Rapid flow-through enzyme immunoassay of progesterone in whole cows' milk

Accepted Manuscript Rapid flow-through enzyme immunoassay of progesterone in whole cows' milk Jeanne V. Samsonova, Valentina A. Safronova, Alexander P. Osipov PII:

S0003-2697(18)30020-4

DOI:

10.1016/j.ab.2018.01.011

Reference:

YABIO 12902

To appear in:

Analytical Biochemistry

Received Date: 19 September 2017 Revised Date:

13 December 2017

Accepted Date: 15 January 2018

Please cite this article as: J.V. Samsonova, V.A. Safronova, A.P. Osipov, Rapid flow-through enzyme immunoassay of progesterone in whole cows' milk, Analytical Biochemistry (2018), doi: 10.1016/ j.ab.2018.01.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Rapid flow-through enzyme immunoassay of progesterone in whole cows’ milk

JeanneV. Samsonovaa,b*, Valentina A. Safronovaa, Alexander P. Osipova,b

Department of Chemical Enzymology, Chemistry Faculty, M.V.Lomonosov Moscow State

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a

University, 119991 Moscow, Russia

National University of Science and Technology “MISiS”, 119991 Moscow, Russia

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b

*Corresponding author: Dr. Jeanne V. Samsonova, Department of Chemical Enzymology,

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Chemistry Faculty, M.V.Lomonosov Moscow State University, 119991 Moscow, Russia

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E-mail: [email protected], [email protected]

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Abstract

A rapid flow-through immunoassay using an enzyme (horseradish peroxidase) as a label for quantitative and semi-quantitative determination of progesterone in whole cows` milk was developed. The flow-through test device consisted of a porous nitrocellulose membrane coated

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with antibodies and an absorbent membrane. The substrate solution containing 3,3′,5,5′ tetramethylbenzidine was used for colour visualization. The detection limit of 0.4 ng/mL P4 was obtained by this method; analysis time did not exceed 15 minutes. To eliminate matrix

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interference a simple sample preparation procedure was used. Results of analysis of whole cows’ milk samples with flow-through method were in good correlation with ELISA results (R=0.96,

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n=34). The developed rapid flow-through test system showed high efficiency for the determination of progesterone level in whole cow's milk and can be used on-site for quick identification of milk samples with low and high progesterone concentration.

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cows` milk

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Keywords: Progesterone; Flow-through enzyme immunoassay; Horseradish peroxidase; Whole

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Introduction

The method of immunofiltration (or vertical flow immunoassay or flow-through immunoassay) is one of express methods of flow solid-phase immunoassay. Simple device based on flow-through principle consists of a porous membrane where specific reagents are

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immobilized and an absorbent membrane, put together into plastic cassette. The reactants flow is carried out in vertical direction through membranes, which significantly reduces the analysis time and simplifies the assay procedure. Unbound labeled reagents are washed by buffer

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solution. On the next step visual or instrumental detection of labeled reagents bound to the support is performed and the detected signal is correlated with target analyte concentration.

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Flow-through devices generally provide qualitative or semi-quantitative assessment of the results. The most frequently used labels for flow-through immunoassay are enzymes such as horseradish peroxidase (HRP) [1], alkaline phosphatase [2] and colloidal gold [3]. Latex particles [4], quantum dots [5] and colloidal dyes (R-3) [6] are also actively used as a label. It was reported that flow-through techniques were implemented with electrochemical [2] and

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fluorescent [7] detection methods. Flow-through immunoassay was developed for a range of high and low molecular weight substances, such as human chorionic gonadotropin [8],

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insecticides [9], mycotoxins [1], sulfonamides [10], antibiotics [11] etc. This method is widely used for the detection of low molecular weight substances and can give an advantage in assay

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sensitivity compared to another express method such as dip-stick immunoassay [10]. Rapid semi-quantitative assays that could be performed outside laboratory are in increasing

demand in veterinary practice. Methods such as lateral flow immunoassay (LFIA) or flowthrough immunoassay can be used for on-site progesterone (P4) analysis in milk as a convenient tool for monitoring of ovarian activity in dairy cows. The reproductive performance of a dairy herd is one of the important factors that determine its profitability. Ideally, the calving interval should be 1 year on average and to achieve such result early detection of pregnancy (less than 90 days after insemination) should be in force [12]. On the 21st day after artificial insemination (end

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of the oestrus cycle) low P4 level in milk sample (up to 3-4 ng/mL) indicates cows’ nonpregnancy. P4 concentration in milk of possibly pregnant cows or animals with some disorders will be around 7-10 ng/mL and higher. Among traditional methods of P4 determination in milk the highly sensitive method of enzyme-linked immunosorbent assay (ELISA) is widely spread

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[13]. However, ELISA is multistage complex process and cannot be conducted under nonlaboratory conditions. In order to screen whole milk samples on farm a portable rapid field test is required [14].

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Lateral flow immunoassay is one of the methods widely used for rapid screening purposes. The first attempt to develop rapid test (LFIA) for P4 determination in cows’ milk was made in

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1996 [15]. However, use of colloidal gold or graphite labels did not help to achieve the required sensitivity and detect P4 concentrations less than 7 ng/mL in real samples and so the developed tests had no practical importance [15,16]. Recently we developed pretreatment-free lateral flow enzyme immunoassay of P4 in whole cows’ milk samples [17]. Utilizing of highly sensitive enzymatic label, horseradish peroxidase, allowed to develop rapid sensitive immunoassay. Limit

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of detection of P4 in whole cows’ milk was 0.8 ng/mL. In terms of use of an enzyme label flowthrough immunoassay is a convenient rapid method where unbound reagents are washed to

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prevent the development of high background signal and unspecific interactions. The main focus of our work was to develop a new express and sensitive flow-through enzyme immunoassay

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system with simple sample preparation for semi-quantitative and quantitative analysis of P4 in whole cows` milk in physiologically important concentration range (0-10 ng/mL).

Materials and methods P4 was purchased from «Sigma» (USA). Inorganic salts, acids and organic solvents were

obtained from Chimmed, Russia. Tween 20 was from Amresco, USA. Ready-to-use TMB substrate solution for membranes (Seramun Blau Prec) was supplied by Seramun Diagnostica GmbH, Germany.

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The following buffers were used: 0.01 M K-phosphate (K2HPO4-KH2PO4), 0.15 M NaCl, pH 7.4 (PBS) and 0.01 M K-phosphate (K2HPO4-KH2PO4), 0.15 M NaCl, 0.05% Tween 20, pH 7.4 (PBST). P4 standard solutions were prepared by dilution of the stock solution (1 mg/mL in ethanol)

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with PBST. Anti-P4 polyclonal rabbit antiserum and P4-HRP conjugate used in the study were described elsewhere [18].

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Analytical nitrocellulose membranes for immunofiltration (CLW-040-SH34 with pore size 0.45 and 0.8 µm, CNJ-X1 with pore size 0.45 µm) and absorbent pad AP080 were used to

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arrange plastic flow-through test cassette (FT-12, dimension - 31x41 mm, inner Ø - 12.6 mm; all MDI, India).

Sample preparation

Whole cows’ milk samples were kindly provided by NPO Nikulino (Moscow region) and

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and vortexed.

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stored for 18-24 hours at 4oC. Before analysis milk samples were incubated for 30 min at 37˚C

Flow-through assay: preparation of analytical membrane

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Analytical nitrocellulose membrane was cut into 2x2 cm pieces. Solution of P4 specific antibodies (20 µg/mL) was applied onto sample spot and anti-HRP antibodies (50 µg/mL) were used to form a control spot (0.5 µL each). The membranes were dried at 37°C for 30 min and then blocked with 1% casein in PBS for another 30 min. Finally, the blocked membranes were dried at 37°C for 45 min and vacuum-packaged in a plastic bag with silica as a moisture adsorbent.

Flow-through enzyme immunoassay procedure

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The flow-through test cassette was assembled of two plastic covers (upper and lower) with analytical porous membrane (2*2 cm) positioned against the round hole of in the center of upper plastic cover and an absorbent layer inside. Sample (whole milk) was diluted 4-fold with PBST supplied with 10% methanol. The mixture of P4 standard solution/sample (100 µL) and

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P4-HRP conjugate (10 µL, working dilution 1:10000) was applied onto the analytical membrane. After solution passed through, the membrane was washed with 100 µL PBST. Finally, assay results were developed with 400 µL of TMB substrate solution. After 5 minutes, instrumental

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and visual signal registration was performed. Scion Image program was used for the analysis of digital images of test and control spots. Spot intensity was corrected by background subtraction

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and was converted to % B/B0 values according to the formula: B/B0 = (I/I0) * 100%, where I is a value of spot intensity for a sample, I0 is the value of spot intensity for 'zero' standard.

ELISA procedure

“ELISA-progesterone-milk” kit (Immunoved, Russia) was used for the determination of P4

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level in whole cows’ milk samples. Aliquots of 10 µl P4 standard solutions/samples were added to wells of a microtiter plate followed by 100 µl enzyme tracer (P4-HRP). After incubation and

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plate washing the substrate solution (100 µl) was added to each well. The colour reaction was stopped after 10-15 min with 100 µl stop solution and the result was evaluated on

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spectrophotometer at 450-620 nm (Anthos 2010, Austria).

Results and discussion Flow-through immunoassay is one of express methods of flow solid-phase immunoassay.

Liquid stream comprising an analyzed sample and reaction components passes through the membrane in transverse direction (perpendicular to the membrane layer). In this work a competitive immunoassay based on antibody-coated format was used with HRP as an enzyme label (Fig. 1). Antibodies specific to P4 (test spot) and specific to HRP (control spot) were

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immobilized on the analytical membrane in two circular zones. To reduce background signal complicating instrumental or visual assessment of the result the analytical membrane was treated with 1% casein after antibodies immobilization step. It was necessary to develop sensitive assay which allows quick identification of cows’ milk samples with high and low P4 concentration. So,

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ideally express flow-through testing of milk sample with low P4 concentration (up to 3-4 ng/mL) should give two bright colour spots in analytical zone (test spot and control spot) whereas under analysis of milk sample with high P4 concentration (around 7-10 ng/mL) only control spot

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should be visible.

For flow-through immunoassay special porous membranes of different chemical nature such

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as nitrocellulose, nylon, glass fiber, etc are usually used as an analytical membrane [19]. In our work a few nitrocellulose membranes with pore size 0.8 and 0.45 µm were utilized. Among them the analytical nitrocellulose membrane with pore size 0.45 µm, which was produced in conjunction with an absorbent membrane, had the best characteristics in terms of flow speed and background signal. Apparently, due to membrane tight fitting to the absorbent layer the

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continuous and steady flow of reactants was provided and as a result the reduced analysis time was achieved. At the same time for this membrane good assay sensitivity was obtained and the

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difference between calibration points corresponded to important physiological P4 concentrations of 3 and 10 ng/mL was the highest (Fig. 2).

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In order to develop quantitative flow-through immunoassay first of all it was necessary to choose reaction conditions under which the colour intensity of the reaction zone (spot) corresponded to amount of enzyme label within conjugate and consequently the measured concentration of P4 in the reaction mixture. The dependence of the test spot intensity on time for different concentrations of P4-HRP conjugate was studied (Fig. 3). For this HRP conjugate solution was applied onto analytical membrane then dried. On the next step TMB (100 µL) was passed through membrane and the developed signal was evaluated by scanner for 60 min. It could be seen that the detected signal was proportional to the amount of the conjugate (enzyme)

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on membrane in the range of investigated concentrations (0 to 250 ng/mL) (Fig. 3B). Analytical signal obtained after staining with TMB substrate solution was stable for 60 min (Fig. 4A). So, the registration of test results could be carried out in 5 minutes after membrane staining or within an hour after staining as the proportionality of results was retained. When 100 µL TMB solution

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was used the gradual colour development in test zone in the form of saturation curve was observed with the spot intensity increasing in about 25% within 30 minutes (Fig. 3A). It was found that 4-fold increase of TMB volume resulted in more stable analytical signal and less

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variable data (Fig. 4B). Application of lager substrate volume increased the colour development of the test and control zones (spots). Therefore, larger substrate volume provided better assay

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conditions when visual as well as instrumental detection of assay results can be performed shortly after staining.

In flow-through immunoassay all reagents are applied onto the test cassette window in particular order, so for this assay appropriate washing step is necessary. In our case the washing step reduced the background signal by removal of unbound enzyme conjugate (P4-HRP). Also,

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under analysis of real samples, washing of the membrane allowed to remove sample components from membrane surface and eliminate matrix effect that could influence further analysis and

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result visualisation. Since the formation of antigen-antibody complex is a reversible process an optimal washing solution and its volume should be chosen. Thus in our work the washing of

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analytical membrane with PBST of 500 µL and larger volume led to the dissociation of antigen antibody complex (Fig. 5A). The effect was not observed while membrane was washed with distilled water or PBS in volume up to 2.5 mL. At the same time the increasing of detergent (Tween 20) content in PBS led to dissociation of antigen-antibody complex when excessive amount of washing solution (~ 2.5 mL) was used (Fig. 5B). As an optimal PBST (100 µL) was chosen as washing solution. As a result of optimization steps a rapid flow-through enzyme immunoassay of P4 was developed. The detection limit of 0.4 ng/mL P4 was obtained by this method; analysis time did

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not exceed 15 minutes. The resulting calibration curve and the visualization of test/control spots at different concentrations are presented on Fig. 6. It can be seen that test spot actually is disappearing at P4 concentration around 7-10 ng/mL so the developed flow-through enzyme immunoassay allows rapid visual identification of samples with high hormone concentration that

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indicates possible cows’ pregnancy. On the next step assay conditions for P4 detection in whole cows` milk were optimized. Flow-through analysis of whole milk samples revealed the problem of slow sample flow rate

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through membrane (about 100 µL per hour). Simultaneously fat film was concentrated on a surface of analytical membrane during sample penetration through it. So, express format of

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analysis was not possible in this case. Whole milk is an emulsion with fat globules ranged from 0.5 to 10 µm. So, to increase sample flow rate a range of sample dilutions was used. It was important to keep the required range of detectable P4 concentrations and assay sensitivity in combination with proper sample flow rate through a membrane to provide a rapid and sensitive test. The optimal dilution of whole milk sample was found to be 4-fold, which increased a

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sample flow rate. At the same time P4 concentration in whole milk samples recovered by flowthrow enzyme immunoassay was lower than that obtained by ELISA, due to poor sensitivity in

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real samples. It is known that about 80% of P4 is contained in milk fat fraction [20]. So, for P4 extraction from milk fat organic solvents such as methanol, acetonitrile etc. were often used [20].

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In our work, the assay sensitivity was increased when methanol was added to the sample dilution buffer. It was found that the presence of methanol in working buffer in concentration up to 10% has no significant effect on assay performance whereas higher percentage of methanol led to sharp decrease of analytical signal, for instance, the test spot intensity decreased for 13, 67 and 96% at methanol concentration 20, 30 and 80%, correspondingly. So the further analysis of whole milk samples was carried out in the presence of the solvent in the sample dilution buffer. It could be assumed that methanol increased the availability of free P4 for specific interaction with an antibody in milk aqueous phase. So, for whole milk sample pretreatment in flow-through

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enzyme immunoassay 4-fold sample dilution with methanol containing buffer (10%) was used. This step provided assay sensitivity in necessary P4 concentration range (0.5-10 ng/mL) and short time of analysis. Thirty eight whole cows’ milk samples were investigated by the developed flow-through

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enzyme immunoassay and results were evaluated visually and instrumentally. Milk samples were collected both from pregnant and non-pregnant cows at different phases of oestrous cycle. Whole milk samples were analysed in laboratory conditions after being transported from a farm and

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stored at 4oC for 12-24 h. So, at cool conditions lipid layer was formed on the top of each sample. To homogenize milk before analysis each sample was warmed up and vortexed. Thus

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for milk samples with high P4 concentration (pregnant cows and non-pregnant cows in the middle of oestrus cycle) one blue spot was observed on the analytical membrane whereas for milk samples with low P4 concentration (non-pregnant cows in the beginning or end of estrous cycle) two blue spots in test zone were indicated (Fig. 7B). The analysis revealed 39.5% samples with high P4 concentration (more than 7 ng/mL) and 60.5% with low. The effectiveness of the

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developed rapid test for the quantitative determination of P4 in whole cows` milk was confirmed by comparison with highly sensitive ELISA (Fig. 7A). Four samples with high P4 concentration

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beyond detectable range of flow-through enzyme immunoassay were not used for correlation data. The data obtained were in good correlation (R=0.96). Therefore, the developed flow-

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through test can be used not only for visual semi-quantitative evaluation of P4 concentration in whole milk but also for quantitative detection of milk P4. The method includes a few simple steps and is more sensitive than developed earlier whole milk P4 lateral-flow enzyme immunoassay [17]. So, for on-site P4 level monitoring ready-to-use test device along with a set of few solutions is provided. Whole milk sample taken directly from a cow should be diluted with special solvent containing buffer, mixed with enzyme tracer then applied onto membrane of flow-through device. After membrane washing the substrate colouring step followed by result assessment is performed. In total assay procedure can be completed in 10-15 min.

ACCEPTED MANUSCRIPT Conclusions The developed method of flow-through enzyme immunoassay allows more accurate visual interpretation of results because of narrow detectable range of P4 concentrations (0.5-10

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ng/mL). Whole milk P4 concentration around 7-10 ng/mL leads to complete disappearance of test spot. Therefore, the use of this method is preferred for semi-quantitative analysis with "yesno" answer. The developed test system can help rapid evaluation of P4 level in cows` milk to

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determine possible cows’ pregnancy and some disorders of cows’ reproductive system. Express method of flow-through enzyme immunoassay includes several simple steps and can be

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performed directly on dairy farms. Analysis of whole milk samples using the developed test does not require special equipment as well as time-consuming, sophisticated sample pretreatment and first of all can be used for rapid identification of whole milk samples with high and low P4 concentration.

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Conflict of interest statement

None of the authors of this paper has a financial or personal relationship with other

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people or organizations that could inappropriately influence or bias the content of the paper.

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Acknowledgements

The work was financially supported by the Ministry of Education and Science of the

Russian Federation in the framework of increase Competitiveness Program of NUST “MISIS”, implemented by a governmental decree dated 16th of March 2013, No. 211 and as a based part of state assignment Organization of scientific researches (project No. 16.6548.2017/BY).

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Figure captions

Fig 1. General scheme of P4 flow-through enzyme immunoassay.

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Fig. 2. The dependence of the test spot intensity on P4 concentration in flow-through enzyme immunoassay for different analytical membranes (1- membrane CNJ-X1, 2 – membrane CLW040-SH34, pore size 0.45 µm, 3 – membrane CLW-040-SH34, pore size 0.8 µm).

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I, RU – colour intensity of the test zone, relative units.

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Fig. 3. The dependence of the spot intensity on measurement time (A) and the P4-HRP conjugate (HRP) concentration on the membrane surface (B) after staining in TMB solution. I, RU – colour intensity of the zone with immobilised P4-HRP conjugate, relative units.

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Fig. 4. The dependence of the spot intensity on measurement time for different volumes of TMB: 100 µL (A), 400 µL (B).

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I, RU – colour intensity of the test zone, relative units.

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Fig. 5. The dependence of PBST volume (A) and Tween 20 concentration in washing buffer (B) on the test spot intensity. I, RU – colour intensity of the test zone, relative units.

Fig. 6. P4 flow-through enzyme immunoassay: calibration curve (A) and its visualization after substrate staining (B).

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Fig. 7. Correlation of the results of P4 detection in whole cows’ milk obtained by ELISA and flow-through enzyme immunoassay and the example of test visualization for non-pregnant and

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pregnant cow.

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