ISSN 00036838, Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 6, pp. 606–612. © Pleiades Publishing, Inc., 2013. Original Russian Text © N.A. Byzova, L.N. Lukhverchik, A.V. Zherdev, N.V. Piven, A.I. Burakovskii, B.B. Dzantiev, 2013, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2013, Vol. 49, No. 6, pp. 606–612.
Development of an Immunochromatographic Test System for the Detection of Human Epidermal Growth Factor N. A. Byzovaa, L. N. Lukhverchikb, A. V. Zherdeva, N. V. Pivenb, A. I. Burakovskiib, and B. B. Dzantieva a
A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, Moscow, 119071 Russia of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich str. 5/2, Minsk, 220141 Belarus email:
[email protected];
[email protected]net.by
b Institute
Received April 15, 2013
Abstract—A method was developed for the rapid detection of human epidermal growth factor based on a sandwichformat immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid flow movement across the membrane components of the test strip, immunochemical reactions, and the formation of colored lines. Requirements on the configuration of the test system in order to achieve the lowest limit of detection were defined in the course of the development of the assay. It was shown that this method enables the detection of human epidermal growth factor within 5 min at concentrations as low as 10 pg/mL in aqueous solutions, urine, and the blood serum and plasma. The developed test system can be used for pointofcare diagnostics. DOI: 10.1134/S0003683813060033
Epidermal growth factor (EGF) is a singlechain globular protein with MW 6.4 kDa consisting of 53 amino acids [1]. This protein is synthesized pre dominantly in salivary glands [2] and is present in all main biological body fluids, such as urine (where it was described as urogastrone), blood, cerospinal fluid, mammary fluid, saliva, gastric juice, and pancreatic juice [3]. Epidermal growth factor stimulates the pro liferation of epidermal and epithelial cells (including fibroplasts, kidney epithelial cells, glial cells, ovarian granulosa cells, and thyroid cells) [4], as well as of embryonic cells, thus enhancing the release of calcium from bone tissue and promoting bone resorption. Epi dermal growth factor substantially contributes to wound healing and angiogenesis and plays an impor tant role in cancerogenesis by inducing, in particular, the expression of cfos and cmyc protooncogenes [5]. A high level of EGF in blood and urine is charac teristic of some cancer diseases [6, 7]. It was shown that the EGF blockade by antibodies inhibits the biding of EGF to the specific receptor, thus retarding tumor growth. This effect is used in the design of antitumor vaccines [8–11]. For instance, the CimaVaxEGF vaccine proved its efficiency in the treatment of nonsmallcell lung cancer. It was found that its therapeutic effect depends on the level of EGF in blood of patients [11]. In medical practice, the concentration of EGF is nowadays determined by Immunochemical methods [12]. Immunoassay microplates commercially avail able from RnD Systems, eBioscience, RayBio, Invit rogen, Thermo Scientific, and Abnova are suitable for the detection of natural and recombinant EGF in cell
culture media, urine, and the blood serum and plasma at concentrations from 1 to 1000 pg/mL. However, due to diffusion limitations, the enzyme linked immunosorbent assay (ELISA) requires the prolonged incubation of reagents [13] and, as a conse quence, the duration of the assay varies from 2 to 4 h. In the past years, conventional formats, such as ELISA, of the immunochemical analysis are actively replaced by rapid diagnostic tests, such as immun osensors, immunochromatographic tests, etc., which require substantially shorter times (5–15 min) for the detection of the tested substances and are less labori ous. In the series of these methods, an immunochro matographic assay (ICA) is of particular interest since it is the most methodologically simple one. All reagents necessary for the assay are immobilized on a test strip (a multimembrane composite). After the contact of the test strip with a fluid sample, the flow movement of the adsorbed fluid across the test strip occurs simultaneously with immunochemical reac tions and the formation of colored lines in particular regions of the test strip, which is indicative of the pres ence (in some cases, of the content) of the tested com pound in the sample [13]. Therefore, ICA is an opera torindependent technique and requires no devices and reagents as supplements to the test strip, due to which this technique is suitable for pointofcare diag nostics and the results can be detected either visually or with the use of portable detection devices, from mobile phone video cameras or office scanners [14] to video digital recorders [15]. Immunochromatographic test systems for the detection of EGF are not described in the scientific
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literature and are not available in the market as com mercial products. In view of the practical significance of the control of the EGF content for the cancer diag nostics and therapy, the aim of the present study was to develop an immunochromatographic test system for the detection of EGF in aqueous solutions, urine, and the blood serum and plasma. For this purpose, we have to solve a number of methodological problems associ ated with the characterization of the interactions of immunoreagents in a crossflow membrane system and the optimization of the conditions for performing immunochromatography. In the present work, we report the result obtained when solving these problems and the characteristics of the developed test system. METHODS Reagents. Recombinant human epidermal growth factor was used. The following chemicals were also used: mouse monoclonal antibodies (MAbs), clone 10827, and goat polyclonal antibodies (PAbs), AB 236NA, antiEGF antibodies (RnD Systems, USA); goat (GAMIss), rabbit (RAMIss), and sheep (SAMIss) antimouse immunoglobulin (Ig) antibod ies, rabbit antigoat Ig antibodies (RAGIss) (Imtek, Moscow, Russia); goat antimouse Ig antibodies (GAMI) and donkey antigoat Ig antibodies (DAGI) (Arista Biologicals, USA); Tris, Triton X100, sodium citrate, sodium azide, hydrochloroauric acid (Sigma, USA); Tween20, bovine serum albumin (BSA) (MP Biomedicals, UK). All auxiliary reagents (salts, acids, alkalis, organic solvents) were of analytical or chemi cal purity. Solutions for the preparation of colloidal gold (CG) and its conjugates were made with the use of deionized water (the resistivity was not higher than 18.2 M cm at 25°С, Simplicity system, Millipore, USA). Immunochromatographic test strips were prepared with the use of mdi Easypack developer’s kits (Advanced Microdevices, India) consisting of a work ing membrane supported on a solid substrate CNPF SN12 with a pore size of 10 µm, a glassfiber pad PT R7 for a colloidal conjugate, a sample pad FR1, and a final adsorbent pad AP045. Preparation of CG by the nitrate method [16]. A 1% hydrochloroauric acid solution (1.0 mL) was added to deionized water (97.5 mL), the reaction mixture was brought to boiling, and then a 1% sodium citrate solu tion (1.5 mL) was added with stirring. The reaction mixture was refluxed for 25 min, cooled, and stored at 4–6°С. Transmission electron microscopy. Colloidal gold preparations were mounted on 300 mesh grids (Pelco International, USA) coated with a support film of polyvinyl formal dissolved in chloroform. Images were obtained with a CX100 electron microscope (Jeol, Japan) under accelerating voltage of 80 kV and an APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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enlargement of 3 300 000 and were analyzed in the dig ital form with the use of the Image Tool software. Synthesis of antibodycolloidal gold conjugates. The preliminary characterization of the conjugation of antibodies to CG was performed according to recom mendations [17]. A CG solution with D520 = 1.0 (1.0 mL) was added to a solution of antibodies (MAbs or PAbs) in water (0.1 mL), the concentration being var ied from 5 to 250 µg/mL. The mixture was stirred and incubated at room temperature for 10 min. Then a 10% sodium chloride solution (0.1 mL) was added and the mixture was stirred for 10 min, after which D580 was measured. The dependence of D580 on the concentra tion of antibodies (a flocculation curve) reflects the aggregation of the conjugate at a high ionic strength of the solution. The antibody to CG ratios were chosen based on the concentration dependences using anti bodies at a concentration 10–15% higher than the point, at which D580 reaches the plateau, as recom mended in [17]. Before the conjugation to CG, the antibodies were dialyzed against a 1000fold volume of 10 mM Tris HCl buffer, ðÍ 9.0, at 4°C for 2 h. Then 0.1 Ì potassium carbonate was added to the CG solution (D520 =1.0) until pH 9.0 followed by the addition of CG to a solu tion of antibodies at a certain concentration. The mix ture was incubated with stirring at room temperature for 30 min, and then an aqueous solution of BSA was added to the final concentration of 0.25%. The colloi dal gold particleimmobilized antibodies were sepa rated from the unreacted antibodies by centrifugation at 8 000 g for 30 min. After the removal of the superna tant, the precipitate was resuspended in 10 mM Tris HCl buffer, ðÍ 9.0, containing 0.25% BSA, 0.05% Tween 20, and 0.05% sodium azide. The conjugate preparations were stored at 4–6°С. Preparation of immunochromatographic test sys tems. Reagents were immobilized on membranes involved in the test system with the use of an IsoFlow automated dispenser (Imagene Technology, USA). The conjugates of CG with MAbs (MAbs–CG) and PAbs (PAbs–CG) were applied on a glassfiber pad at a dilution corresponding to D520 = 6.0 (16 µL per cen timeter of the glassfiber pad width). The test zone was formed with the use of MAbs or PAbs (0.5 mg/mL in 50 mM potassium phosphate buffer, ðÍ 7.4, containing 0.1 M sodium chloride, 0.5% BSA, and 0.1% sodium azide, PBSBA); the control zone, with the use of the GAMI, GAMIss, RAMIss, and SAMIss antibodies (0.5 mg/mL in PBSBA) for the MAbs–CG conjugate or the RAGIss and DAGI antibodies (0.3 mg/mL in PBSBA) for the PAbs–CG conjugate. Then 2.0 µL of the solution was applied per centimeter of the working pad width. The glassfiber and working pads thus pre pared were dried in air at 20–22°C for at least 20 h. A multimembrane composite was assembled from these two pads, the sample pad and the final adsorbent pad, and was then cut into 3.5mmwide test strips with an Index Cutter1 automated guillotine cutter (APoint
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Fig. 1. Dependence of the color intensity of the test line (arb. units) on the concentration of EGF (ng/mL) for four types of immunochromatographic test systems: either PAbs (curves 1 and 3) or MAbs (curves 3 and 4) are immo bilized on the test zone; either MAbs (curves 1 and 4) or PAbs (curves 2 and 3) are conjugated to colloidal gold.
Technologies, USA). These test strips were hermeti cally packed in laminated aluminum foil bags contain ing silica gel as the desiccant with the use of a FR900 miniconveyor (Wenzhou dingli packing machinery, China). The splitting and packing were carried out at 20–22°C in a special room with a relative humidity under 30%. The packed test strips were stored at room temperature. Immunochromatographic assay and recording of its results. The assays were carried out at room tempera ture. The bag was opened, the test strip was taken out, its lower end was vertically submerged into an aliquot of the sample (50 µL) for 1 min, and then the test strip was placed on a horizontal surface. The results were controlled 5 min after the beginning of the assay by taking a digital image of the test strip with CanoScan LiDE 90 scanner (Canon) and calculating the color intensity of the lines on the pad with the use of the TotalLab software as was described in [18] or with the use of a Reflecom video digital recorder (OctaMedica, Russia) [15]. RESULTS AND DISCUSSION Preparation and characterization of reagents used for immunochromatography. Taking into account the size of the EGF molecule, it would be expected that it contains several nonoverlapping antigenbinding sites. Due to this fact, it becomes possible to use the twosite sandwich immunoassay for the detection of
EGF based on the formation of the complex (anti EGF antibodies immobilized on the test zone) : (EGF in the sample) : (antiEGF antibodies conjugated to CG) in the course of the assay. To perform this assay, we synthesized the preparation of CG and specific antibodyCG conjugates. The sizes and homogeneity of CG particles synthe sized by the citrate method were determined by trans mission electron microscopy. It was shown that the synthesized CG preparation does not contain aggre gates and is characterized by the average particle diameter of 34 nm (based on the results of 90 measure ments). The average maximum value of the axis of the particles in the images was 37 ± 8 nm; the minimum axis was 30 ± 5 nm. Based on the flocullation curves, the MAbs–CG and PAbs–CG conjugates were synthesized using antibodies at concentrations of 12 (MAbs) and 9 (PAbs) µg/mL. For these conditions of the conjugate synthesis, the antibody : CG molar ratio in the reac tion mixture was 380 : 1 for MAbs and 290 : 1 for PAbs, which is excessive relative to the ratio achieved in the case of the singlelayer immobilization [19]. An excess of the unreacted antibodies was removed together with the supernatant using the precipitation of the conju gates by centrifugation. Selection of the completion of the immunochro matographic test system. Four types of immunochro matographic test systems were manufactured using two preparations of antibodies specific to EGF (Fig. 1). In the first and third types of test systems, PAbs were immobilized on the test zone; MAbs were used for the immobilization in the second and fourth types of test systems. In the first and fourth types of test systems, MAbs were conjugated to CG. In the second and third types of test systems, PAbs were used for the conjuga tion to CG. The dependence of the color intensity of the test line on the concentration of EGF in the sam ple was measured for each configuration of the test system. As can be seen from Fig. 1, the first and second types of test systems enable the detection of EGF at concentrations as low as 10 pg/mL; the third test sys tem, at concentrations down to 30 pg/mL; the fourth test system, at concentrations as low as 1 ng/mL. The maximum color intensity of the test line for the first test system is 1.7, 4, and 12 times higher than that for the second, third, and fourth test systems, respectively. Therefore, the first test system is preferable. In this test system, PAbs are immobilized on the test zone, and MAbs are conjugated to CG. Optimization of the selected test system. To provide the best reliability of the results of testing and to detect EGF at minimum concentrations, we optimized the test system, including the choice of the type and con centration of the reagents in the control, test, and starting zones of the test strip. Based on the results of our previous studies [18, 19], the reagents were immobilized on the control and test zones from a volume of 2.0 µL per centimeter of
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Fig. 2. Dependences of the color intensity of the test line (arb. units) of the immunochromatographic test system (arb. units) on the concentration of EGF (ng/mL) obtained for the immobilization of PAbs from solutions with concentrations of 0.5, 0.25, and 0.125 mg/mL (curves 1–3, respectively).
Fig. 3. Dependence of the color intensity of the test line (arb. units) on the concentration of EGF (ng/mL) for the detection of EGF in PBS using the immunochromato graphic test system, which was prepared according to the optimized procedure.
the pad width, which provides the uniform immobili zation and, correspondingly, the minimum discrep ancy between the test strips prepared in one series with respect to the degree of binding of the colored marker in the course of the assay. The MAbs–CG conjugate was immobilized from a solution with D520 = 6.0 at a rate of 16.0 µL per centi meter of the glassfiber pad width. This provided the formation of intensely colored lines in the course of the assay combined with the completeness of washing out of the reagent from the starting zone and the absence of nonspecific staining of the working pad. The binding of the MAbs–CG conjugate in the immunochromatography was compared for a series of preparations of goat antimouse Ig antibodies immo bilized on the control zone. At saturating concentra tions of the reagents (see the Methods), the color intensity of the control line was as follows: for goat antibodies, 80 arb. units (GAMIss) and 87 arb. units (GAMI); for rabbit antibodies (RAMIss), 51 arb. units; for sheep antibodies (SAMIss), 73 arb. units. Goat antibodies GAMI (Arista Biologicals) were taken for further use in assays because they provide the best binding of the colored marker. For the formation of the test zone, the amount of immobilized PAbs was varied. Figure 2 shows the ICA calibration curves for EGF obtained for the immobili zation of PAbs on the test zone from solutions with concentrations of 0.5, 0.25, and 0.125 mg/mL. It was
found that at 0.5 and 0.25 mg/mL concentrations of immobilized PAbs, the limit of detection of EGF is 10 pg/mL; at a 0.125 mg/mL concentration, the limit of detection is 100 pg/mL. At the 16 ng/mL concen tration of EGF in the sample, the color intensity of the test line is 120, 93, and 72 arb. units for the concentra tions of PAbs equal to 0.5, 0.25, and 0.125 mg/mL, respectively. Based on these data and for the purpose of achieving high reliability of the diagnostics, we further employed the procedure for the preparation of test sys tems involving the immobilization of PAbs on the test zone from a solution with a concentration of 0.5 mg/mL. Characteristics of the developed immunochromato graphic test system. Based on the results of the optimi zation, we prepared test strips and analyzed their use for the detection of EGF in standard solutions, urine, and the blood serum and plasma. Figure 3 presents the results of testing of samples with different concentrations of EGF in a buffer (50 mM potassium phosphate buffer, pH 7.4, 0.1 MNaCl; PBS). The limit of detection of EGF is 10 g/mL. The accuracy of the detection (rms deviation of the recorded color intensity) is 5–7%. Then we assessed the detection of EGF in the blood serum and plasma. As can be seen from Figs. 4 and 5, where the result of testing and the concentra tion dependences are presented, the limit of detection of EGF in such samples is also 10 pg/mL. The absence
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Fig. 4. Immunochromatographic detection of EGF in the blood serum: à, a view of test strips after their use in the assay (I, the control zone; II, the test zone); 1–9, the concentrations of EGF are 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 ng/mL; b, the depen dence of the color intensity of the test line (arb. units) on the concentration of EGF (ng/mL).
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Fig. 5. Immunochromatographic detection of EGF in the blood plasma: à, a view of test strips after their use in the assay (I, the control zone; II, the test zone); 1–9, the concentrations of EGF are 0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 30 ng/mL; b, the depen dence of the color intensity of the test line (arb. units) on the concentration of EGF (ng/mL).
of the adverse effect of the matrix on the results of test ing eliminates the necessity of the additional dilution of the samples and simplifies the assay procedure. The accuracy of the detection in the testing of plasma and serum samples is 3–7% and 10%, respectively. Let us note that the color intensity monotonously depends on the concentration of EGF throughout the diagnos tically significant concentration range down to 10 ng/mL (a slight decrease is observed only for the detection of EGF at concentrations higher than 10 ng/mL in serum samples). Therefore, the selected concentrations of immunoreagents exclude the so
called hook effect—a decrease in the signal at high concentrations of antigens [20]—and enable the reli able quantitative detection of EGF. The binding of the conjugate to the reagent immobilized on the test line does not interfere with the quality control of the test system based on the interaction between the unreacted conjugate and antispecies antibodies in the control line. In the case of both the blood serum and plasma, the coloration of the control line is reliably detected over the whole concentration range of EGF. To preliminarily assess the possibilities of the detection of EGF in urine, a dilution series of the bio
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Fig. 6. Immunochromatographic detection of EGF in urine. Sample 1 corresponds to the undiluted sample; samples 2–8 correspond to 2, 4, 8, 16, 32, 64, and 128fold dilutions, respectively, of urine in PBS; samples 9 and 10 correspond to undiluted urine with the addition of EGF at concentrations of 10 (9) and 20 (10) ng/mL.
sample (bars 2–8 in Fig. 6) was characterized and the results of the addition of standard EGF to the undi luted urine sample (bars 9 and 10 in Fig. 6) were ana lyzed. The correlation between the color intensity and the EGF content (endogenous or added) confirms the possibility of the detection of EGF in this matrix. Since the sample matrix potentially can influence the flow rate of the fluid front and immunoreagents across the test strip, we experimentally studied the dynamics of the coloration of the test and control lines for serum samples. As can be seen from Fig. 7, 3 min after the beginning of the movement of the fluid front, the color intensity of the control and test lines is 81 and 48% respectively, of the maximum value; after 5 min, 93 and 78%; after 7 min, 97 and 88%, respec tively. The formation of the detected complexes is not slowed down under the influence of the sample matrix and occurs at the same rate as that observed for stan dard EGF solutions, which enables the very rapid test ing. Based on these results, the recommended dura tion of the detection of EGF with the use of the devel oped test system is 5 min. The above results show that the developed system can be used for the detection of EGF over the physio logical concentration range (down to 10 pg/mL). The EGF content can be quantitatively determined by measuring the color intensity of the test line. The duration of the assay is 5 min. It should be noted that the testing of biosamples (plasma, serum, urine) does not require the preliminary sample preparation. A nontrivial result is that the developed test system is ver APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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Fig. 7. Dynamics of the coloration of the control (1) and test (2) zones in the immunochromatographic test system for the detection of EGF during testing of the blood serum con taining EGF at a concentration of 1 ng/mL. One arbitrary unit of the color intensity measured with a Reflecom ana lyzer corresponds to 35 arb. units obtained with a scanner.
satile. Thus, the same configuration of the test system proved to be suitable for the testing of both urine and blood samples. The newly developed immunochro matographic system is a promising analytical tool in the diagnosis of cancer and the design and control of therapeutic measures. The work was supported by the Federal Target Pro grams “Scientific and ScientificPedagogical Person nel of Innovative Russia for 2009–2013” (Govern ment Contract 14.740.11.1065, May 24, 2011). We are grateful to S.M. Pridvorova (A.N. Bañh Institute of Biochemistry, Russian Academy of Sci ences) for performing electron microscopy measure ments of a colloidal gold preparation. REFERENCES 1. Boonstra, J., Rijken, P.J., Humbel, B., Cremers, F., Verkleij, A.J., and van Bergen en Henegouwen, P.M.P., Cell Biol. Intern., 1995, vol. 19, no. 5, pp. 413–430. 2. Gresik, E.W., Microsc. Res. Tech., 1994, vol. 27, no. 1, pp. 1–24. 3. Jorgensen, P.E., Danish Med. Bull., 1997, vol. 44, no. 2, pp. 111–124. 4. Duffy, M.J., O’Donovan, N., and Crown, J., Cancer Treatment Rev., 2011, vol. 37, no. 2, pp. 151–159. 5. Aaronson, S.A., Science, 1991, vol. 254, no. 5035, pp. 1146–1153.
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13. Dzantiev, B.B. and Zherdev, A.V., Biokhimicheskie metody analiza (Biochemical Methods of Analysis), Dzantiev, B.B., Ed., Moscow: Nauka, 2010, pp. 303– 332. 14. Breslauer, D.N., Maamari, R.N., Switz, N.A., Lam, W.A., and Fletcher, D.A., PLoS ONE, 2009, vol. 4, no. 7, p. e6320. 15. Starovoitova, T.A., Zaiko, V.V., Steriopolo, N.A., Mar tynkina, L.P., Kutvitskii, V.A., Tugolukov, A.E., Voloshchuk, S.G., Toguzov, R.T., and Vengerov, Yu.Yu., 2 Biomed. Zh. Medline.ru, 2010, vol. 11, pp. 44–62. 16. Frens, G., Nat. Phys. Sci., 1973, vol. 241, no. 105, pp. 20–22. 17. Hermanson, G.T., Bioconjugate Techniques, Amster dam: Acad. Press, Elsevier, 2008. 18. Byzova, N.A., Zvereva, E.A., Zherdev, A.V., Eremin, S.A., and Dzantiev, B.B., Talanta, 2010, vol. 81, no. 3, pp. 843–848. 19. Byzova, N.A., Zvereva, E.A., Zherdev, A.V., and Dzan tiev, B.B., Appl. Biochem. Microbiol., 2011, vol. 47, no. 6, pp. 685–693. 20. Schweers, B.A., Old, J., Boonlayangoor, P.W., and Reich, K.A., Forensic Sci. Intern.: Genetics, 2008, vol. 2, no. 3, pp. 243–247.
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