HYBRIDOMA Volume 30, Number 2, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/hyb.2010.0090
Development of a Colloidal Gold-based Immunochromatographic Test Strip for Screening of Microalbuminuria Kobra Omidfar,1 Solmaz Kia,2 and Bagher Larijani1
A rapid immunochromatography (ICG) assay based on antibody colloidal gold nanoparticles specific to human serum albumin (HSA) was developed, and its applications for primary screening of HSA in the urine were evaluated. A monoclonal antibody (MAb) specific to HSA was produced from cloned hybridoma cells (EMRC1) and used to develop an ICG strip. The nanocolloidal gold, with an average particle diameter of 20 nm, was synthesized and labeled MAb as the detection reagent. An antibody colloidal gold probe was applied on the conjugate pad, and HSA antigen was immobilized to a nitrocellulose membrane as the capture reagent to prepare the ICG strip test. This test required only 10 min to accomplish a semiquantitative detection of albumin. The sensitivity to urinary albumin was found to be approximately 20 mg/mL, and the analytical range was 20– 25 mg/mL. The reliability of the testing procedures was examined by carrying out the ICG strip test with 40 urine samples and comparing the results of these tests with those obtained via immunoturbidimetry. The ICG strip was adequately sensitive and accurate for a rapid screening of HSA in the urine.
Introduction
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uman serum albumin (HSA) is the most abundant plasma protein synthesized exclusively in the liver, with a molecular mass of 66 kDa. In a healthy person, only a small amount of the total protein excreted in the urine is albumin. The upper normal limit of albumin excretion, generally measured through specific methods such as highperformance liquid chromatography and immunoassays, is 30 mg/24 h (or 20 mg/min). Microalbuminuria (MAU) is defined as an abnormal urinary excretion rate of albumin between 20 and 200 mg/min (30–300 mg/day), which indicates a high probability of renal damage and is one of the earliest indicators of diabetic nephropathy among 30–40% of type 1 and type 2 diabetic patients. In addition, MAU has diagnostic implications in pregnancy as a predictive marker of preeclampsia and may play a role in identifying higher risks of developing complications from cardiovascular diseases even in non-diabetic patients (1,2) The level of albumin and its fragments in biological samples can be measured by means of several methods such as dye-binding assays, high-performance liquid chromatography (HPLC), and immunochemical methods.(3–11) HPLC offers good detection limits, but it is time-consuming and requires the use of expensive instruments.(5,6)
Compared with HPLC, immunoassays are demonstrated to be simple, specific, sensitive, and cost-effective for MAU measurements in small sample volumes. Immunoturbidimetry (IT), fluoroimmunoassay (FIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA) have all been well established for the quantitative measurement of the urine albumin. Nonetheless, these methods often require a long reaction time, special equipment, and reagent, and involve multiple steps.(7–11) In 2004, Choi and co-workers(3) introduced a fluorescence immunochromatography (ICG) assay for the quantitative measurement of albumin in the urine. Their method was based on a fluorescence immunochromatography assay (FICA) test strip in a disposable cartridge, a fluorescently labeled detector, and a laser fluorescence reader. The utilization of this assay is, however, confined to laboratories equipped with tools and devices for analysis. In recent years, there has been a growing interest in the ICG strip for a rapid detection of analytes because of its convenient use and visual end-point. It has practical advantages both in clinical and basic sciences and has been widely used as a popular diagnostic tool in clinical chemistry for detecting tumor markers,(12) hormones,(13,14) viruses,(15–17) bacteria, and parasitic antigens,(18–22) as well as for the detection of drug and toxins.(23–26) Therefore, the test has become convenient and speedy due to a novel concept of ICG that depends on the transportation of a reactant to its
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Endocrinology and Metabolism Research Center, Tehran University of Medical Sciences, Tehran, Iran. Department of Biology, School of Sciences, Razie University, Kermanshah, Iran.
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binding partner immobilized on a membrane surface. This recently developed technique, often called strip assay or lateral-flow assay, is based on an immunochromatographic procedure that utilizes antigen antibody properties and provides a rapid detection of an analyte. It combines several benefits, including a user-friendly format, short assay time, long-term stability over a wide range of climates, and costeffectiveness. These characteristics make it ideally suited for on-site screening by unskilled persons. Thus, developing a convenient detection method for a rapid measurement of albumin in the urine is extremely desirable, and colloidal nanogold appears to be the most attractive method at the moment. In the present study, MAb against HSA was produced and conjugated with colloidal nanogold to develop a rapid, onestep ICG test strip for the screening and also semiquantitative or quantitative measuring of MAU in the early detection of diabetic and non-diabetic nephropathy.
electron microscope were prepared, similar to the procedure described by Zhang and colleagues but with minor modifications.(28) For this purpose, the chloroauric acid solution (100 mL) was heated with electric heating to boiling point in an Erlenmeyer flask with a magnetic stirrer, and then 3 mL of the aqueous 1% sodium citrate solution were added to the flask while stirring rapidly; the mixing speed was 1000 rpm. The reaction time was 2 min, during which the color of the solution changed from light yellow to wine red. After continued boiling for another 6 min to complete the reduction of the colloidal gold, the nanogold particle solution was cooled to room temperature, pH was adjusted to 8.5 using 0.01 M potassium carbonate, and sodium azide was added to a final concentration of 0.01% (w/v) before it was stored at 48C in a dark-colored glass bottle until use. The obtained colloidal gold solution could be stored at 48C for several months.
Materials and Methods
The MAb EMRC1 was conjugated with the colloid gold nanoparticles according to the following method. Before conjugation, the optimal amount of the MAb concentration for conjugation with the colloidal gold solution was determined by the following steps. The MAb solution, with a concentration of 0.01–0.05 mg/mL, was added to a series of 2 mL tubes containing 1 mL pH-adjusted colloidal gold nanoparticle solution. The mixtures were incubated for 1 h at room temperature, and thereafter 500 mL of 10% NaCl were added to each tube and stirred for another minute. After 5 min, absorption at 520 nm and 580 nm was measured (A520–A580). A minimum amount of MAb was evaluated by color change from reddish to blue. The optimum concentration of MAb for colloidal gold labeling was the lowest concentration of MAb solution that did not change color. For conjugation, 600 mg of HSA MAb (30 mg /mL, in phosphate buffer [pH 7.5]) was added to 20 mL pH-adjusted colloid gold solution. The mixture was gently mixed for 3 h, and subsequently 4 mL of 10% BSA solution were added to block the residual surface of the colloidal gold nanoparticles. The mixture was then incubated for 20 min at room temperature before being centrifuged three times at 13,000 rpm for 45 min at 48C. After the last centrifugation, the gold pellets were suspended in 2 mL dilution phosphate-buffer [10 mM buffer (pH 7.2) containing 1% (w/v) BSA and 0.05% sodium azide], and the optical density was adjusted to 8.0 at 520 nm with dilution buffer. This anti-HSA IgG-coated colloidal gold probe was stored at 48C before use.
Materials Chloroauric acid (HAuCl4), RPMI 1640, fetal calf serum (FCS), streptomycin, penicillin, bovine serum albumin (BSA), HSA, hemoglobin, anti-mouse IgG-horseradish peroxidase (HRP), protein G column, and sodium citrate were all obtained from Sigma Chemical Company (St. Louis, MO). ELISA plates (96 wells) and other plasticware were obtained from Nunc (Max-Isorp, Roskilde, Denmark). High-flow nitrocellulose membranes, glass fibers, and absorption pads were obtained from Schleicher and Schell (Dassel, Germany) and Whatman (Fairfield, NJ). All other chemicals used in the present study were either analytically pure or of highest quality. Production and purification of monoclonal antibody Hybridomas producing mouse monoclonal antibodies to HSA have been described in detail elsewhere.(27) Briefly, HSA was used to immunize BALB/c mice, and hybridomas were generated by the fusion of spleen cells to SP2/0 myeloma cells. Hybridoma supernatants were tested for reactivity to HSA via ELISA, using HSA as a specific and hemoglobin as a non-specific capture antigen. We selected anti-HSA MAbs through two successive limiting dilutions. The hybridomas showing strong reactivity against HSA via ELISA and having no cross-reactivity with other related molecules were expanded for large-scale production of MAbs. The hybridoma cells were grown in 50 mL in a 3% FCS-containing medium, and the antibodies were purified from the culture supernatants via affinity chromatography on HiTrap protein G columns (Sigma Aldrich, St. Louis, MO). The eluted antibodies were dialyzed against phosphate-buffered saline (PBS) and concentrated in Ulterafree-15 centrifugal filter units (Millipore, Billerica, MA). EMRC1 was utilized for designing an ICG assay kit in the experiments described here. Synthesis of colloidal gold An aqueous solution of chloroauric acid [0.01% (w/v) HAuCl4] and a sodium citrate solution [1% (w/v)] in twice distilled water were prepared. Colloidal gold nanoparticles with a mean diameter of 20 nm checked with a transmission
Preparation of colloidal gold-MAb conjugate
Characterization of conjugates UV-vis spectroscopy analysis. The formation of the colloid gold nanoparticles and antibody-coated colloidal gold was monitored by UV-vis spectroscopy (200–700 nm) using a double-beam spectrophotometer (Shimadzo-uv-3100) operated at 1 nm. The gold solutions were monitored immediately after the centrifugation and resuspension of the conjugates in appropriate buffers and after agglutination with goat antimouse antibody. Thermal stability and activity analysis. The sensitivity, activity, and thermal stability of the conjugate probe and free antibody were determined using an uncompetitive indirect ELISA after different incubation times at 378C (up to 100 h),
COLLOIDAL GOLD-BASED IMMUNOCHROMATOGRAPHIC TEST STRIP similar to the procedure described by us previously.(29,30) For this purpose, the wells of the microtiter plates were coated with 100 mL of HSA (1mg per well) in phosphate-buffered saline overnight at 378C. Following the blockade of the nonspecific binding sites, the wells were treated with the colloidal gold anti-HSA MAb conjugate and free antibody at a concentration of 1 mg/well. This was incubated as before at 378C (bovine serum albumin was used as non-specific binding [NSB]). The residual binding was detected with anti-mouse HRP-conjugated antibody and expressed as a percentage of the optical density value given by a control. To assess the ability of the conjugate probe and free antibody to withstand irreversible thermal denaturization, 50 mg/mL of antibody diluted in PBS were incubated for 4 h at a range of tempera-
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tures (48C, 408C, 508C, 608C, and 708C) before its introduction to an ELISA plate coated with 1 mg/100mL of purified concentration of antigen. The rest of the experiments were performed as was described above. Furthermore, in the other parts of this study, the thermostability of the immunostrips treated with various concentrations of sucrose (3%, 5%, and 8%) was determined for 1 month after storage at 608C and at room temperature for 1 year and was compared with that prepared in the absence of sucrose. Preparation of immunochromatography test strip The lateral flow test strip was composed of a sample pad, a detector conjugated pad, an absorption pad, and one
Schematic of the test strip showing its several components.
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nitrocellulose membrane with test and control lines. Figure 1 depicts the preparation of the ICG test strip. Using a dispenser, 1 mL of different concentrations of HSA (1, 3, 5, and 10 mg/mL) diluted in PBS (0.01 mol/L, pH 7.2) was immobilized on the nitrocellulose membrane as a test line capture reagent to bind the HSA/colloidal nanogold conjugated MAb. Goat anti-mouse antibody diluted to 1mg/mL in PBS was also immobilized on the nitrocellulose membrane as a control line capture reagent to confirm the success of the test. These capture reagents were dispensed using the BioJet Quanti3000 dispenser onto a nitrocellulose membrane as two discrete zones, one for control and the other for tests. The residual surface on the membrane was blocked by incubation with 1% BSA in PBS and 0.05% sodium azide for 30 min at room temperature. The strips were washed once with PBS and then dried. A gold-labeled antibody probe (4 mL/strip and without dilution) was added onto the glass fiber and dried at 458C. The conjugate pad was prepared by adding the colloidal gold probe particles coated with anti-HSA MAb onto a glass fiber at the site of 15 mm from the bottom end and then dried. The jetted nitrocellulose, absorption pad, and conjugated pad
components were dried and assembled as the test strips. The laminated sheet was attached onto a plastic backing plate and then cut into individual strips (3.5 mm-wide strips), using a strip-cutter (model CM4000, Biodot, Irvine, CA). The strips were then sealed in a self-seal plastic bag with desiccant gel and stored at 378C. Principle of immunochromatography test strip and microalbuminuria determination The assay was based on the competitive reaction theory (Fig. 1). Ninety mL of a standard solution or sample were added onto the sample pad of the test strip. The strip was then placed flat to allow the solution to migrate up the membrane. The reaction between the colloidal gold MAb (EMRC1) and analyte immediately took place and moved into the nitrocellulose membrane with the immobilized binder. The analyte and the immobilized HSA on the test line complete their binding with a limited amount of colloidal gold-labeled anti-HSA MAb. The more HSA that was present in the sample, the weaker the test line appeared. The excess of the
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FIG. 2. (A) Light absorption of colloidal gold at different amounts of antibody concentration (0.005–0.05 mg/mL). (B) The UV-spectra of the colloidal gold solution before conjugation, antibody–colloidal gold conjugate only, and after agglutination with goat anti-mouse antibody. Dashed line, colloidal gold; solid line, Ab-gold conjugate; dashed-dotted line, agglutinated sample.
COLLOIDAL GOLD-BASED IMMUNOCHROMATOGRAPHIC TEST STRIP labeled anti-HSA MAb migrated further and was trapped by the goat anti-mouse IgG antibodies forming the control line. As soon as all the solution reached the top of the IC-test strip (usually in less than 10 min), a visible color appeared at the control line and/or result line in the strip. Finally, immunoturbidimetry assay (Randox) was employed to measure the level of albumin in the 40 different urine samples, and their results were compared with the nanogold particles ICtest strip. Results Production and characterization of antibodies to HSA Designing an ICG assay kit for screening MAU in the early detection of diabetic and non-diabetic nephropathy required large quantities of MAbs. We, therefore, sought to develop our own source of anti-HSA MAb. Hybridomas were obtained by the fusion of spleen cells from immunized mice with mouse myeloma cell line (SP2) as described above. After limiting dilutions, three clones producing antibodies were
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designed as EMRC1-3, which displayed different patterns of fine specificity for HSA and low cross-reaction with other proteins as elucidated by the inhibition ELISA. These clones were found to be of immunoglobulin G (IgG) class with k light chain. Subclass determination showed that all the three MAbs secreted the IgG1 type of antibody. The results of affinity purification for the two selected clones (EMRC1 and EMRC3) displayed high affinity with no cross-reactivity with any of the related protein molecules. The stable hybridomas secreting anti-HSA (EMRC1) were expanded in 50 mL flasks for a large-scale production of the required antibodies. The standard curves were constructed with a sensitivity of 10 pg/well covering up to 100 ng/well. Characterization of colloidal gold particles Nanogold colloid particles were synthesized by chemical condensation using the reduction of chloroauric acid (HAuCl4). Chloroauric acid was reduced to gold atoms by sodium citrate, and many of the gold atoms accumulated into
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Temperature (∞C ) FIG. 3. (A) Antigen-binding activity of free antibody and conjugated probe measured after up to 100 h incubation at 378C. (B) Stability analysis of free antibody and conjugated probe to irreversible thermal denaturation. Residual antigen-binding activity with respect to a non-treated control was determined following incubation at a range of temperatures for 4 h. All assays were performed in duplicate.
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FIG. 4. Test results of working standards with different concentrations of HSA in urine using the strips. Lane 1, 10 mg/mL; lane 2, 15 mg/mL; lane 3, 20 mg/mL; lane 4, 65 mg/ mL; lane 5, 80 mg/mL; lane 6, 150 mg/mL; lane 7, 180 mg/mL; lane 8, 200 mg/mL. The concentration of coating protein is 1 mL of 1 and 5 mg/mL for C-line and T-line, respectively. nanogold particles. The diameter of the colloidal gold particles obtained by UV-vis measurements and examination under the transmission electron microscope was 20 nm. Figure 2A shows the light absorption of the conjugate at different antibody concentrations. In our experiment, 30 mg of antibody was determined as the minimal antibody concentration for the stabilization of the colloidal gold. The spectra of the colloidal gold solution before conjugation and antibody–colloidal gold conjugate only and after agglutination with goat anti-mouse antibody can also be observed in Figure 2B. The UV-vis spectroscopic analysis showed a maximum absorbance at 520 nm, and the transmission electron microscope image revealed that the average diameter of colloidal gold particles was 20 nm. Theses results indicated that the colloidal gold particles provided the basis requirement for probe preparation and signal generation in test strips. In vitro thermal stability and activity The procedure described previously(29,30) was followed to test the thermo behavior and stability of the conjugate probe and free antibody. In this procedure, the remaining antigenbinding activity of the purified anti-HSA antibody and con-
The sensitivity of the test strip was determined by testing the HSA standard samples. The standard compounds were prepared by diluting the albumin stock solution (1 mg/mL) with the normal urine samples to final concentrations of 10, 15, 20, 65, 80, 150, 180, and 200 mg/mL. These standard samples were thereafter examined and judged with the naked eye. The dose-response phenomenon (the color intensity in the result line) of the newly developed ICG strip test was determined using standard samples. The color intensity in the result line decreased in the dose-response manner of HSA (Fig. 4). This differential color intensity represented the concentration of HSA and would be applicable as a marker for the quantification of the results. In this test strip, the coating concentration of 5 mg/mL for HSA was optimized. The albumin concentration range was considered to be 200 mg/L)
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Immunoturbidimetry Immunochromatographic strip
All results were judged 10 min after the test. Data are the mean of triplicates.
COLLOIDAL GOLD-BASED IMMUNOCHROMATOGRAPHIC TEST STRIP of the above substances interfered with the results of the semiquantitative microalbuminuria test kit. The ICG strips were used to test 25 positive urine samples as well as 15 negative controls. As demonstrated in Table 1, 23 patients with albuminuria tested positive and two patients with MAU in the range of 20–25 mg/mL tested negative according to the test strips. For the control urine samples of the healthy individuals, two bands were observed: the lower band (test line) had more intensity than did the upper band (control line) on the strips. Comparison of immunochromatographic strip test with immunoturbidimetry assay The reliability of the test strip was also examined by carrying out the ICG strip test with the 40 urine samples and comparing the results of these tests with those obtained via IT (Table 1). The results revealed that the two methods had significant concordance in diagnostic judgment. The results also demonstrated that this test strip had a sensitivity of 92%, specificity of 83%, positive predictive value of 100%, and negative predictive value of 83%. Discussion Simple, near-patient, decentralized, point-of-care, and on-site rapid testing is emerging as a tool for more efficient diagnosis and patient evaluation. Technological innovations based on lateral flow assays have enabled a move to bring testing closer to the patient and provide a simple, easy-toread, rapid, and convenient diagnostic method. The feasibility to execute such diagnostic tests at a location far from the laboratory would be highly utilitarian as regards the pace and economic costs. The ICG assay or simple form strip assay has been in use for a while. This technique is based on an immunochromatographic method that employs antigen-antibody in a new manner to provide a rapid detection of the analyte. Colloidal nanogold particles have been successfully applied for the development of a one-step strip test. Organic molecules such as antibody could be directly attached to gold nanoparticle by non-covalent reaction, including London-van der Waals force and hydrophobic interaction. The balance between electrostatic repulsion and London-van der Waals attraction among the particles results in the formation of a colloidal gold solution. The size of the colloidal gold particles is directly dependent on the amount of sodium citrate as the ionic substances used in its preparation process. The strength of color is closely related to the size and quality of the colloidal gold particles. When ionic substance is added, the attracting force becomes greater than the counteraction and leads to an aggregation and color change from red (lmax & 520 nm, A 520) to blue (lmax A 580).(31) This instability of the colloidal gold particles could be avoided or prevented by coating the colloidal surfaces with protein molecules such as antibody. A stable conjugate of colloidal gold and MAb can be provided through a minimum amount of antibody. Therefore, preliminary serial dilution experiments are performed to quantitatively determine the minimal antibody concentration sufficient for having a strong adsorption between the gold and antibody conjugate. The nanoscale surfaces, provided by the colloidal gold particles, can accelerate the antibody-antigen reaction sufficiently and amplify the signal for immunoassay.
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In the present study, we constructed an immunochromatographic lateral flow test strip for the screening of urinary albumin. This one-step assay is based on the principle of the competitive immunochemical reaction between the albumin in urine samples and albumin immobilized on the membrane for the limited antibody site. The albumin present in the urine binds with the colloidal nanogold particles, and the antigen-antibody binding complex transports into the next nitrocellulose membrane via the flow caused by capillary action. Through the visual recognition of a test line, this strip test affords an easy and clear detection of the albumin in the urine within 10 min, and the appearance of a control line verifies the validity and performance of the test. At low levels of albumin in the sample, these nanogolds bind to the test line and produce a red-colored band on the membrane. As the amount of the albumin in the urine increases, more colloidal nanogolds pass through the first band (test line) and are bound by the second band (control line). After 10 min, the darkness or intensity of the two red bands on the nitrocellulose membrane is compared. If the color intensity of the test line is darker than or equal to that of the control line, the test is assumed to be negative and vice versa. We constructed this newly rapid test successfully in our laboratory using MAb EMRC1 conjugated with 20 nm colloidal gold particles, and the test exhibited high sensitivity for the screening of MAU. The detection limit of the test strip was approximately 20 mg/mL. High reproducibility, as well as high sensitivity and specificity of the rapid test, was evaluated and confirmed using samples from various patients as well as healthy controls (Table 1). The test strip showed no cross-reactivity with other human proteins at the biological ranges. Furthermore, in the presence of various human proteins and common medications, and also other substances that are likely to be present in the urine, no interference in the detection of albumin was observed. These results suggest that the ICG test is simple, reliable, and highly specific for the screening of urinary albumin at the range of MAU. Acknowledgment This research was supported by the Endocrinology and Metabolism Research Center and the Medical Nanotechnology Research Center at Tehran University of Medical Sciences. References 1. Redon J: Measurement of microalbuminuria—what the nephrologists should know. Nephrol Dial Transplant 2006;21: 573–576. 2. Romundstad S, Holmen J, Hallan H, Kvenild K, Kruger Q, and Midthjell K: Microalbuminuria, cardiovascular disease and risk factors in a nondiabetic nonhypertensive population. J Intern Med 2002;252:164–172. 3. Choi S, Choi EY, Kim HS, and Oh SW: One-site quantification of human urinary albumin by a fluorescence immunoassay. Clin Chem 2004;50:1052–1055. 4. Kessler MA, Meinitzer A, Petek W, and Wolfbeis OS: Microalbuminuria and borderline increased albumin excretion determined with a centrifugal analyzer and the albumin blue 580 fluorescence assay. Clin Chem 1997;43:996–1002.
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Address correspondence to: Dr. Kobra Omidfar Endocrinology and Metabolism Research Center Tehran University of Medical Sciences P.O. Box 14395/1179 Tehran, Iran E-mail:
[email protected] Received: August 21, 2010 Accepted: September 24, 2010
