Biosensors and Bioelectronics 54 (2014) 1–6
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Gold nanoparticle based Tuberculosis immunochromatographic assay: The quantitative ESE Quanti analysis of the intensity of test and control lines Phumlani Mdluli n, Phumlani Tetyana, Ndabenhle Sosibo, Hendriëtte van der Walt, Mbuso Mlambo, Amanda Skepu, Robert Tshikhudo Nanotechnology Innovation Centre, Advanced Materials Division, Mintek, Private Bag X3015, Randburg 2125, Gauteng, South Africa
art ic l e i nf o
a b s t r a c t
Article history: Received 2 August 2013 Received in revised form 30 September 2013 Accepted 10 October 2013 Available online 30 October 2013
A rapid dual channel lateral flow assay for the detection of Mycobacterium Tuberculosis antibodies (MTB 38 kDa monoclonal antibody) in human blood was developed. The MTB 6–14–38 kDa fusion antigen and anti-Protein A were used as the capture proteins for test and control lines respectively. Protein A labeled 40 nm gold nanoparticles were used as the detection conjugate. Whole blood and serum were spiked with MTB 38 kDa monoclonal antibody to make a positive sample model. The developed lateral flow was used to test MTB 38 kDa monoclonal antibody, and a detection limit of 5 ng/ml was used as a cut-off concentration of the analytes. The effect of the analyte concentration on the MTB lateral flow assay was studied using the variation of the intensity obtained from a ESE Quanti reader. There was a direct correlation between the analyte (MTB 38 kDa monoclonal antibody) concentration and the intensity of the test line. The intensity increased with an increase in the concentration of MTB 38 kDa monoclonal antibody, while in contrast, an increase in analyte concentration decreased the intensity of the control line. & 2013 Elsevier B.V. All rights reserved.
Keywords: Mycobacterium tuberculosis ESE Quanti reader Gold nanoparticles
1. Introduction Tuberculosis (TB), a bacterial infection caused by Mycobacterium Tuberculosis (MTB), poses a major health risk throughout the world. The disease has been observed to predominate in countries with poor health services and a low per capita income (Harries et al., 2001). It is known to co-exist with various fatal diseases, such as Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS; Winkler et al., 2005). Some Tuberculosis strains may become resistant to the current medication used for the treatment; as a result there is an increase of challenges in dealing with the scourge of TB (Nagel et al., 2008). TB remains the leading cause of the fatality compared to other curable and airborne diseases despite the availability of short-course inexpensive and effective therapy (Harries et al., 2001, Hendrickson et al., 2000; Chan et al., 2000; Daniel et al., 1994; Dolin et al., 1994, Jackett et al., 1988). The clinical management of TB in developing countries is hampered by the lack of a simple and effective diagnostic test. An improved diagnosis of TB is thus required for early screening which will lead to an effective treatment, reduced transmission and the control of the
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Corresponding author. Tel.: þ 27 11 7094002; fax: þ 27 11 7094480. E-mail address: [email protected] (P. Mdluli).
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development of drug resistance TB. It is thus imperative to design a fast, cheap and highly sensitive test device for the early detection of the disease (Thanyani et al., 2008). Various tests, including enzymelinked immunosorbent assay (ELISA) developed for the detection of TB, have fallen out of favor due to the long analysis times, laborious operation, decreased sensitivity, low specificity and high cost (Cousins et al., 1992; Miller et al., 1994; Noordhoek et al., 1994; Shawar et al., 1993). Blood-based serodiagnostic tests constitute a promising and alternative approach for detection of Tuberculosis (Andersen et al., 2000; Lyashchenko et al., 2006). Though some methods such as Tuberculin-induced lymphocyte transformation were good in early studies of TB diagnosis (Baram et al., 1971; Chaparas et al., 1975), this type of diagnosis was never developed into real point-of-care diagnostic tools. There are many further studies that evaluated the in vitro gamma interferon assay in experimental and natural infections for TB diagnosis (Garcia et al., 2004; Vervenne et al., 2004). The antibody serological diagnostic tests are simple, rapid, accurate, and relatively inexpensive. There are many reported attempts that had been made to develop a rapid serodiagnostic test for human TB and most of them were apparently inadequate (Okuda et al., 2004; Perkins et al., 2003). However, serodiagnostics has been successfully demonstrated in animals (Greenwald et al., 2003; Lyashchenko et al., 2006). This paper reports the design of a
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rapid diagnostic test for the detection of TB antibodies in whole blood and serum. In this paper the application of an ESE Quanti reader is used to evaluate the effects of MTB 38 kDa monoclonal antibody on the intensities of test and control lines. The performance of these strips was further evaluated for their use as potential rapid diagnostic testing devices in both serum and whole blood.
2. Materials and methods 2.1. Sera, biomarkers, membranes and pads M. Tuberculosis fusion antigen, MTB 6–14–38 kDa, was purchased from CTK Biotech (USA). The monoclonal Anti-MTB 38 kDa antibody from mouse was purchased from Biocom Biotech (USA). The polyclonal anti-Protein A from rabbit, gold tetrachloride hydrate and Protein A were all purchased from Sigma-Aldrich South Africa. The backing cards, nitrocellulose Whatman AE 98 fast membrane and Millipore GF041 conjugate pads were purchased from Diagnostic Consulting Network (DCN USA). The human serum and whole blood samples were purchased from the South African National Blood Service (SANBS). All antibodies and proteins were used without further purification. 2.2. Instrumentation and analysis High purity or ultrapure water with resistivity of 18.1 Ω m was obtained from a Milli-Q Advantage water system purchased from Millipore (USA) and was used in all the experiments. A PerkinElmer Lambda 20 UV–visible spectrophotometer was used to carry out the optical measurements. Transmission electron microscopy (TEM) images were obtained using a JEM-2100F at 200 kV. The TEM grids were prepared by depositing approximately 10 μl of the solution obtained after centrifugation and allowed to dry in air. The analysis was done in silica cuvettes (1 cm path length), using water as the reference solvent. The ESE Quanti reader lateral flow studio was used to determine the lines' intensities and designed to do the analysis of test strips with dimension of 5 100 mm (b l). A Biodot XYZ Series dispensing system was used to immobilize proteins at a rate of 1 ml/cm and the conjugate at 10 ml/cm. 2.3. Preparation of the detection conjugate The as-synthesized and purified gold nanoparticles were characterized using UV–visible and TEM as discussed in the Supporting information; detailed images showing UV–visible and TEM are shown in Figs. S1 and S2 (Supporting document). A dialyzed Protein A (100 μl, 10 mg/ml in water) solution was added to gold nanoparticles (50 ml, pH 8) at room temperature. The reaction mixture swelled for 10 min immediately after the introduction of the protein and the resultant conjugate was purified through centrifugation (12,000 rpm, 10 min and 22 1C) and washed with water three times. The conjugate was finally redispersed in borate buffer (5 ml, OD¼10) and stored at 4 1C until further use. 2.4. Preparation of conjugate pads The MTB 38 kDa monoclonal antibody standard concentration of 1 mg/ml was diluted to standard analyte concentrations from 5 to 300 ng/ml. The Millipore GF041 conjugate pad was used to immobilize the detection conjugate on the immunochromatographic test strip. The conjugate and sample pads were first blocked with 30% Bovine Serum Albumin (BSA), 10% Polyvinylpyrrolidone (PVP) and 2.5% Triton X-100 for 2 min and dried at 37 1C for 30 min. The detection conjugates (300 ml with 20% sucrose) at
different concentrations were sprayed onto the conjugate pads at a rate of 10 ml/cm. After drying for 30 min at 37 1C, the conjugate pads were stored at room temperature in sealed aluminum foil pouches containing 1 g desiccant. 2.5. Preparation of membrane The MTB 6–14–38 kDa fusion antigen (1 mg/ml) in phosphate buffered saline (PBS) was used as the capture antigen on the immunochromatographic tests nitrocellulose membrane. The MTB fusion 38 kDa antigen (30 ml, 1 mg/ml) and anti-Protein A (30 ml, 1 mg/ml) were dispensed onto the nitrocellulose membrane as the test and control lines respectively. After drying the membrane for 30 min at 37 1C, it was blocked with a membrane blocking buffer (MBB) that consisted of 1% sucrose, 1% BSA, 2.5% PVP and 0.1 M NaH2PO4. The membrane was then dried for another 30 min at 37 1C. 2.6. Assembly of the strip 2.6.1. Basic strip assembly (single channel) All strip components were assembled onto the plastic backing support along with the analytical membrane with the immobilized capture proteins as indicated in Fig. S3(a) (Supporting document). The dried conjugate pad was attached to the backing support with a 1–2 mm overlap onto the analytical membrane on the side of the test line. The absorbent pad was attached to the support at the other end of the membrane with a 1–2 mm overlap. The assembled single channel was then cut into 5 mm thick strips and stored at room temperature in a sealed microarray storage aluminum pouch. 2.6.2. Dual channel strip assembly All strip components were assembled onto the plastic backing support following procedure discussed in single channel strip assembling with the exception of the additional secondary conjugate pad. The primary conjugate pad was attached directly to the backing support with a 2 mm overlap onto the analytical membrane on the side of the test line. The secondary conjugate pad (sample pad) was attached on top of the primary conjugate pad with a simple polymer separator as illustrated in Fig. S3(b) (Supporting information). The absorbent pad was attached as discussed for single channel assembling. The assembled dual channel card was cut into 5 mm thick strips and stored at room temperature in a sealed microarray storage aluminum pouch. 2.7. Experimental testing procedure for single and dual channels The MTB 38 kDa antibody at each concentration from 5 to 300 ng/ml reacts with the gold nanoparticle–Protein A detection conjugate and forms a gold nanoparticle–Protein A–antibody complex. The complex binds to the capture antigen on the membrane and forms a bright red colored band at the test line which is the characteristic of a positive test result. The absence of the antibody leads to the absence of the red band on the test line as a symbol of a negative test result. To test the immunochromatographic test strips, whole blood or serum samples were spiked with the MTB 38 kDa antibody and applied to the sample pad. The blood or serum was allowed to migrate towards the membrane along with 100 ml Tris-buffered saline (TBS) at pH 7.2. After the sample had migrated from the sample pad and reached the test line of the analytical membrane, the detection conjugate was migrated through the application of an additional 100 ml TBS onto the conjugate pad. The test was left to run for an additional 5 min and the results interpreted.
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3. Results and discussion The design of the better and improved diagnostic test is crucial to eliminate false-negative and false-positive which are very common in TB diagnostics and can lead to devastating TB outbreaks. Lateral flow technology gives crucial features that symbolize it to be a most attractive and user-friendly serodiagnostic format. Lateral flow diagnostics can contribute to early diagnostic of diseases such as deadly Tuberculosis. The introduction of Protein A, an immunoglobulin-binding protein, onto the surface of the citrate-capped gold nanoparticles was conducted through the electrostatic approach. Electrostatic immobilization of biomolecules onto the surface of nanoparticles offers one of the simplest bio-functionalization methods that can be performed under mild conditions (Niemeyer, 2001; Rajh et al., 1999; Meziani and Sun, 2003). The structural assessment and dispersion of the Protein A conjugates were obtained through the use of TEM measurements and an image is presented in Fig. S1(c) (Supporting information). The conjugates were well dispersed and there was no apparent aggregation observed. The optical absorption measurements, shown in Fig. S1(d) (Supporting information), of the conjugates under various electrolytic concentrations using sodium chloride (NaCl) were performed to test nanoparticle stability. The SPR peaks were all centered at 523 nm and showed no shifts with NaCl concentrations of 0.1–1.0 M. A better UV–visible spectrum which shows the centered surface Plasmon peak of gold nanoparticle conjugate at 523 nm is clearly shown in Fig. S2 (Supporting information). Protein A bioconjugates showed remarkable stability and could be stored for months without losing their functional activity in a suitable buffer. 3.1. Immunochromatographic assay 3.1.1. Dual channel immunochromatographic assay principle The general principle of dual channel immunochromatographic test strips is shown in Fig. 1(A). The analyte of interest in the sample tested is first allowed to bind to the detection antigen to
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form antibody–antigen complex on the test line. This is subsequently followed by the capillary movement of the gold–Protein A conjugate along the membrane to the test line where an antibody– antigen complex has accumulated. The gold–Protein A conjugate binds to the antibody–antigen complex at the test line and results in the display of a red colored line. The antibody therefore serves as an anchor that links the detection conjugate to the capture antigen on the test line. The gold–protein conjugate further moves and binds to anti-protein A, where a second red line appears as a control, thus indicating the validity of the test strip. When the analyte of interest is not present in the tested sample, the detection conjugate will not bind the analyte and will continue to migrate freely along the membrane, surpass the test line, without binding and reach the control line where it binds to the immobilized protein as shown in Fig. 1(A). 3.1.2. Single channel design The basic single channel test strip allows the analyte to react with the immobilized protein on the test line before the conjugate is filtered through the nitrocellulose membrane. Although this allows for reaction of the analyte with a specific target protein, the specificity of the binding protein limits the efficiency of the immunochromatographic assay. As illustrated in Fig. 1(B), the probability of the binding protein to cross-react with a variety of analytes present in the test sample, especially with the use of whole blood or serum samples, is increased with the use of a protein such as Protein A. This reduces efficiency of the assay, thus limiting its use as an effective diagnostic device. 3.1.3. Single channel testing The greatest challenge in current test devices is the use of whole blood without prior modification. The use of whole blood in test devices allows tests to be conducted almost anywhere without the use of sophisticated equipment and lengthy processes. A direct immunochromatographic method was used to test for the TB antibodies as shown in the strips in Fig. 2(A)–(D). The negative and positive tests are clearly distinguishable in the buffered
Positive Negative After migration of blood After migration of positive blood and conjugate
Positive
Negative After migration of conjugate
Antigen Protein A Antiprotein A Antibodies in blood
Negative
Gold Conjugate Fig. 1. Diagram showing the principle of the dual and single pathway immunochromatographic test strips compared with the standard immunochromatographic test strip. (A) Dual channel immunochromatographic test strip when blood is used. (B) Single channel process that occurs during the use of blood in the single path immunochromatographic test strip.
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Fig. 2. Image shows the test strips constructed for use with direct immunochromatographic assay. The strips were tested on both buffer (A, B) and whole blood (C, D) at an analyte concentration of 5–100 ng/ml (A, C, Negative test; B, D, Positive test). Images showing the test strips constructed using the double conjugate pad technique. Strips were tested on both buffer (E, F) and whole blood (G, H) (E, G, Negative test; F, H, Positive test).
system as shown in Fig. 2(A) and (B), while the direct use of whole blood greatly decreases the test performance as shown in Fig. 2 (C) and (D). When whole blood was used (Fig. 2(C) and (D)), the negative (C) and positive (D) tests on the immunochromatographic test strips showed the same negative result. The results observed in the single channel showed the degree of cross-reactivity, thus prompting the development of a simple dual channel test strip that shall eliminate the interaction of test sample (blood and serum) with conjugate. The dual channel will allow the MTB 38 kDa antibody to interact with the antigen in the test line before Protein A conjugate thus reducing the cross-reactivity.
3.1.4. Two channel immunochromatographic assay redesign For more effective detection of TB in a whole blood sample, an improved test strip design was developed and patented (Tshikhudo et al., 2012). The basic design shown in Fig. S3(b) (Supporting information) allows the specific binding of Protein A to the analyte of choice by eliminating the possibility of cross reaction with related analytes. The dual channel assays design utilizes a plastic membrane to prevent non-specific binding of the Protein-A conjugate. Dual path immunoassay has been reported by Esfandiari (2007), a lateral flow with two strips connected into T-shaped. In his invention, the first strip was used for the flow path of the sample and the second for running buffer. However, this invention made the cassette to be larger to allow for fabrication of these two strips; as a result the cost of the whole fabrication process rises up. Our design is in line with this invention that an analyte sample must be allowed to react with the test line before a running conjugate is run through. Our design proves to be simpler and cost effective as both the strips were overlaid and separated by a plastic; as a result there was no need of designing larger plastic cassettes for these tests. The dual channel strip proposed in Fig. S3(b) (Supporting information) was put to the test and compared to the standard immunochromatographic test strip method. Fig. 3 illustrates the test strips used during the experiment. With the use of whole blood (Fig. 3), the strip performance increased dramatically. The background was minimized, with the test and control lines clearly visible. The intensities of the test and control lines increased compared to the single channel, indicating an increase in visual detection. This may be due to a decrease in non-binding analytes on the membrane surface, which prevents the complete transfer of the analyte to the test and control lines. The test was designed to eliminate the non-specific binding that may be caused by the contents of blood or plasma. It can be noted that Protein A
can bind to any immunoglobulin antibodies, especially the IgGs in either blood or serum. To eliminate this non-specific binding, the dual channel design used in this test allowed the interaction of the test sample (blood or serum) with test line before the Protein A conjugate was run through. This design is in line with the previously reported serodiagnostic test by Greenwald, which was useful to demonstrate the application of TB serodiagnosis in various host species (Greenwald et al., 2003; Lyashchenko et al., 2006). However, one of the common limitations of serodiagnostic methods compared to cell-mediated immune response assays, such as the intradermal test, is the fact that in TB infection, the antibody response develops at the latter stage (Andersen et al., 2000; Lyashchenko et al., 2004). Thus, the dual channel design can be easily distinguished between the false positive due to nonspecific binding and the actual presence of the antibody response due to TB infection. The effect of a change in the concentration of the TB antibody, used as an analyte on the test and control lines in a TB lateral flow assay, was investigated. Fig. 3(A) shows the immunochromatographic test strips used in the experiment. It shows the appearance of both the test and control lines due to the reaction of the MTB 38 kDa antibody with the respective lines with increasing concentration (0–100 ng/ml). The intensity of the test line increases with an increase in the concentration of the TB antibody as shown in Table S1 (Supporting information). As the antibody concentration increases, the amount of antibody that binds to the Protein A–gold complex increases, thus increasing the amount of the antibody–Protein A–gold complex. The increased amount of complex then binds to the immobilized capture antigen, producing a deeper or more intense line color. In combination, as the concentration of the antibody increases, the intensity of the control line decreases. The control line intensity depends on the concentration of the antibody–unbound Protein A–gold complex; an increase in the concentration of the antibody would decrease the amount of free Protein A–gold complex available for binding to the control line. This would thus decrease the intensity of the control line with an increase in the antibody concentration. The contrary would also hold; a decrease in antibody concentration would lead to an increase in control line intensity. The relationship between the intensity and concentration is plotted in Fig. S6 (Supporting information). The intensity and concentration of an analyte are linearly proportional as obviously graphically demonstrated in Fig. S6 (Supporting information). The intensities of the test and control lines were measured with an ESE Quanti lateral flow assay strip reader to determine the intensities of the test and control lines. The intensity values of the test and control lines are shown in Table 1S (Supporting
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Fig. 3. (A) Images of the test strips showing the effect of the analyte concentration on the intensities of the test and control lines in a TB lateral flow device. The MTB 38 kDa antibody was used as an analyte at a concentration range of 0–100 ng/ml. (B) Graphs showing the change in the intensities of the test and control lines in lateral flow assay with respect to change in the concentration of the MTB 38 kDa antibody (analyte).
information) and are associated with the magnitude of the areas of the resultant peaks in Fig. 3(B). An increase in the peak area is related to an increase in the color intensity of the lines. At 0 ng/ml, the test line has some intensity which has been treated as the background referred to as control line in Fig. S5 (Supporting information); this is shown by the appearance of the test line only indicating a negative test. The area of the peaks increased with an increase in the MTB 38 kDa antibody concentration. There is almost no change in peak area between 80 ng/ml and 100 ng/ml concentrations as can be seen in Table 1S (Supporting information). This may indicate the saturation of the test at these concentrations. The Protein A–gold conjugate is either completely used up or all the available conjugate molecules have reacted with the analyte in the test sample resulting in a limit on the reactable complexes. This is highly unlikely as the unreacted Protein A–gold complex reacts with the control line. As a control line is visible, there had to have been unreacted complex available for this reaction to occur. The maximum amount of antigen–antibody– Protein A–gold complexes could have been formed on the test line in contrast. In this case, the antibody–Protein A–gold complex has reacted with the entire available antigen/capture antibody on the test line, resulting in maximum detection capability for the
specific capture antibody concentration on the test line. Further studies were conducted in serum samples spiked with MTB antibodies, and the results are shown in Figs. S4 and S5 (Supporting information). It was found that both test and control lines were clearly visible and they both increase with increasing concentration of the MTB antibodies from 0 to 300 ng/ml spiked into the serum samples. The intensities of serum samples are shown in Fig. S5 (Supporting information). It was established that as the concentration of MTB antibodies increased from 0 to 300 ng/ml, both control and test lines were visible for the concentration of 10–300 ng/ml, and as expected the control line was only observed at zero analyte concentration which showed that the assay was valid. The visibility of the both test line and control line was further evaluated for their intensities, and it was found that as the concentration increased the intensity linearly increased as well (Fig. S5, Supporting information).
4. Conclusion Dual channel serodiagnostic assay was successfully developed. It was clear from experimental results that the use of a common
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single channel immunochromatographic test would not be effective for the detection of the MTB 38 kDa antibody. A modified dual channel immunochromatographic test proved to be more effective with clear distinction between the positive and negative tests with the use of whole blood and serum. The use of an ESE Quanti reader confirmed the increase in the intensity of the test line on the immunochromatographic strip with an increase in antibody concentration. The maximum detection for the immobilized capture antibody on the test line was established with no increase in line intensity observed at antibody concentrations of 80 and 100 ng/ml. The present work does not address whether the experimental infections models used for test development and evaluation adequately reflect naturally occurring Tuberculosis infections. The detection limit of 5 ng/ml does not represent the actual TB antibody titer in blood or serum; it was used to evaluate the effectiveness and to mimic the actual samples. Thus, additional work is in progress in our laboratories to probe the sensitivity and selectivity of these serological tests in real samples which were acquired from the World Health Organization (WHO). These samples from WHO shall assist us in evaluating the minimum antibody titer that can be detected using the prepared lateral flow test devices. Acknowledgment The authors would like to thank Mintek/NIC for permission to publish this work and for financial support. PSM would also like to thank the NRF for financial support through Thuthuka Post-PhD programme Grant number: TTK1206051037. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.10.019. References Andersen, P., Munk, M.E., Pollock, J.M., Doherty, T.M., 2000. Lancet 356, 1099–1104. Baram, P., Soltysik, L., Condoulis, W., 1971. Lab. Anim. Sci. 21, 727–733. Chan, E.D., Reves, R., Belisie, J.T., Brennan, P.J., Hahn, W.E., 2000. Am. J. Respir. Crit. Care Med. 161, 1713–1719.
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Short communication
Gold nanoparticle based Tuberculosis immunochromatographic assay: The quantitative ESE Quanti analysis of the intensity of test and control lines Phumlani Mdluli n, Phumlani Tetyana, Ndabenhle Sosibo, Hendriëtte van der Walt, Mbuso Mlambo, Amanda Skepu, Robert Tshikhudo Nanotechnology Innovation Centre, Advanced Materials Division, Mintek, Private Bag X3015, Randburg 2125, Gauteng, South Africa
art ic l e i nf o
a b s t r a c t
Article history: Received 2 August 2013 Received in revised form 30 September 2013 Accepted 10 October 2013 Available online 30 October 2013
A rapid dual channel lateral flow assay for the detection of Mycobacterium Tuberculosis antibodies (MTB 38 kDa monoclonal antibody) in human blood was developed. The MTB 6–14–38 kDa fusion antigen and anti-Protein A were used as the capture proteins for test and control lines respectively. Protein A labeled 40 nm gold nanoparticles were used as the detection conjugate. Whole blood and serum were spiked with MTB 38 kDa monoclonal antibody to make a positive sample model. The developed lateral flow was used to test MTB 38 kDa monoclonal antibody, and a detection limit of 5 ng/ml was used as a cut-off concentration of the analytes. The effect of the analyte concentration on the MTB lateral flow assay was studied using the variation of the intensity obtained from a ESE Quanti reader. There was a direct correlation between the analyte (MTB 38 kDa monoclonal antibody) concentration and the intensity of the test line. The intensity increased with an increase in the concentration of MTB 38 kDa monoclonal antibody, while in contrast, an increase in analyte concentration decreased the intensity of the control line. & 2013 Elsevier B.V. All rights reserved.
Keywords: Mycobacterium tuberculosis ESE Quanti reader Gold nanoparticles
1. Introduction Tuberculosis (TB), a bacterial infection caused by Mycobacterium Tuberculosis (MTB), poses a major health risk throughout the world. The disease has been observed to predominate in countries with poor health services and a low per capita income (Harries et al., 2001). It is known to co-exist with various fatal diseases, such as Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS; Winkler et al., 2005). Some Tuberculosis strains may become resistant to the current medication used for the treatment; as a result there is an increase of challenges in dealing with the scourge of TB (Nagel et al., 2008). TB remains the leading cause of the fatality compared to other curable and airborne diseases despite the availability of short-course inexpensive and effective therapy (Harries et al., 2001, Hendrickson et al., 2000; Chan et al., 2000; Daniel et al., 1994; Dolin et al., 1994, Jackett et al., 1988). The clinical management of TB in developing countries is hampered by the lack of a simple and effective diagnostic test. An improved diagnosis of TB is thus required for early screening which will lead to an effective treatment, reduced transmission and the control of the
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Corresponding author. Tel.: þ 27 11 7094002; fax: þ 27 11 7094480. E-mail address: [email protected] (P. Mdluli).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.019
development of drug resistance TB. It is thus imperative to design a fast, cheap and highly sensitive test device for the early detection of the disease (Thanyani et al., 2008). Various tests, including enzymelinked immunosorbent assay (ELISA) developed for the detection of TB, have fallen out of favor due to the long analysis times, laborious operation, decreased sensitivity, low specificity and high cost (Cousins et al., 1992; Miller et al., 1994; Noordhoek et al., 1994; Shawar et al., 1993). Blood-based serodiagnostic tests constitute a promising and alternative approach for detection of Tuberculosis (Andersen et al., 2000; Lyashchenko et al., 2006). Though some methods such as Tuberculin-induced lymphocyte transformation were good in early studies of TB diagnosis (Baram et al., 1971; Chaparas et al., 1975), this type of diagnosis was never developed into real point-of-care diagnostic tools. There are many further studies that evaluated the in vitro gamma interferon assay in experimental and natural infections for TB diagnosis (Garcia et al., 2004; Vervenne et al., 2004). The antibody serological diagnostic tests are simple, rapid, accurate, and relatively inexpensive. There are many reported attempts that had been made to develop a rapid serodiagnostic test for human TB and most of them were apparently inadequate (Okuda et al., 2004; Perkins et al., 2003). However, serodiagnostics has been successfully demonstrated in animals (Greenwald et al., 2003; Lyashchenko et al., 2006). This paper reports the design of a
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rapid diagnostic test for the detection of TB antibodies in whole blood and serum. In this paper the application of an ESE Quanti reader is used to evaluate the effects of MTB 38 kDa monoclonal antibody on the intensities of test and control lines. The performance of these strips was further evaluated for their use as potential rapid diagnostic testing devices in both serum and whole blood.
2. Materials and methods 2.1. Sera, biomarkers, membranes and pads M. Tuberculosis fusion antigen, MTB 6–14–38 kDa, was purchased from CTK Biotech (USA). The monoclonal Anti-MTB 38 kDa antibody from mouse was purchased from Biocom Biotech (USA). The polyclonal anti-Protein A from rabbit, gold tetrachloride hydrate and Protein A were all purchased from Sigma-Aldrich South Africa. The backing cards, nitrocellulose Whatman AE 98 fast membrane and Millipore GF041 conjugate pads were purchased from Diagnostic Consulting Network (DCN USA). The human serum and whole blood samples were purchased from the South African National Blood Service (SANBS). All antibodies and proteins were used without further purification. 2.2. Instrumentation and analysis High purity or ultrapure water with resistivity of 18.1 Ω m was obtained from a Milli-Q Advantage water system purchased from Millipore (USA) and was used in all the experiments. A PerkinElmer Lambda 20 UV–visible spectrophotometer was used to carry out the optical measurements. Transmission electron microscopy (TEM) images were obtained using a JEM-2100F at 200 kV. The TEM grids were prepared by depositing approximately 10 μl of the solution obtained after centrifugation and allowed to dry in air. The analysis was done in silica cuvettes (1 cm path length), using water as the reference solvent. The ESE Quanti reader lateral flow studio was used to determine the lines' intensities and designed to do the analysis of test strips with dimension of 5 100 mm (b l). A Biodot XYZ Series dispensing system was used to immobilize proteins at a rate of 1 ml/cm and the conjugate at 10 ml/cm. 2.3. Preparation of the detection conjugate The as-synthesized and purified gold nanoparticles were characterized using UV–visible and TEM as discussed in the Supporting information; detailed images showing UV–visible and TEM are shown in Figs. S1 and S2 (Supporting document). A dialyzed Protein A (100 μl, 10 mg/ml in water) solution was added to gold nanoparticles (50 ml, pH 8) at room temperature. The reaction mixture swelled for 10 min immediately after the introduction of the protein and the resultant conjugate was purified through centrifugation (12,000 rpm, 10 min and 22 1C) and washed with water three times. The conjugate was finally redispersed in borate buffer (5 ml, OD¼10) and stored at 4 1C until further use. 2.4. Preparation of conjugate pads The MTB 38 kDa monoclonal antibody standard concentration of 1 mg/ml was diluted to standard analyte concentrations from 5 to 300 ng/ml. The Millipore GF041 conjugate pad was used to immobilize the detection conjugate on the immunochromatographic test strip. The conjugate and sample pads were first blocked with 30% Bovine Serum Albumin (BSA), 10% Polyvinylpyrrolidone (PVP) and 2.5% Triton X-100 for 2 min and dried at 37 1C for 30 min. The detection conjugates (300 ml with 20% sucrose) at
different concentrations were sprayed onto the conjugate pads at a rate of 10 ml/cm. After drying for 30 min at 37 1C, the conjugate pads were stored at room temperature in sealed aluminum foil pouches containing 1 g desiccant. 2.5. Preparation of membrane The MTB 6–14–38 kDa fusion antigen (1 mg/ml) in phosphate buffered saline (PBS) was used as the capture antigen on the immunochromatographic tests nitrocellulose membrane. The MTB fusion 38 kDa antigen (30 ml, 1 mg/ml) and anti-Protein A (30 ml, 1 mg/ml) were dispensed onto the nitrocellulose membrane as the test and control lines respectively. After drying the membrane for 30 min at 37 1C, it was blocked with a membrane blocking buffer (MBB) that consisted of 1% sucrose, 1% BSA, 2.5% PVP and 0.1 M NaH2PO4. The membrane was then dried for another 30 min at 37 1C. 2.6. Assembly of the strip 2.6.1. Basic strip assembly (single channel) All strip components were assembled onto the plastic backing support along with the analytical membrane with the immobilized capture proteins as indicated in Fig. S3(a) (Supporting document). The dried conjugate pad was attached to the backing support with a 1–2 mm overlap onto the analytical membrane on the side of the test line. The absorbent pad was attached to the support at the other end of the membrane with a 1–2 mm overlap. The assembled single channel was then cut into 5 mm thick strips and stored at room temperature in a sealed microarray storage aluminum pouch. 2.6.2. Dual channel strip assembly All strip components were assembled onto the plastic backing support following procedure discussed in single channel strip assembling with the exception of the additional secondary conjugate pad. The primary conjugate pad was attached directly to the backing support with a 2 mm overlap onto the analytical membrane on the side of the test line. The secondary conjugate pad (sample pad) was attached on top of the primary conjugate pad with a simple polymer separator as illustrated in Fig. S3(b) (Supporting information). The absorbent pad was attached as discussed for single channel assembling. The assembled dual channel card was cut into 5 mm thick strips and stored at room temperature in a sealed microarray storage aluminum pouch. 2.7. Experimental testing procedure for single and dual channels The MTB 38 kDa antibody at each concentration from 5 to 300 ng/ml reacts with the gold nanoparticle–Protein A detection conjugate and forms a gold nanoparticle–Protein A–antibody complex. The complex binds to the capture antigen on the membrane and forms a bright red colored band at the test line which is the characteristic of a positive test result. The absence of the antibody leads to the absence of the red band on the test line as a symbol of a negative test result. To test the immunochromatographic test strips, whole blood or serum samples were spiked with the MTB 38 kDa antibody and applied to the sample pad. The blood or serum was allowed to migrate towards the membrane along with 100 ml Tris-buffered saline (TBS) at pH 7.2. After the sample had migrated from the sample pad and reached the test line of the analytical membrane, the detection conjugate was migrated through the application of an additional 100 ml TBS onto the conjugate pad. The test was left to run for an additional 5 min and the results interpreted.
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3. Results and discussion The design of the better and improved diagnostic test is crucial to eliminate false-negative and false-positive which are very common in TB diagnostics and can lead to devastating TB outbreaks. Lateral flow technology gives crucial features that symbolize it to be a most attractive and user-friendly serodiagnostic format. Lateral flow diagnostics can contribute to early diagnostic of diseases such as deadly Tuberculosis. The introduction of Protein A, an immunoglobulin-binding protein, onto the surface of the citrate-capped gold nanoparticles was conducted through the electrostatic approach. Electrostatic immobilization of biomolecules onto the surface of nanoparticles offers one of the simplest bio-functionalization methods that can be performed under mild conditions (Niemeyer, 2001; Rajh et al., 1999; Meziani and Sun, 2003). The structural assessment and dispersion of the Protein A conjugates were obtained through the use of TEM measurements and an image is presented in Fig. S1(c) (Supporting information). The conjugates were well dispersed and there was no apparent aggregation observed. The optical absorption measurements, shown in Fig. S1(d) (Supporting information), of the conjugates under various electrolytic concentrations using sodium chloride (NaCl) were performed to test nanoparticle stability. The SPR peaks were all centered at 523 nm and showed no shifts with NaCl concentrations of 0.1–1.0 M. A better UV–visible spectrum which shows the centered surface Plasmon peak of gold nanoparticle conjugate at 523 nm is clearly shown in Fig. S2 (Supporting information). Protein A bioconjugates showed remarkable stability and could be stored for months without losing their functional activity in a suitable buffer. 3.1. Immunochromatographic assay 3.1.1. Dual channel immunochromatographic assay principle The general principle of dual channel immunochromatographic test strips is shown in Fig. 1(A). The analyte of interest in the sample tested is first allowed to bind to the detection antigen to
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form antibody–antigen complex on the test line. This is subsequently followed by the capillary movement of the gold–Protein A conjugate along the membrane to the test line where an antibody– antigen complex has accumulated. The gold–Protein A conjugate binds to the antibody–antigen complex at the test line and results in the display of a red colored line. The antibody therefore serves as an anchor that links the detection conjugate to the capture antigen on the test line. The gold–protein conjugate further moves and binds to anti-protein A, where a second red line appears as a control, thus indicating the validity of the test strip. When the analyte of interest is not present in the tested sample, the detection conjugate will not bind the analyte and will continue to migrate freely along the membrane, surpass the test line, without binding and reach the control line where it binds to the immobilized protein as shown in Fig. 1(A). 3.1.2. Single channel design The basic single channel test strip allows the analyte to react with the immobilized protein on the test line before the conjugate is filtered through the nitrocellulose membrane. Although this allows for reaction of the analyte with a specific target protein, the specificity of the binding protein limits the efficiency of the immunochromatographic assay. As illustrated in Fig. 1(B), the probability of the binding protein to cross-react with a variety of analytes present in the test sample, especially with the use of whole blood or serum samples, is increased with the use of a protein such as Protein A. This reduces efficiency of the assay, thus limiting its use as an effective diagnostic device. 3.1.3. Single channel testing The greatest challenge in current test devices is the use of whole blood without prior modification. The use of whole blood in test devices allows tests to be conducted almost anywhere without the use of sophisticated equipment and lengthy processes. A direct immunochromatographic method was used to test for the TB antibodies as shown in the strips in Fig. 2(A)–(D). The negative and positive tests are clearly distinguishable in the buffered
Positive Negative After migration of blood After migration of positive blood and conjugate
Positive
Negative After migration of conjugate
Antigen Protein A Antiprotein A Antibodies in blood
Negative
Gold Conjugate Fig. 1. Diagram showing the principle of the dual and single pathway immunochromatographic test strips compared with the standard immunochromatographic test strip. (A) Dual channel immunochromatographic test strip when blood is used. (B) Single channel process that occurs during the use of blood in the single path immunochromatographic test strip.
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Fig. 2. Image shows the test strips constructed for use with direct immunochromatographic assay. The strips were tested on both buffer (A, B) and whole blood (C, D) at an analyte concentration of 5–100 ng/ml (A, C, Negative test; B, D, Positive test). Images showing the test strips constructed using the double conjugate pad technique. Strips were tested on both buffer (E, F) and whole blood (G, H) (E, G, Negative test; F, H, Positive test).
system as shown in Fig. 2(A) and (B), while the direct use of whole blood greatly decreases the test performance as shown in Fig. 2 (C) and (D). When whole blood was used (Fig. 2(C) and (D)), the negative (C) and positive (D) tests on the immunochromatographic test strips showed the same negative result. The results observed in the single channel showed the degree of cross-reactivity, thus prompting the development of a simple dual channel test strip that shall eliminate the interaction of test sample (blood and serum) with conjugate. The dual channel will allow the MTB 38 kDa antibody to interact with the antigen in the test line before Protein A conjugate thus reducing the cross-reactivity.
3.1.4. Two channel immunochromatographic assay redesign For more effective detection of TB in a whole blood sample, an improved test strip design was developed and patented (Tshikhudo et al., 2012). The basic design shown in Fig. S3(b) (Supporting information) allows the specific binding of Protein A to the analyte of choice by eliminating the possibility of cross reaction with related analytes. The dual channel assays design utilizes a plastic membrane to prevent non-specific binding of the Protein-A conjugate. Dual path immunoassay has been reported by Esfandiari (2007), a lateral flow with two strips connected into T-shaped. In his invention, the first strip was used for the flow path of the sample and the second for running buffer. However, this invention made the cassette to be larger to allow for fabrication of these two strips; as a result the cost of the whole fabrication process rises up. Our design is in line with this invention that an analyte sample must be allowed to react with the test line before a running conjugate is run through. Our design proves to be simpler and cost effective as both the strips were overlaid and separated by a plastic; as a result there was no need of designing larger plastic cassettes for these tests. The dual channel strip proposed in Fig. S3(b) (Supporting information) was put to the test and compared to the standard immunochromatographic test strip method. Fig. 3 illustrates the test strips used during the experiment. With the use of whole blood (Fig. 3), the strip performance increased dramatically. The background was minimized, with the test and control lines clearly visible. The intensities of the test and control lines increased compared to the single channel, indicating an increase in visual detection. This may be due to a decrease in non-binding analytes on the membrane surface, which prevents the complete transfer of the analyte to the test and control lines. The test was designed to eliminate the non-specific binding that may be caused by the contents of blood or plasma. It can be noted that Protein A
can bind to any immunoglobulin antibodies, especially the IgGs in either blood or serum. To eliminate this non-specific binding, the dual channel design used in this test allowed the interaction of the test sample (blood or serum) with test line before the Protein A conjugate was run through. This design is in line with the previously reported serodiagnostic test by Greenwald, which was useful to demonstrate the application of TB serodiagnosis in various host species (Greenwald et al., 2003; Lyashchenko et al., 2006). However, one of the common limitations of serodiagnostic methods compared to cell-mediated immune response assays, such as the intradermal test, is the fact that in TB infection, the antibody response develops at the latter stage (Andersen et al., 2000; Lyashchenko et al., 2004). Thus, the dual channel design can be easily distinguished between the false positive due to nonspecific binding and the actual presence of the antibody response due to TB infection. The effect of a change in the concentration of the TB antibody, used as an analyte on the test and control lines in a TB lateral flow assay, was investigated. Fig. 3(A) shows the immunochromatographic test strips used in the experiment. It shows the appearance of both the test and control lines due to the reaction of the MTB 38 kDa antibody with the respective lines with increasing concentration (0–100 ng/ml). The intensity of the test line increases with an increase in the concentration of the TB antibody as shown in Table S1 (Supporting information). As the antibody concentration increases, the amount of antibody that binds to the Protein A–gold complex increases, thus increasing the amount of the antibody–Protein A–gold complex. The increased amount of complex then binds to the immobilized capture antigen, producing a deeper or more intense line color. In combination, as the concentration of the antibody increases, the intensity of the control line decreases. The control line intensity depends on the concentration of the antibody–unbound Protein A–gold complex; an increase in the concentration of the antibody would decrease the amount of free Protein A–gold complex available for binding to the control line. This would thus decrease the intensity of the control line with an increase in the antibody concentration. The contrary would also hold; a decrease in antibody concentration would lead to an increase in control line intensity. The relationship between the intensity and concentration is plotted in Fig. S6 (Supporting information). The intensity and concentration of an analyte are linearly proportional as obviously graphically demonstrated in Fig. S6 (Supporting information). The intensities of the test and control lines were measured with an ESE Quanti lateral flow assay strip reader to determine the intensities of the test and control lines. The intensity values of the test and control lines are shown in Table 1S (Supporting
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Fig. 3. (A) Images of the test strips showing the effect of the analyte concentration on the intensities of the test and control lines in a TB lateral flow device. The MTB 38 kDa antibody was used as an analyte at a concentration range of 0–100 ng/ml. (B) Graphs showing the change in the intensities of the test and control lines in lateral flow assay with respect to change in the concentration of the MTB 38 kDa antibody (analyte).
information) and are associated with the magnitude of the areas of the resultant peaks in Fig. 3(B). An increase in the peak area is related to an increase in the color intensity of the lines. At 0 ng/ml, the test line has some intensity which has been treated as the background referred to as control line in Fig. S5 (Supporting information); this is shown by the appearance of the test line only indicating a negative test. The area of the peaks increased with an increase in the MTB 38 kDa antibody concentration. There is almost no change in peak area between 80 ng/ml and 100 ng/ml concentrations as can be seen in Table 1S (Supporting information). This may indicate the saturation of the test at these concentrations. The Protein A–gold conjugate is either completely used up or all the available conjugate molecules have reacted with the analyte in the test sample resulting in a limit on the reactable complexes. This is highly unlikely as the unreacted Protein A–gold complex reacts with the control line. As a control line is visible, there had to have been unreacted complex available for this reaction to occur. The maximum amount of antigen–antibody– Protein A–gold complexes could have been formed on the test line in contrast. In this case, the antibody–Protein A–gold complex has reacted with the entire available antigen/capture antibody on the test line, resulting in maximum detection capability for the
specific capture antibody concentration on the test line. Further studies were conducted in serum samples spiked with MTB antibodies, and the results are shown in Figs. S4 and S5 (Supporting information). It was found that both test and control lines were clearly visible and they both increase with increasing concentration of the MTB antibodies from 0 to 300 ng/ml spiked into the serum samples. The intensities of serum samples are shown in Fig. S5 (Supporting information). It was established that as the concentration of MTB antibodies increased from 0 to 300 ng/ml, both control and test lines were visible for the concentration of 10–300 ng/ml, and as expected the control line was only observed at zero analyte concentration which showed that the assay was valid. The visibility of the both test line and control line was further evaluated for their intensities, and it was found that as the concentration increased the intensity linearly increased as well (Fig. S5, Supporting information).
4. Conclusion Dual channel serodiagnostic assay was successfully developed. It was clear from experimental results that the use of a common
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single channel immunochromatographic test would not be effective for the detection of the MTB 38 kDa antibody. A modified dual channel immunochromatographic test proved to be more effective with clear distinction between the positive and negative tests with the use of whole blood and serum. The use of an ESE Quanti reader confirmed the increase in the intensity of the test line on the immunochromatographic strip with an increase in antibody concentration. The maximum detection for the immobilized capture antibody on the test line was established with no increase in line intensity observed at antibody concentrations of 80 and 100 ng/ml. The present work does not address whether the experimental infections models used for test development and evaluation adequately reflect naturally occurring Tuberculosis infections. The detection limit of 5 ng/ml does not represent the actual TB antibody titer in blood or serum; it was used to evaluate the effectiveness and to mimic the actual samples. Thus, additional work is in progress in our laboratories to probe the sensitivity and selectivity of these serological tests in real samples which were acquired from the World Health Organization (WHO). These samples from WHO shall assist us in evaluating the minimum antibody titer that can be detected using the prepared lateral flow test devices. Acknowledgment The authors would like to thank Mintek/NIC for permission to publish this work and for financial support. PSM would also like to thank the NRF for financial support through Thuthuka Post-PhD programme Grant number: TTK1206051037. Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.10.019. References Andersen, P., Munk, M.E., Pollock, J.M., Doherty, T.M., 2000. Lancet 356, 1099–1104. Baram, P., Soltysik, L., Condoulis, W., 1971. Lab. Anim. Sci. 21, 727–733. Chan, E.D., Reves, R., Belisie, J.T., Brennan, P.J., Hahn, W.E., 2000. Am. J. Respir. Crit. Care Med. 161, 1713–1719.
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