITeA), an Alternaria mycotoxin, in food samples. The ITeA was derivatized with hydrazine hydrate to produce the antigen (E)-3-(1-hydrazonoethyl)-4-hydroxy-5-isobutyl-1H-pyrrol-2(5H)-one (ITeAH) which was further reacted with glyoxalic acid to generate the hapten (E)-2-((Z)-(1-(4-hydroxy-5-isobutyl-2-oxo-2,5-dihydro-1H-pyrrol-3-yl)ethylidene) (ITeAHGA) which was used as an immunogen after conjugation to bovine serum albumin (BSA). A highly specific monoclonal antibody selectively binding to ITeAH was generated via the hybridoma technique and subsequently used to construct a heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) using ITeAH as the competitive antigen for the detection of ITeA with a limit of detection (LOD) of 0.5 ng/mL. Under the optimum conditions, the developed icELISA is highly sensitive (IC₅₀ = 7.8 ng/mL) with recovery rates ranged from 82.3 to 109.8% for spiked food samples. The comparative analysis of results revealed a good correlation between the icELISA and the standard HPLC-MS/MS method, confirming the suitability of the developed icELISA for screening and detection of mycotoxin ITeA in food samples.     

Development of Sandwich Double-Competitive ELISA for Sulfonamides. Comparative Analytical Characteristics and Matrix Effect Resistance

Sandwich enzyme-linked immunosorbent assay (ELISA) of sulfonamides based on double-competitive interaction between haptenized protein as a captured antigen and analyte for binding to immobilized and enzyme-labeled antibodies was developed. This experimental assay format was examined in analytical properties and matrix effect resistance in comparison with usual ones: indirect, direct antigen-coated and antibody-coated ELISAs. All four assay formats were designed on the basis of interactions between the previously prepared monoclonal antibody and immunizing hapten, 4-(4-(4-aminophenylsulfonamido) phenyl)butanoic acid, providing the uniform output optical signal for correct comparison of each assay characteristic. The sensitivity (IC₅₀) of the developed competitive sandwich assay was rather below 100 ng/ml for 11 sulfonamides which was suitable for their determination in milk, muscles and animal sera at established maximum residue limit concentration. Comparative examination did not reveal changes in assay specificity, and advantages in sensitivity, matrix effect resistance, and procedure duration before commonly used assay formats. Moreover, new design of assay was shown to be three-fold more consumable in antibody reagents in comparison with direct assay formats.     

Development of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Analysis of Diclazuril in Chicken Tissues

A highly sensitive and specific monoclonal antibody (Mab) against diclazuril was produced. The hapten with diclazuril coupled to diazotised 4-aminobenzoic acid was synthesized and conjugated to bovine serum albumin by the active ester method to form an immunogen for antibody generation. A novel diclazuril carboxymethyloxime derivative used in an ovalbumin conjugate was applied as a heterologous coating antigen and was expected to improve the immunoassay sensitivity. A sensitive and simple indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the Mab for the determination of diclazuril was developed. Under the optimized conditions, the icELISA for diclazuril showed a half maximum inhibition concentration (IC₅₀) value of 1.8 ng/mL, with limit of detection of 0.24 ng/mL and negligible cross-reactivities with other coccidiostat compounds including toltrazuril, robenidine, nicarbazin, halofuginone, amprolium, monensin, and maduramycin. The icELISA was successfully applied to diclazuril residue analysis in spiked chicken tissues. The average recoveries, intra-assay, and inter-assay coefficients of variation were in a range from 77.6 to 103.7 %, 3.7 to 13.0 %, and 5.6 to 18.3 %, respectively.     

Indirect competitive enzyme-linked immuno-sorbent assay (ELISA) for nitroimidazoles in food products

Ronidazole was used as the starting material to prepare an immunogen and coating antigen. An anti-nitroimidazole monoclonal antibody was produced and an indirect competitive ELISA was established to detect nitroimidazole compounds in food products. The IC₅₀ values were determined to be 0.20?ng/ml for metronidazole, 4.0?ng/ml for tinidazole, 0.17?ng/ml for dimetridazole and 0.24?ng/ml for ornidazole. Considering that nitroimidazoles were commonly used as veterinary drugs, nitroimidazole residues in food products of animal origin were detected by the method. The coefficient of variation for nitroimidazoles determination in contaminated chicken, chicken liver and shrimp were all <14% and the recovery rate was in the range 74.0–90.6%. The results proved that the developed method was successful in detecting nitroimidazoles in food products.     

Development of an indirect competitive immunoassay for parathion in vegetables

An indirect competitive immunoassay for the insecticide parathion has been optimized and characterized. This assay is based on a monoclonal antibody (2H₉) produced from an immunogen, a bovine thyroglobulin (BTG) conjugate wherein the reduced form of parathion was multiply bound to the carrier protein via diazo bonds. Assay was performed in the parathion-HSA coated (0.25 μg/ml) ELISA format in which antibody was diluted 1:2000. Several physicochemical factors (pH, ionic strength, BSA concentrations and organic solvent) that influence assay performance were studied and optimized. Finally, the assay was applied to the analysis of parathion in spiked vegetable samples. The sensitivity, estimated as the IC₅₀ value, was 360 ng/ml, with a practical working range between 47 and 6000 ng/ml, a limit of detection of 26 ng/ml, and inter-assay and intra-assay variations less than 10%. The average recovery of parathion added to potato, celery and Chinese cabbage were 173 ± 34%, 108 ± 15% and 98 ± 6%, respectively.     

Development of an Immunochromatographic Test Strip for Rapid Simultaneous Detection of Enrofloxacin and Ofloxacin in Tissue of Chicken Muscle and Pork

ENR and OFL are the most consumed quinolones on livestock in China. In this work, we developed a rapid immunochromatographic lateral flow test strip for simultaneous detection of the residues of enrofloxacin and ofloxacin in chicken muscle and pork. We screened an anti-ENR and OFL monoclonal antibody. The IC₅₀ of anti-ENR and anti-OFL were 6.67 and 7.13 ng/ml, respectively. The present immunochromatographic lateral flow test strip is a one-step assay and required much less professional personnel and experimental instruments. In the present study, the decision limit (CCα) of the test strip was calculated to be 0.089 ng/mL and detection capability (CCβ) was 0.217 ng/mL with the scanner. The limit of detection was estimated to be 10 ng/mL. According to parallel HPLC analysis with 47 blind samples, coincidence rate is 100 % when the contents of ENR and OFL were more than 10 ng/mL. Results indicated that the strip test we developed had good reliability, and the strip test gave neither false positive nor false negative results. It will provide results within 20 min without special equipment. Therefore, the test strip is very useful as a screening method for semi-quantitative or qualitative detection of enrofloxacin and ofloxacin in chicken muscle and pork.     

Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors

A phage display library of single chain variable fragment (scFv) antibodies was constructed to screen specific scFv antibodies against amatoxins. One recombinant scFv antibody with high specificity for amatoxins, termed scFv-A4, was isolated from the library by biopanning. SDS-PAGE analysis showed that the soluble scFv-A4 protein was expressed successfully, and the protein posed relatively good specificity with an IC₅₀ of 77.48 ng/mL and IC₁₅ of 1.91 ng/mL using indirect competitive ELISA (ic-ELISA). On the basis of the expressed scFv-A4 protein, a rapid and simple new immunoassay using surface plasmon resonance (SPR) sensor for the detection of amatoxins was developed. The IC₅₀ and IC₁₅ of new immunoassay were 7.66 and 0.17 ng/mL. The sensitivity of immunoassay using SPR sensor was about 10 times that of ic-ELISA. These results showed the potential usefulness and advantages of new immunoassay using SPR sensor for the detection of toxins.     

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for the Determination of Thiacloprid in Agricultural Samples

An enzyme-linked immunosorbent assay (ELISA) was developed based on a polyclonal antibody for the analysis of thiacloprid in agricultural samples. Thiacloprid hapten was synthesized and conjugated to bovine serum albumin to produce an immunogen and ovalbumin to produce a coating antigen. Polyclonal antibodies were obtained from immunized New Zealand white rabbits. Under optimal conditions (5 % methanol, 0.1 mol/L Na⁺, pH 5.5), the ELISA showed a 50 % inhibitory concentration (IC₅₀) value of 0.01 mg/L and a limit of detection (IC₁₀) of 0.47 μg/L. No obvious cross-reaction with the other structural analogs of neonicotinoid insecticides showed that the polyclonal antibodies had a high specificity for thiacloprid. The average recoveries from spiked water, soil, pear, and tomato were in the range of 80 to 119 %. The results of the ELISA were confirmed by high-performance liquid chromatography, and the correlation of the results from the two methods had a high correlation coefficient of 0.99 (n?=?3) in spiked samples (soil, pear, and tomato). The proposed ELISA could successfully be applied to the determination of thiacloprid residues in agricultural samples.     

Development of a sensitive monoclonal-based enzyme-linked immunosorbent assay for monitoring T-2 toxin in food and feed

The consumption of food or feed contaminated with high levels of T-2 toxin may cause adverse health effects in humans and other animals. In this study, to monitor T-2 toxin rapidly in food and feed, a sensitive and specific monoclonal antibody (mAb) against T-2 toxin was generated and a simple and rapid indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) developed. T-2 toxin was first converted to T-2-hemisuccinate (T-2HS) and T-2-hemiglutarate (T-2HG), which were then conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare an immunogen and coating antigen, respectively. After the inoculation of female Balb/c mice and cell fusions, one cell line, 4D8, with the IgG1 isotype was obtained. The 4D8 antibody exhibited the ability specifically to recognise T-2 toxin with IC₅₀ 1.46 µg l^?1. Based on this 4D8 mAb, an optimised ic-ELISA protocol was developed using only methanol–water (7:3, v/v) in feed and cereal samples and ethyl acetate in muscle samples. The limits of detection of T-2 toxin in various sample matrices varied from 0.07 to 15.8 µg kg^?1; the recoveries ranged from 50.3% to 113.6%; and the CVs were less than 19.0%. These results suggest that the prepared mAb and the developed ic-ELISA method will be a useful tool for detecting T-2 toxin in foods and feeds.     

Immunoaffinity removal and immunoassay for rhodamine B in chilli powder

A rapid and simple immunoassay and a sol–gel‐based immunoaffinity chromatography (IAC) purification method for Rhodamine B (RB) were developed using spiked chilli powder. A polyclonal antibody against RB was generated by immunisation of rabbits with an immunogen hapten. An enzyme‐linked immunosorbent assay (ELISA) was developed that achieved a 50% inhibition (IC₅₀) value of 0.94 ± 0.05 ng mL^?1. The limit of detection of the ELISA method was 1 ng g^?1 in chilli powder. For the IAC, recovery was 68.1–86.2% at 1 ng g^?1 and 72.6–89.3% at 5 ng g^?1 in spiked chilli powder. Fortified samples were analysed by high performance liquid chromatography–mass spectrometry (HPLC–MS) after IAC purification, and the results showed a good agreement between the two methods. The ELISA could be a convenient tool for screening RB residue in foods, and the IAC cleanup procedure coupled with HPLC–MS could be an effective alternative method for the determination of RB in various substances.     

Development of an Indirect Competitive ELISA for Analysis of Alternariol in Bread and Bran Samples

Alternariol (AOH) is one of the major mycotoxins produced by various species of Alternaria fungi. Natural occurrences of AOH have been reported in various foods, including fruits; processed fruit products such as apple juice, tomato products; wheat and other grains; oilseeds and products thereof, such as sunflower seeds, oilseed rape meal, and flax seed/linseed; and pecans. In this study, AOH-specific polyclonal antibodies were generated and developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for monitoring AOH in bread and bran samples. The assay was very sensitive with a limit of detection (LOD) of 2.4?±?0.6 ng/g and a half maximal inhibitory concentration (IC₅₀) of 15.2?±?2.6 ng/g in bread and a LOD of 8.4?±?1.2 ng/g and IC₅₀ of 52.8?±?10.8 ng/g in bran extract. The assay was very specific to AOH and showed no cross-reactivity to alternariol monomethyl ether, altertoxin, altenuene, tentoxin, or tenuazonic acid. The effect of organic solvents on the assay was tested. The ELISA system tolerated methanol and acetonitrile as co-solvents at up to 5% content without significant loss of IC₅₀ value. Recoveries in all cases were greater than 75%, and the results using this method were comparable to those obtained from mass spectrometry methods. We conclude that this method is suitable for rapid detection of AOH in bread and bran samples, without expensive analytical equipment or time-consuming sample preparation.     

Determination of Amaranth in Beverage by Indirect Competitive Enzyme-linked Immunosorbent Assay (ELISA) Based on Anti-amaranth Monoclonal Antibody

Amaranth (E 123) is a member of azo dyes, and it is allowed to use in various foods. The acceptable maximum addition of amaranth is strictly fixed because of its potential risk to physical health. The objective of this study was to prepare a specific anti-amaranth monoclonal antibody and develop an indirect competitive ELISA for amaranth quantification analysis. The immunogen and the coating antigen were designed by introducing a carboxyl group into amaranth for the conjugation with carrier proteins. Based on the immunogen, the monoclonal antibody exhibits satisfactory performances and the proposed ELISA shows an IC₅₀ of 20.33 ng mL^?1. The limit of detection is as low as 3.35 ng mL^?1, and the linear standard curve of the method ranges from 3.0 to 243.0 ng mL^?1. Additionally, the antibody reflects minimal cross-reactivity (<1 %) with six related food dyes (erythrosine, ponceau 4R, allura red, tartrazine, sunset yellow FCF, and brilliant blue). The recoveries of amaranth spiked beverage samples are in the range of 85.8–100.7 % with low coefficient of variation values (<11.5 %). The data shows that the developed ELISA provides a simple, sensitive, specific, and accurate alternative for amaranth determination and monitoring. Furthermore, it is the first time that icELISA of amaranth is developed based on monoclonal antibodies.     

Determination of folic acid in milk, milk powder and energy drink by an indirect immunoassay

BACKGROUND: Folic acid (FA) is essential for healthy people (reference daily intake 400 µg day^?1) and pregnant women (600 µg day^?1). Insufficient intake of FA will increase the risk of neural tube defects in newborns. In this study an indirect enzyme‐linked immunosorbent assay was developed for rapid and convenient detection of FA in vitamin‐fortified foods. RESULTS: A carbodiimide‐modified active ester method was used to synthesise the immunogen (FA–bovine serum albumin (BSA) conjugate) to raise polyclonal antibodies for FA. The coupling ratio of FA with BSA was determined to be 14:1 (molar ratio). The detection limit of the immunoassay was 3.0 ng mL^?1 in buffer, 3.52 ng mL^?1 in energy drink, 11.91 ng mL^?1 in milk and 16.50 ng mL^?1 in milk powder. Intra‐ and inter‐assay variability ranged from 6.6 to 15.1%. Analytical recoveries of FA‐spiked samples were 88.3–108.9%. CONCLUSION: The immunoassay developed in this study can be used as a simple, rapid and accurate method for fast semi‐quantitative and quantitative on‐site analysis of FA in food products. Copyright © 2012 Society of Chemical Industry     

Development of a polyclonal antibody-based indirect competitive ELISA for determination of sterigmatocystin in wheat and corn flours

Sterigmatocystin (STC) is a toxic secondary metabolite produced by more than 50 fungal species, including Aspergillus flavus, A. parasiticus, A. nidulans, and A. versicolor. The Joint FAO/WHO Expert Committee on Food Additives concluded that sterigmatocystin is genotoxic and carcinogenic with the critical effect determined to be carcinogenicity. The present study describes a simple method to prepare hapten and immunogens in order to generate polyclonal antibodies against this metabolite. We developed a sensitive and specific polyclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ciELISA) for monitoring STC in wheat and corn flours without the need for derivatisation of STC or clean-up of samples by immunoaffinity chromatography for quantification. The half inhibitory concentration (IC₅₀) of the established method was 4.52 ± 0.81 ng mL^?1, with the limit of detection (IC₁₀) being 0.19 ± 0.04 ng mL^?1 in wheat and corn flour matrices with the coefficient of variation of less than 22%.The assay was very specific to STC and showed no cross-reactivity with its analogue structures. The ELISA allowed for up to 5% methanol without significant influence on the IC₅₀ value. Validation of the assay was performed by spiking STC into a blank flour matrix and the recoveries were in the range of 75.3 % to 104.5 % with a coefficient of variation less than 15%. A small retail survey was conducted by purchasing wheat (n = 8) and corn flours (n = 2) from local grocery stores. All of these retail samples were negative for STC using the developed ELISA method and were confirmed by LC-MS/MS. We demonstrated a rapid, simple, and reliable method for screening STC in wheat and corn flours.     

A monoclonal antibody-based sensitive enzyme-linked immunosorbent assay (ELISA) for the analysis of the organophosphorous pesticides chlorpyrifos-methyl in real samples

Chlorpyrifos-methyl hapten, O-methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1), was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. Then H1–OVA conjugate was used as coating antigen, while H1–BSA conjugate was used as immunogen for producing monoclonal antibody. After optimisation, a monoclonal antibody-based effective competitive indirect enzyme-linked immunsorbent assay (ELISA) was developed and applied for determination of chlorpyrifos-methyl with a novel combination of antibody/antigen, I₅₀ of which was 75.22 ng/ml, limit detection (LD) was 0.32 ng/ml, and there was relative high cross-reactivity (CR) only with chlorpyrifos (1.4%), and CRs with other tested pesticides were all below 1% and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to real samples, including grape, Chinese cabbages, water and soil were all from 82.4% to 110.2%. Therefore, the optimised ELISA might become a convenient and satisfied analytical tool for monitoring chlorpyrifos-methyl residues in agriculture ecosystem.     

1-芘丁酸人工抗原的制备及鉴定

为制备及鉴定1-芘丁酸（1-pyrenebutyric acid）人工抗原。采用碳化二亚胺（EDC）法将1-芘丁酸与牛血清白蛋白（BSA）和鸡卵清白蛋白（OVA）进行偶联,合成人工免疫原PBA-BSA和人工检测抗原PBA-OVA;紫外扫描及SDS-聚丙烯凝胶电泳（SDS-PAGE）结果显示抗原制备成功;鼠源多抗血清的效价均已超过1 000,IC₅₀=14.31 ng/mL;与多环芳烃1-芘甲醇、1-芘甲醛、芘的交叉反应率小于14.40%,与菲、萘、苯并芘、BSA以及OVA交叉反应率均小于0.05%。作者成功制备出了PBA-BSA抗原,并且得到了敏感性良好的鼠源多抗血清,为后期单克隆抗体的制备及免疫学快速检测方法的建立奠定基础。     

An immunogen synthesis strategy for the development of specific anti-deoxynivalenol monoclonal antibodies

An immunogen synthesis strategy was designed to develop anti-deoxynivalenol (DON) monoclonal antibodies with low cross-reactivity against structurally similar trichothecenes. A total of eight different DON immunogens were synthesised, differing in the type and position of the linker on the DON molecule. After immunisation, antisera from mice immunised with different DON immunogens were checked for the presence of relevant antibodies. Then, both homologous and heterologous enzyme-linked immunosorbent assays (ELISAs) were performed for hybridoma screening. Finally, three monoclonal antibodies against DON and its analogues were generated. In addition, monoclonal antibody 13H1 could recognise DON and its analogues in the order of HT-2 toxin > 15-acetyldeoxynivalenol (15-ADON) > DON, with IC₅₀ ranging from 1.14 to 2.13 µg ml^–1. Another monoclonal antibody 10H10 manifested relatively close sensitivities to DON, 3-acetyldeoxynivalenol (3-ADON) and 15-ADON, with IC₅₀ values of 22, 15 and 34 ng ml^–1, respectively. Using an indirect ELISA format decreases the 10H10 sensitivity to 15-ADON with 92%. A third monoclonal antibody 2A9 showed to be very specific and sensitive to 3-ADON, with IC₅₀ of 0.38 ng ml^–1. Using both 2A9 and 10H10 monoclonal antibodies allows determining sole DON contamination.     

间接竞争酶联免疫法测定鱼体中的亚甲基蓝

目的 本实验旨在建立快速检测鱼体中亚甲基蓝的间接竞争酶联免疫分析方法。方法 以BSA载体蛋白,以天青C(Azure C)为半抗原,利用戊二醛法合成免疫原Azure C-BSA,免疫新西兰大白兔,制备多克隆抗体。通过对包被浓度、抗体浓度和二抗浓度等一系列实验条件的优化,建立检测鱼体中亚甲基蓝的间接竞争酶联免疫吸附方法(ic-ELISA)。结果 获得的多克隆抗体特异性强、灵敏度高,在5 -500 ng/mL范围内,线性良好,线性回归方程为y=0.3658x-0.1867 (R²=0.98),IC50为75.4 ng/mL,检测限为8.3 ng/mL,样品添加回收率为77.2%-79.3%,RSD为5.3 %-8.1 %。结论 该方法灵敏度高、特异性强,可用于鱼体中亚甲基蓝的快速检测。     

A Sensitive Immunoaffinity Column-Linked Indirect Competitive ELISA for Ochratoxin A in Cereal and Oil Products Based on a New Monoclonal Antibody

A new sensitive monoclonal antibody (mAb) 1H2 against ochratoxin A (OTA) was reported herein. This mAb belonged to the immunoglobulin G1 (k chain) isotype. In the optimized indirect competitive enzyme-linked immunosorbent assay (icELISA), 1H2 showed a 50 % inhibition concentration (IC₅₀) value of 0.058 ng/mL and a detection limit (IC₁₀) of 0.001 ng/mL. The cross-reactivity of 1H2 with ochratoxin B, aflatoxins, deoxynivalenol, zearalenone, T-2 toxin, or fumonisins was below 0.3 %. Based on this mAb, an immunoaffinity column (IAC)-linked icELISA was developed for OTA detection in the cereal and oil products. The working range of the assay for solid sample was 0.36–16 μg/kg. The recoveries from spiked samples of IAC-linked icELISA ranged from 83 to 101 %. These recoveries were much higher than those of icELISA (21–78 %) and in good agreement with those obtained by using the standard high-performance liquid chromatography method (87–110 %). The results indicated that the mAb 1H2 had the values for studies of OTA in the crude agricultural products.     

Chemical composition of essential oil and antioxidant activity of leaves and stems of Phlomis lurestanica

Essential oils from leaves and stems of Phlomis lurestanica at the flowering stage were extracted by hydrodistillation. The essential oils were analyzed using Gas Chromatography and Gas Chromatography-Mass Spectrometry. Twenty-eight and twenty-five compounds were identified in the stems and leaves, respectively. The main compounds of stems were α-pinene (12.40%), γ-cadinene (10.92%), and γ-elemene (6.46%). Hexadecane (8.97%), 2-dodecnenal (6.57%), and heptadecane (6.32%) were the major constituents in the essential oils of the leaves. The methanol extracts of stems and leaves were evaluated for their antioxidant properties using 2,2-diphenyl-1-picrylhydrazyl and β-carotene/linoleic acid tests. In the 2,2-diphenyl-1-picrylhydrazyl assay, the leaves (IC₅₀ = 1168.9 µg/ml) have stronger antioxidant activity than the stems (IC₅₀ = 1563.6 µg/ml). The extracts of the leaves and stems showed remarkable antioxidant activity in β-carotene/linoleic acid assay (96.2% and 95%, respectively). The total phenol and flavonoid contents of the species were determined using Folin-Ciocalteu and AlCl₃ assays, respectively. The phenol and flavonoid contents of the leaves (301.0 µg/ml and 45.2 µg/ml, respectively) were observed more than the stems (172.3 µg/mg and 18.8 µg/mg, respectively).