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Specific detection of tetanus toxoid using an aptamer-based matrix
Accepted Manuscript Title: Specific detection of tetanus toxoid using an aptamer-based matrix Author: Harshvardhan B. Modh Ankan K. Bhadra Kinjal A. Patel Rajeev K. Chaudhary Nishant K. Jain Ipsita Roy PII: DOI: Reference:
S0168-1656(16)31503-6 http://dx.doi.org/doi:10.1016/j.jbiotec.2016.09.004 BIOTEC 7664
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
13-7-2016 30-8-2016 12-9-2016
Please cite this article as: Modh, Harshvardhan B., Bhadra, Ankan K., Patel, Kinjal A., Chaudhary, Rajeev K., Jain, Nishant K., Roy, Ipsita, Specific detection of tetanus toxoid using an aptamer-based matrix.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Specific detection of tetanus toxoid using an aptamer-based matrix Harshvardhan B. Modha, Ankan K. Bhadrab, Kinjal A. Patel, Rajeev K. Chaudharyc, Nishant K. Jaind and Ipsita Roy*
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India Running title: ALISA of tetanus toxoid
a
Present address Institut für Technische Chemie Callinstrasse 5, D 30167 Hannover, Germany b Present address Department of Cell Biology & Physiology Washington University School of Medicine St. Louis, MO 63110, USA c Present address Sun Pharma Advanced Research Company (SPARC Ltd.) Baroda 390020, India d Present address Pfizer, Inc. San Diego, CA 92121, USA
* Author to whom correspondence should be addressed at Tel: 91-172-229 2061 Fax: 91-172-221 4692 Email: [email protected]
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Highlights High affinity aptamers with distinct recognition sites on tetanus toxoid were identified An antibody-less sandwich ALISA protocol was designed for detection of tetanus toxoid The assay was as sensitive as antibody-based ELISA in detecting tetanus toxoid In vitro synthesized aptamers provide a more robust platform than antibodies
Abstract Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwichALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid. Abbreviations ALISA (Aptamer-linked immobilized sorbent assay); ELISA (Enzyme linked immunosorbent assay); Nucleic acid aptamers Keywords ALISA (aptamer-linked immobilized sorbent assay); Aptamers; ELISA (enzymelinked immunosorbent assay); Tetanus toxoid
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1. Introduction Vaccines have played an unambiguous role in the success of various global immunization programmes. Sporadic failures may result from the denaturation of the vaccine when exposed to stress conditions. Thus, stability of the vaccine during production, transport and storage is a major concern. Lower potency of the vaccine may lead to inadequate protection and require re-vaccination (PATH, 2010; Centers for Disease Control and Prevention, 2012). Downstream processing of biologicals leads to a lot of variation in product characteristics among different batches (Alahamad et al., 2010; Rustichelli et al., 2013; Oliveira et al., 2014). Quality control of various batches is thus an important issue. The testing and retesting of these batches at the level of the manufacturer and the regulatory authorities require a large number of laboratory animals. Although no precise figures are available, it is generally assumed that numbers exceed 15% of total animals used in biomedical research (Hendricksen, 2006; 2007; Schiffelers et al., 2007). In vaccine research and development, laboratory animals are used for a wide range of purposes, such as adjuvant selection, for testing immunogenicity, immunokinetics and safety of the selected antigen components (Stickings et al., 2011). Animals are also used for batch release testing so there is no variation remaining in the final product. Apart from ethical issues, use of animals for such studies is also expensive and time consuming. Potency testing of vaccines has been a highlight of the so-called 3Rs research (reduction, refinement and replacement of animals used in research and testing). According to this, the protective antibody response is estimated by an in vitro method. In case of tetanus toxoid, for example, the method of choice is ELISA (enzyme-linked 3
immunosorbent assay) or the toxin binding inhibition assay. Testing the antigenic purity of tetanus toxoid by ELISA has now become a standard method of analysis (Determan et al., 2006; Matejtschuk et al., 2009; Stickings et al., 2011; Tiernay et al., 2011; Jain et al., 2013; Jetani et al., 2014; Juan-Giner et al., 2014; Lockyer et al., 2015). Its application in quality testing in the manufacturing process of tetanus toxoid is limited by the sensitivity of the assay. Since minor differences among batches can result in a vaccine losing its potency, the sensitivity of the assay needs to be high enough to detect minor changes in the antigenicity of the toxoid. The major problem with any antibody-based diagnostic technique is the availability and stability of the antibody itself, especially in tropical countries where variation in temperature and moisture may result in denaturation of the antibody ligand. Aptamers are short, single-stranded DNA or RNA sequences which recognize their targets on the basis of shape complementarity with high degree of specificity and affinity. Aptamers have been selected against a wide variety of targets such as proteins like growth and clotting factors, cell-surface proteins, cancer cells, small molecules such as nucleotides, antibiotics, organic dyes, cofactors, sugars, amino acids, etc. They have been used in fields as varied as drug discovery and development, target validation, analysis, diagnostics, etc. (Kaur and Roy, 2008; Cerchia and de Franciscis, 2010; Keefe et al., 2010; Kim et al., 2010; Hu et al., 2014; Ma et al., 2015). Aptamers have often been referred to as ‘chemical antibodies’ because of the high affinity that they exhibit for their targets. In contrast to antibodies, however, they are easier to handle and manipulate since they are synthesized in vitro and can be selected against self or toxic targets. They have been able to substitute antibodies in almost all the 4
areas where the latter are used and in some cases, have also overtaken the application of antibodies (Keefe et al., 2010; Lollo et al., 2014; McConnell et al., 2014). One such application is that of ELISA. We have earlier selected specific RNA aptamers against tetanus toxoid, which bind to the protein with high affinity and stabilize the toxoid against aggregation when exposed to different stress conditions (Jain et al., 2013; Jetani et al., 2014). Thus, they have been referred to as ‘universal stabilizers’ of proteins. We report here that some of these nucleic acid sequences bind to different ‘aptatopes’ on the toxoid and hence can be employed as capture-detection pair in a diagnostic assay by replacing antibodies in the traditional format of the in vitro measurement. This approach will minimize the use of biological reagents and improve the robustness of the assay. 2. Materials and Methods Deoxyribonucleotides (dNTPs), ribonucleotides (rNTPs), ribonuclease A, Corning® 96-well plates (catalogue no. 3590, 3912 and 3925) and aluminium hydroxide gel (13 mg/ml, AlhydrogelTM, Cat. No. A8222) were purchased from Sigma-Aldrich, Bangalore, India. RNase free DNase I, T7 RNA polymerase and yeast inorganic pyrophosphatase were purchased from Fermentas Inc., Maryland, USA. RNaseOUT was obtained from Invitrogen Corporation, California, USA. GoTaq® Flexi DNA polymerase and PCR Clean-Up System were obtained from Promega Corporation, Madison, USA. Fluorescein RNA labeling mix was obtained from Roche Applied Science, Mumbai, India. Mouse anti-tetanus toxoid monoclonal antibody (HYB 27801, raised against full length formaldehyde inactivated tetanus toxoid) was obtained
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from Santa Cruz Biotechnology, Inc., California, USA. All other reagents and chemicals used were of analytical grade or higher. 2.1 Synthesis and purification of RNA aptamers RNA sequences showing high affinity for tetanus toxoid (Table 1) were selected earlier by SELEX (sequential evolution of ligands by exponential enrichment), an iterative selection process (Jain et al., 2013). Glycerol stocks of five different clones (named as TT-13, TT-17, TT-20, TT-23 and TT-24) were inoculated in 10 ml of Luria bertani (LB) media containing ampicillin (100 µg/ml), incubated at 37 °C with shaking at 200 rpm overnight. Grown cells were harvested by centrifugation. Plasmid DNA was extracted by alkaline lysis method (Sambrook and Russell, 2001). The isolated plasmids were subjected to polymerase chain reaction (PCR) using the primers and conditions described earlier (Jain et al., 2013). In vitro transcription was carried out with the amplified product and the transcribed product was purified by 8 % urea denaturing polyacrylamide gel electrophoresis. 2.2 Displacement assay Dot blot assay was carried out to check RNA-protein interaction. Tetanus toxoid (100 nM) was added in the SELEX buffer (50 mM phosphate buffer, pH 7.4 containing 150 mM NaCl and 3 or 4 mM MgCl2). A constant amount (70 ng) of fluorescein labelled and different amounts of unlabelled RNA (in increasing order) were incubated in a reaction volume of 50 l for 2 h at 25 °C. Samples were filtered through a pre-wetted PVDF membrane (0.45 m) using a 96-well vacuum filtration manifold (WhatmanBiometra, Goettingen, Germany). The membrane was washed with SELEX buffer and dried between folds of filter papers. Fluorescence intensity of the retained RNA6
protein complex was measured using an image scanner (Typhoon Trio, GE Healthcare) in the fluorescence mode. 2.3 Aptamer-linked immobilized sorbent assay (ALISA) Unlabelled aptamer (0.5-2 ng/l, in SELEX buffer) or mouse anti-tetanus toxoid monoclonal antibody (2-4 ng/l, in 50 mM carbonate buffer, pH 9.6) was coated on a 96-well microtitre plate for 14 h at 24 °C with shaking at 300 rpm. Unbound aptamer/antibody was removed by washing with SELEX buffer. Unbound sites in wells were blocked with 0.2 M glycine for 30 min at 24 °C, followed by washing with SELEX buffer. Non-specific binding was eliminated by blocking with 2 % BSA for 6 h, followed by washing with SELEX buffer. Tetanus toxoid prepared in SELEX buffer was added to the wells and incubated for 2 h at 24 °C, 300 rpm. Unbound protein was removed by washing with SELEX buffer. Fluorescein-labelled aptamer (0.7 ng/l) was added to each well and incubated for 2 h at 24 °C. Wells were washed with SELEX buffer and the fluorescence intensity in the wells was read at 526 nm, using an excitation wavelength of 488 nm. For direct ALISA, increasing amounts of tetanus toxoid were coated on a 96-well microtitre plate for 18 h at 24 °C. Unbound toxoid was removed by washing with SELEX buffer. Unreacted sites were blocked with glycine and BSA, as before. Labelled aptamer (0.7 ng/l) was added to each well and incubated for 2 h at 24 °C. Wells were washed with SELEX buffer and the fluorescence intensity in the wells was read at 526 nm, using an excitation wavelength of 488 nm. 2.4 Exposure of adjuvanted tetanus toxoid to stress conditions
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Tetanus toxoid was adsorbed on alumina as described earlier (Solanki et al., 2011, 2012; Jetani et al., 2014). Adsorbed toxoid was subjected to thermal stress by incubating the preparation at 50 °C with mild shaking for 150 min. Mechanical stress was administered by agitating the samples at 300 rpm for 2 h at 37 °C. The adjuvanted preparation was also subjected to freeze-thawing stress by five cycles of incubation at -20 °C for 12 h, followed by 2 h in a water bath at 37 °C. Details of stress conditions have been described earlier (Jetani et al., 2014). In each case, after completion of stress exposure, the suspensions were centrifuged at 500 g for 3 min, the pellet was resuspended in 10 mM sodium phosphate buffer, pH 7.4 and capture ALISA was performed to determine residual antigenicity of the adsorbed toxoid, as described above. 2.5 Enzyme-linked immunosorbent assay (ELISA) After immobilizing tetanus toxoid on a microtitre plate (catalogue no. CLS3590) as described above, mouse anti-tetanus toxoid monoclonal antibody (1:5000) was used as the primary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:3000) was used as the secondary antibody. Tetanus toxoid was detected after addition of tetramethyl benzidine/H2O2 (TMB/H2O2) as the substrate for HRP. The absorbance of solution in the wells was measured at 450 nm after terminating the reaction with 0.02 N H2SO4 (Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014). 3. Results 3.1 Displacement assay
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In order to determine the binding specificities of the aptamers, i.e. whether they bind at the same or different sites on tetanus toxoid, displacement studies were carried out by dot blot assay. For this, labelled and unlabelled aptamers were added to tetanus toxoid in a reaction mixture and incubated for 2 h at 25 °C. The amount of labelled aptamer was constant and the amount of unlabelled aptamer was varied. If both aptamers bind at the same site on tetanus toxoid, as the amount of unlabelled aptamer is increased, it will displace the labelled aptamer and the fluorescence intensity of the complex retained on the membrane will be reduced. If the aptamers bind to different sites, no significant change in fluorescence intensity would be observed upon increase in concentration of the unlabelled aptamer. In order to validate the displacement assay, labelled TT-24 was allowed to bind to tetanus toxoid and challenged with unlabelled TT-24. As equilibrium is set up between the labelled aptamer and tetanus toxoid leading to the formation of proteinRNA complex, increasing the amount of the unlabelled aptamer will lead to the displacement of the labelled aptamer from the surface of the protein surface. This will result in reduction in the fluorescence intensity of the protein-RNA complex retained on the membrane. As the amount of unlabelled RNA (TT-24) was increased, the intensity of the retained complex decreased on the membrane in a concentrationdependent manner (Figure 1A). Thus, this positive control validates the use of displacement assay in this work. Next, constant amounts of different labelled aptamers (TT-13, TT-14, TT-20 and TT23) were mixed with increasing amounts of unlabelled TT-24 and incubated with tetanus toxoid. The fluorescence intensity of the protein-RNA complex retained on the 9
membrane was determined by densitometry. The fluorescence intensity of the proteinRNA complex with labelled TT-17 was found to decrease with increasing amounts of unlabelled TT-24 (Figure 1A). Thus, TT-24 was able to displace TT-17 from the protein surface, indicating that these two aptamers probably bind to the same site on the toxoid. Similarly, the fluorescence intensity of the protein-RNA complex with labelled TT-20 was found to decrease with increasing amounts of unlabelled TT-24 (Figure 1A), although not to the same extent as with labelled TT-17, showing that TT20 and TT-24 recognize overlapping regions on the surface of the toxoid. A similar result was seen with labelled TT-13-protein complex. With increasing amount of unlabelled TT-24, the fluorescence intensity of the protein-RNA complex was found to decrease (Figure 1A), although the reduction was much less than with TT-17. Thus, TT-13 and TT-24 may partially share binding site on the surface of the protein. No significant difference in fluorescence intensity of the protein-RNA complex was observed with labelled TT-23 when challenged with increasing amounts of unlabelled TT-24 (Figure 1A). Thus, TT-24 was not able to displace TT-23 from the protein surface, indicating that these two aptamers bind to different regions of the toxoid. Similar comparative measurements were carried out with other aptamer pairs. With increasing amount of unlabelled TT-23, significant reduction in fluorescence intensity of protein-RNA complex in case of labelled TT-13 and TT-17 was observed (Figure 1B), demonstrating the partial displacement of TT-13 and TT-17 by TT-23 from the surface of the toxoid. On the other hand, unlabelled TT-23 was not able to displace labelled TT-20 (Figure 1B). Thus, it is likely that TT-13, TT-17 and TT-23 may share binding sites on tetanus toxoid. It is probable that TT-20 binds to a different site on 10
the protein. On being tested with increasing amounts of unlabelled TT-13, only partial reduction in fluorescence intensity of the protein-RNA complex was seen with labelled TT-17 and TT-20 (Figure 1C). This confirms that TT-13, TT-17 and TT-20 partially share the ‘aptatope’ on the toxoid. Similar overlapping recognition of the toxoid surface was seen with partial displacement of labelled TT-20 by unlabelled TT17 too (Figure 1D). 3.2 Aptamer-linked immobilized sorbent assay (ALISA) Five monoclonal aptamer sequences were selected which have high affinity and selectivity towards tetanus toxoid (Jain et al., 2013). In displacement studies, it was found that two pairs of aptamers were not able to displace each other while the remaining pairs could displace each other from the surface of the protein (tetanus toxoid) (Figure 2A). Thus, the aptamer pairs belonging to the first category (with ≥80 % retention of fluorescence intensity) were not able to displace each other and presumably bound to different regions on the protein. These could be probable candidates to carry out sandwich ALISA. The other aptamers would be suitable candidates for detection of tetanus toxoid, either alone (direct ALISA), or when paired with tetanus toxoid-specific monoclonal antibody, if the latter does not bind to the same epitope as the aptamer. Aptamers TT-20 and TT-23 were shown to bind to different regions on tetanus toxoid (Figure 1B). Hence these were chosen as capture and detection pair for sandwich ALISA. Unlabelled aptamer TT-23 was used as the capture aptamer and labelled aptamer TT-20 was used as the detection aptamer. The capture aptamer was immobilized on three different plates (Table 2), followed by addition of different 11
amounts of the analyte (tetanus toxoid) and a constant amount of the labelled detection aptamer. Detectable and reproducible results could be obtained only with Corning® plate (catalogue no. CLS3912) (Figure 2B) and this plate was used for further studies. The specificity of the test was confirmed by running a control. In this case, the unlabelled (capture) aptamer was not coated on the wells. Tetanus toxoid (5 ng) was added to the well after blocking the unreacted sites in the well with glycine followed by BSA, as described in the Methods section, and was quantified using labelled (detection) aptamer. Negligible fluorescence intensity was observed in this case as compared to coating the well with the capture aptamer (Figure 2C). No toxoid was detected in the washings in the latter case showing that all the protein added could be ‘captured’ by the aptamer. Thus, the increase in fluorescence intensity observed with increasing amounts of tetanus toxoid (Figure 2B) was due to the detection of the toxoid by the sandwich pair and not due to non-specific adsorption of the protein on the well. In order to optimize the amount of capture aptamer, different amounts of the unlabelled aptamer TT-23 were used to coat the wells. With 0.25 ng/l of the aptamer, the resultant fluorescence intensity was found to be quite low (Figure 3A). With 0.5 or 1 ng/l of the capture aptamer, the fluorescence intensity was found to be in the linear range. No significant difference in fluorescence intensity could be observed between the two cases, indicating that 0.5 ng/l of the capture aptamer is sufficient to bind to the highest amount of the toxoid (100 ng). Surprisingly, with 2 ng/l of the capture aptamer, the fluorescence intensity was found to be saturated at higher amounts of the toxoid (Figure 3A). In order to determine the sensitivity of the assay, the experiment 12
was repeated with lower amounts of tetanus toxoid. At lower amounts, both 1 ng/l and 2 ng/l of the capture aptamer showed linear curves over the whole range, i.e. till 5 ng toxoid (Figure 3B). Thus, the signal intensity is linear at lower amounts of the analyte but reaches saturation at higher amounts with higher amounts of the capture aptamer. Next, we compared the use of mouse anti-tetanus toxoid monoclonal antibody with unlabelled TT-23 as the capture reagent. Labelled TT-20 was used as the detection aptamer in both cases. At a lower concentration of the antibody (2 ng/l), the fluorescence intensity of detection was lower while the signal output was higher with a higher concentration of the antibody (4 ng/l) (Figure 4A). Thus, the signal intensity of response did not show any difference irrespective of whether the monoclonal antibody or the aptamer was used as the capture reagent. As the aptamer pair TT-23 and TT-24 was shown to bind to different regions on the toxoid (Figures 1A and 2A), the above experiment was repeated with unlabelled TT24 (2 ng/l) as the capture aptamer and labelled TT-23 as the detection aptamer. This pair was also shown to exhibit reproducible and linear curve with increasing amounts of the analyte (1-5 ng) (Figure 4B). At higher amounts of the toxoid (5-20 ng), the curve for fluorescence intensity was found to reach saturation (Figure 4B), similar to the other pair (TT-20 and TT-23) (Figure 3A). Thus, the method described here is reproducible and not limited to a single pair of aptamers being used for detection of the analyte. 3.3 Direct ALISA
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We repeated the conventional protocol of capture ELISA (Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014) by replacing the antibody with the aptamer for detection. Increasing amounts of tetanus toxoid (1-5 ng) were coated on the walls of the wells. Detection was carried out with labelled TT-23 and TT-24 (0.7 ng/l). Curves obtained with both of the aptamers were found to be linear (Figure 5A) at lower amounts of the analyte and were found to saturate at higher amounts (Figure 5B). Using mouse anti-tetanus toxoid monoclonal antibody as the detection agent (Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014) confirmed that there was no reduction in sensitivity of detection upon using aptamers (Figure 5A). 3.4 Exposure of adjuvanted tetanus toxoid to stress conditions We have shown earlier that when exposed to stress conditions, adjuvanted tetanus toxoid is partially desorbed from the matrix (Jetani et al., 2014). The residual adsorbed toxoid is partially inactive, as determined by measurement of antigenicity by ELISA. In order to determine whether the method reported here has broad applicability, we subjected adjuvanted tetanus toxoid to three stress conditions, viz. thermal, mechanical and freeze-thawing, and determined the residual antigenicity of the adsorbed toxoid by ALISA using unlabelled TT-23 as the capture aptamer and labelled TT-20 as the detection aptamer. In all cases, loss of antigenicity of the adsorbed toxoid was observed (Table 3). Comparison with antigenicity values reported by ELISA [13] showed that there was no significant difference between the measurements using antibodies or aptamers as ligands (Table 3). As exposure to accelerated stress conditions leads to changes in the native conformation of tetanus toxoid (Solanki et al., 2011; 2012), our results show that the selected aptamers are 14
capable of discriminating between antigenically active and inactive forms of tetanus toxoid. Thus, the method developed here can measure changes in antigenicity of tetanus toxoid occurring during the production, storage and transport steps and has comparable sensitivity to conventional ELISA. Thus, it may replace the antibody in the analytical measurement. 4. Discussion Potency of vaccines is confirmed by in vivo assays. These assays are approved by the European Pharmacopoeia and World Health Organization (WHO). In these assays, multiple dilutions of a reference and test preparations are required which leads to the need of a large number of animals (Hendriksen et al., 1987; Hendriksen, 2006; 2007; Schiffelers et al., 2007; Council of Europe, 2013; 2014). The collaborative studies done by National Institute for Biological Standards and Control (NIBSC) and the European Pharmacopoeia have led to the validation of serological methods used for testing the potency of tetanus vaccine (Stickings et al., 2011). These studies validated a correlation between the results obtained by animal challenge and in vitro analysis methods including ELISA and the toxin neutralization test (TNT) assays for checking the potency of testing of vaccines. In order to decrease the use of animals, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) are promoting alternative methods that will help in reduce, refine (less pain and distress) and replace animal use in testing the vaccines. The primary objective of this work was to develop a method which will replace animal testing, 15
minimize the use of antibodies from biological sources and thus reduce batch-to-batch variation in the toxoid production process. For detection of hVEGF (human vascular endothelial growth factor) ELISA method has LOD (limit of detection) of 10 pg but in case of aptamer, the LOD is 100 fg (Yoshida et al., 2009). The aptamer-based assay was able to detect 1 ng of tetanus toxoid, which was the same as with ELISA. Lower concentrations of capture aptamers (0.25 ng/l) were able to detect the maximum amount of analyte although the fluorescence readout was low. This problem could be solved with using marginally higher concentrations (0.5 ng/l) of the aptamers. A diagnostic tool with a pair of aptamers (and no antibody) was recently reported for the detection of Escherichia coli O157:H7 (Wu et al., 2015). Detection was however based on a coupled amplification step. Some of the aptamer pairs, which were found to bind to different sites on the surface of the toxoid, were used for sandwich ALISA. Some of the aptamers were used for direct ALISA for detection of tetanus toxoid. Two pairs of aptamers [(TT-20 and TT-23) and (TT-23 and TT-24)] were confirmed to bind at different positions on the surface of tetanus toxoid. They were further used for development of sandwich ALISA. They were able to detect 1 ng tetanus toxoid. Sensitivity of ELISA for tetanus toxoid was also confirmed. 1 ng of tetanus toxoid could be determined by ELISA. A major drawback of the use of aptamers is their nuclease-instability during in vivo administration (Cerchia and de Franciscis, 2010; Keefe et al., 2010). In the present case, this is not a limiting factor as the application envisaged is in vitro analysis of batch-to-batch variation of the toxoid during production. 5. Conclusion 16
Aptamers are selected by an in vitro evolution process such that they exhibit very high specificity and selectivity for their target molecule. They possess several advantages over antibodies which make them suitable for use in diagnostic and clinical applications. Their in vitro synthesis and amenability to chemical modification for improved stability make them attractive ligands for any binding/inhibitions studies where antibodies could potentially be employed (Jayasena, 1999; Keefe et al., 2010; Lollo et al., 2014; McConnell et al., 2014). Reproducibility of antibody generation protocols in cell cultures or polysera, especially for large-scale production, introduces additional variables. The main purpose of this study was to establish a wholly antibody-less aptamer-linked immobilized sorbent assay (ALISA) for detection of tetanus toxoid. We have shown earlier that the selected aptamers bind specifically to tetanus toxoid and do not show affinity for other proteins with similar properties (Jain et al., 2013). Our developed method matches the sensitivity of the present gold standard method for detection of tetanus toxoid. Complete replacement of antibodies with aptamers makes this protocol less susceptible to adverse environmental conditions and reduces the loss of reproducibility commonly associated with the use of antibodies.
6. Acknowledgements Tetanus toxoid was obtained as a gift from Shantha Biotechnics Ltd., Hyderabad, India. This work was partially supported by Indian Council of Medical Research (ICMR). AKB, KAP, RKC and NKJ acknowledge the award of senior research fellowships from DST-INSPIRE programme, Department of Biotechnology, ICMR 17
and Council for Scientific and Industrial Research, respectively. The funding agency had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
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McConnell, E.M., Holahan, M.R., DeRosa, M.C., 2014. Aptamers as promising molecular recognition elements for diagnostics and therapeutics in the central nervous system. Nucleic Acid Ther 24, 388-404. Oliveira, C., Teixeira, J.A., Domingues, L., 2014. Recombinant production of plant lectins in microbial systems for biomedical application - the frutalin case study. Front. Plant Sci. 5, 390. PATH, 2010. Immunization logistics and supply systems: From vision to action. Seattle: PATH. Rustichelli, D., Castiglia, S., Gunetti, M., Mareschi, K., Signorino, E., Muraro, M., Castello, L., Sanavio, F., Leone, M., Ferrero, I., Fagioli, F., 2013. Validation of analytical methods in compliance with good manufacturing practice: a practical approach. J. Transl. Med. 11, 197. Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratory manual, third ed. New York: Cold spring harbor laboratory press. Schiffelers, M.J., Blaauboer, B.J., Fentener van Vlissingen, J.M., Kuil, J. et al., 2007. Factors stimulating or obstructing the implementation of the 3Rs in the regulatory process. ALTEX 24, 271-278. Solanki, V.A., Jain, N.K., Roy, I., 2011. Stabilization of tetanus toxoid formulation containing aluminium hydroxide adjuvant against freeze-thawing. Int. J. Pharm. 414, 140-147. Solanki, V.A., Jain, N.K., Roy, I., 2012. Stabilization of tetanus toxoid formulation containing aluminium hydroxide adjuvant against agitation. Int. J. Pharm. 423, 297302. 21
Stickings, P., Rigsby, P., Coombes, L., Hockley, J., Tierney, R., Sesardic, D., 2011. Animal refinement and reduction: alternative approaches for potency testing of diphtheria and tetanus vaccines. Procedia Vaccinol. 5, 200-212. Tierney, R., Stickings, P., Hockley, J., Rigsby, P., Iwaki, M., Sesardic, D. 2011. Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed. Biologicals 39, 404-416. Wu W., Zhao, S., Mao, Y., Fang, Z., Lu, X., Zeng, L., 2015. A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification. Anal. Chim. Acta 861, 62-68. Yoshida, Y., Horii, K., Sakai, N., Masuda, H., Furuichi, M., Waga, I., 2009. Antibody-specific aptamer-based PCR analysis for sensitive protein detection. Anal. Bioanal. Chem. 395, 1089-1096.
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Figure 1. Displacement assay was carried out to determine the overlap of binding site of the aptamers on the surface of the toxoid. Densitometric analysis of the retained fluorescence intensity of the RNA-protein complex was carried out using ImageQuantTL software (GE Healthcare). Results are shown for increasing amounts of unlabelled (A) TT-24, (B) TT-23, (C) TT-13 and (D) TT-17 aptamers, in the presence of constant amount (0.7 ng/l) of fluorescein labelled TT-13 (cross), TT-17 (triangle), TT-20 (circle), TT-23 (square) and TT-24 (star). In each case, duplicate pairs are avoided. The fluorescence intensity of the RNA-protein complex in the absence of any unlabelled aptamer has been arbitrarily assigned a value of 100 % in each case. Values shown are mean±s.e.m. of three independent experiments.
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Figure 2. Standardization of conditions for carrying out ALISA. (A) Summary of binding results obtained in Figure 1. The table shows the retention of fluorescence intensity (in %) when increasing amounts of unlabelled (UL) aptamer is added to a constant amount of labelled (L) aptamer complexed with tetanus toxoid. Pairs which showed ≥80 % retention of fluorescence intensity on the PDVF membrane were assumed to bind to distinct sites on the surface of the protein and were considered to be suitable for sandwich ALISA. (B) ALISA was carried out with three different Corning® 96-well plates. Features of the plates are described in Table 2. Values shown are mean±s.e.m. of three independent experiments. (C) Specificity of the assay was confirmed by incubating tetanus toxoid on the blocked plate in the presence (sample) or absence (control) of labelled capture aptamer (TT-23). The fluorescence intensity of the well in the presence of the capture aptamer has been arbitrarily assigned a value of 100 % in each case. Values shown are mean±s.e.m. of three independent experiments. 24
Figure 3. Standardization of ALISA conditions was carried out at (A) higher and (B) lower amounts of tetanus toxoid as the analyte. TT-23 was used as the capture aptamer and labelled TT-20 was used as the detection aptamer. Values shown are mean±s.e.m. of three independent experiments.
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Figure 4. Validation of sandwich ALISA. (A) The experiments were repeated using mouse anti-tetanus toxoid monoclonal antibody (solid lines) as the capture agent. Results with TT-23 as the capture agent (dashed lines) are shown for comparison. In both cases, labelled TT-20 was used as the detection agent. (B) The experiment was also repeated using unlabelled TT-24 (2 ng/l) as the capture agent and labelled TT23 (0.7 ng/l) as the detection agent. Values shown are mean±s.e.m. of three independent experiments.
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Figure 5. Comparison of aptamers with antibody for detection of tetanus toxoid by direct measurement. (A) Lower and (B) higher amounts of tetanus toxoid were immobilized on microtitre plate (Sigma catalogue no. CLS3590) wells. Capture ELISA was carried out using mouse monoclonal antibody (diamond, dashed line) and detection of absorbance was carried out at 450 nm (secondary axis). Capture ALISA was carried out using 1 ng/l of labelled TT-23 (triangle, solid line) or TT-24 (circle, solid line). Values shown are mean±s.e.m. of three independent experiments.
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Table 1. RNA aptamers selected against tetanus-toxoid (Jain et al., 2013) and used in this work. Values represent mean±s.e.m. of three independent experiments. Designation TT-13 TT-17 TT-20 TT-23 TT-24
Length of sequence 97 bp 97 bp 97 bp 97 bp 97 bp
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Kd, nM 142±22 271±11 224±25 223±22 181±40
Table 2. Properties of Corning® 96-well plates used in this work. Catalog No. Properties* (Sigma) High binding surface binds medium (>10kD) and large biomolecules CLS3590 that possess ionic groups and/or hydrophobic regions Not treated (or medium binding) polystyrene surface is hydrophobic in nature and binds biomolecules through passive interactions. White CLS3912 microplates enhance luminescent signals and have low background luminescence and fluorescence. High binding surface is capable of binding medium (>10kD) and large biomolecules that possess ionic groups and/or hydrophobic regions. CLS3925 Black microplates have low background fluorescence and minimize light scattering. * As per information available at the company website
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Table 3. Comparison of ALISA in detecting changes in antigenicity of tetanus toxoid. Adjuvanted tetanus toxoid was subjected to thermal, mechanical and freeze-thawing stress as already described. Antigenicity of the toxoid retained on the matrix was determined using ALISA. In both cases, 100 % antigenicity refers to the antigenicity of the adsorbed tetanus toxoid which was incubated at 4 °C for the same time period as the stress condition. Values shown are mean±s.e.m. of three independent experiments.
Stress Thermal Mechanical Freeze-thawing
Residual antigenicity, % Antibody Aptamer (Jetani et al., 2014) 63.40 ± 1.30 71.18 ± 4.16 50.73 ± 2.09 46.69 ± 3.30 24.54 ± 3.07 31.46 ± 6.10
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p-value 0.148 0.294 0.368
S0168-1656(16)31503-6 http://dx.doi.org/doi:10.1016/j.jbiotec.2016.09.004 BIOTEC 7664
To appear in:
Journal of Biotechnology
Received date: Revised date: Accepted date:
13-7-2016 30-8-2016 12-9-2016
Please cite this article as: Modh, Harshvardhan B., Bhadra, Ankan K., Patel, Kinjal A., Chaudhary, Rajeev K., Jain, Nishant K., Roy, Ipsita, Specific detection of tetanus toxoid using an aptamer-based matrix.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Specific detection of tetanus toxoid using an aptamer-based matrix Harshvardhan B. Modha, Ankan K. Bhadrab, Kinjal A. Patel, Rajeev K. Chaudharyc, Nishant K. Jaind and Ipsita Roy*
Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India Running title: ALISA of tetanus toxoid
a
Present address Institut für Technische Chemie Callinstrasse 5, D 30167 Hannover, Germany b Present address Department of Cell Biology & Physiology Washington University School of Medicine St. Louis, MO 63110, USA c Present address Sun Pharma Advanced Research Company (SPARC Ltd.) Baroda 390020, India d Present address Pfizer, Inc. San Diego, CA 92121, USA
* Author to whom correspondence should be addressed at Tel: 91-172-229 2061 Fax: 91-172-221 4692 Email: [email protected]
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Highlights High affinity aptamers with distinct recognition sites on tetanus toxoid were identified An antibody-less sandwich ALISA protocol was designed for detection of tetanus toxoid The assay was as sensitive as antibody-based ELISA in detecting tetanus toxoid In vitro synthesized aptamers provide a more robust platform than antibodies
Abstract Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwichALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid. Abbreviations ALISA (Aptamer-linked immobilized sorbent assay); ELISA (Enzyme linked immunosorbent assay); Nucleic acid aptamers Keywords ALISA (aptamer-linked immobilized sorbent assay); Aptamers; ELISA (enzymelinked immunosorbent assay); Tetanus toxoid
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1. Introduction Vaccines have played an unambiguous role in the success of various global immunization programmes. Sporadic failures may result from the denaturation of the vaccine when exposed to stress conditions. Thus, stability of the vaccine during production, transport and storage is a major concern. Lower potency of the vaccine may lead to inadequate protection and require re-vaccination (PATH, 2010; Centers for Disease Control and Prevention, 2012). Downstream processing of biologicals leads to a lot of variation in product characteristics among different batches (Alahamad et al., 2010; Rustichelli et al., 2013; Oliveira et al., 2014). Quality control of various batches is thus an important issue. The testing and retesting of these batches at the level of the manufacturer and the regulatory authorities require a large number of laboratory animals. Although no precise figures are available, it is generally assumed that numbers exceed 15% of total animals used in biomedical research (Hendricksen, 2006; 2007; Schiffelers et al., 2007). In vaccine research and development, laboratory animals are used for a wide range of purposes, such as adjuvant selection, for testing immunogenicity, immunokinetics and safety of the selected antigen components (Stickings et al., 2011). Animals are also used for batch release testing so there is no variation remaining in the final product. Apart from ethical issues, use of animals for such studies is also expensive and time consuming. Potency testing of vaccines has been a highlight of the so-called 3Rs research (reduction, refinement and replacement of animals used in research and testing). According to this, the protective antibody response is estimated by an in vitro method. In case of tetanus toxoid, for example, the method of choice is ELISA (enzyme-linked 3
immunosorbent assay) or the toxin binding inhibition assay. Testing the antigenic purity of tetanus toxoid by ELISA has now become a standard method of analysis (Determan et al., 2006; Matejtschuk et al., 2009; Stickings et al., 2011; Tiernay et al., 2011; Jain et al., 2013; Jetani et al., 2014; Juan-Giner et al., 2014; Lockyer et al., 2015). Its application in quality testing in the manufacturing process of tetanus toxoid is limited by the sensitivity of the assay. Since minor differences among batches can result in a vaccine losing its potency, the sensitivity of the assay needs to be high enough to detect minor changes in the antigenicity of the toxoid. The major problem with any antibody-based diagnostic technique is the availability and stability of the antibody itself, especially in tropical countries where variation in temperature and moisture may result in denaturation of the antibody ligand. Aptamers are short, single-stranded DNA or RNA sequences which recognize their targets on the basis of shape complementarity with high degree of specificity and affinity. Aptamers have been selected against a wide variety of targets such as proteins like growth and clotting factors, cell-surface proteins, cancer cells, small molecules such as nucleotides, antibiotics, organic dyes, cofactors, sugars, amino acids, etc. They have been used in fields as varied as drug discovery and development, target validation, analysis, diagnostics, etc. (Kaur and Roy, 2008; Cerchia and de Franciscis, 2010; Keefe et al., 2010; Kim et al., 2010; Hu et al., 2014; Ma et al., 2015). Aptamers have often been referred to as ‘chemical antibodies’ because of the high affinity that they exhibit for their targets. In contrast to antibodies, however, they are easier to handle and manipulate since they are synthesized in vitro and can be selected against self or toxic targets. They have been able to substitute antibodies in almost all the 4
areas where the latter are used and in some cases, have also overtaken the application of antibodies (Keefe et al., 2010; Lollo et al., 2014; McConnell et al., 2014). One such application is that of ELISA. We have earlier selected specific RNA aptamers against tetanus toxoid, which bind to the protein with high affinity and stabilize the toxoid against aggregation when exposed to different stress conditions (Jain et al., 2013; Jetani et al., 2014). Thus, they have been referred to as ‘universal stabilizers’ of proteins. We report here that some of these nucleic acid sequences bind to different ‘aptatopes’ on the toxoid and hence can be employed as capture-detection pair in a diagnostic assay by replacing antibodies in the traditional format of the in vitro measurement. This approach will minimize the use of biological reagents and improve the robustness of the assay. 2. Materials and Methods Deoxyribonucleotides (dNTPs), ribonucleotides (rNTPs), ribonuclease A, Corning® 96-well plates (catalogue no. 3590, 3912 and 3925) and aluminium hydroxide gel (13 mg/ml, AlhydrogelTM, Cat. No. A8222) were purchased from Sigma-Aldrich, Bangalore, India. RNase free DNase I, T7 RNA polymerase and yeast inorganic pyrophosphatase were purchased from Fermentas Inc., Maryland, USA. RNaseOUT was obtained from Invitrogen Corporation, California, USA. GoTaq® Flexi DNA polymerase and PCR Clean-Up System were obtained from Promega Corporation, Madison, USA. Fluorescein RNA labeling mix was obtained from Roche Applied Science, Mumbai, India. Mouse anti-tetanus toxoid monoclonal antibody (HYB 27801, raised against full length formaldehyde inactivated tetanus toxoid) was obtained
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from Santa Cruz Biotechnology, Inc., California, USA. All other reagents and chemicals used were of analytical grade or higher. 2.1 Synthesis and purification of RNA aptamers RNA sequences showing high affinity for tetanus toxoid (Table 1) were selected earlier by SELEX (sequential evolution of ligands by exponential enrichment), an iterative selection process (Jain et al., 2013). Glycerol stocks of five different clones (named as TT-13, TT-17, TT-20, TT-23 and TT-24) were inoculated in 10 ml of Luria bertani (LB) media containing ampicillin (100 µg/ml), incubated at 37 °C with shaking at 200 rpm overnight. Grown cells were harvested by centrifugation. Plasmid DNA was extracted by alkaline lysis method (Sambrook and Russell, 2001). The isolated plasmids were subjected to polymerase chain reaction (PCR) using the primers and conditions described earlier (Jain et al., 2013). In vitro transcription was carried out with the amplified product and the transcribed product was purified by 8 % urea denaturing polyacrylamide gel electrophoresis. 2.2 Displacement assay Dot blot assay was carried out to check RNA-protein interaction. Tetanus toxoid (100 nM) was added in the SELEX buffer (50 mM phosphate buffer, pH 7.4 containing 150 mM NaCl and 3 or 4 mM MgCl2). A constant amount (70 ng) of fluorescein labelled and different amounts of unlabelled RNA (in increasing order) were incubated in a reaction volume of 50 l for 2 h at 25 °C. Samples were filtered through a pre-wetted PVDF membrane (0.45 m) using a 96-well vacuum filtration manifold (WhatmanBiometra, Goettingen, Germany). The membrane was washed with SELEX buffer and dried between folds of filter papers. Fluorescence intensity of the retained RNA6
protein complex was measured using an image scanner (Typhoon Trio, GE Healthcare) in the fluorescence mode. 2.3 Aptamer-linked immobilized sorbent assay (ALISA) Unlabelled aptamer (0.5-2 ng/l, in SELEX buffer) or mouse anti-tetanus toxoid monoclonal antibody (2-4 ng/l, in 50 mM carbonate buffer, pH 9.6) was coated on a 96-well microtitre plate for 14 h at 24 °C with shaking at 300 rpm. Unbound aptamer/antibody was removed by washing with SELEX buffer. Unbound sites in wells were blocked with 0.2 M glycine for 30 min at 24 °C, followed by washing with SELEX buffer. Non-specific binding was eliminated by blocking with 2 % BSA for 6 h, followed by washing with SELEX buffer. Tetanus toxoid prepared in SELEX buffer was added to the wells and incubated for 2 h at 24 °C, 300 rpm. Unbound protein was removed by washing with SELEX buffer. Fluorescein-labelled aptamer (0.7 ng/l) was added to each well and incubated for 2 h at 24 °C. Wells were washed with SELEX buffer and the fluorescence intensity in the wells was read at 526 nm, using an excitation wavelength of 488 nm. For direct ALISA, increasing amounts of tetanus toxoid were coated on a 96-well microtitre plate for 18 h at 24 °C. Unbound toxoid was removed by washing with SELEX buffer. Unreacted sites were blocked with glycine and BSA, as before. Labelled aptamer (0.7 ng/l) was added to each well and incubated for 2 h at 24 °C. Wells were washed with SELEX buffer and the fluorescence intensity in the wells was read at 526 nm, using an excitation wavelength of 488 nm. 2.4 Exposure of adjuvanted tetanus toxoid to stress conditions
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Tetanus toxoid was adsorbed on alumina as described earlier (Solanki et al., 2011, 2012; Jetani et al., 2014). Adsorbed toxoid was subjected to thermal stress by incubating the preparation at 50 °C with mild shaking for 150 min. Mechanical stress was administered by agitating the samples at 300 rpm for 2 h at 37 °C. The adjuvanted preparation was also subjected to freeze-thawing stress by five cycles of incubation at -20 °C for 12 h, followed by 2 h in a water bath at 37 °C. Details of stress conditions have been described earlier (Jetani et al., 2014). In each case, after completion of stress exposure, the suspensions were centrifuged at 500 g for 3 min, the pellet was resuspended in 10 mM sodium phosphate buffer, pH 7.4 and capture ALISA was performed to determine residual antigenicity of the adsorbed toxoid, as described above. 2.5 Enzyme-linked immunosorbent assay (ELISA) After immobilizing tetanus toxoid on a microtitre plate (catalogue no. CLS3590) as described above, mouse anti-tetanus toxoid monoclonal antibody (1:5000) was used as the primary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:3000) was used as the secondary antibody. Tetanus toxoid was detected after addition of tetramethyl benzidine/H2O2 (TMB/H2O2) as the substrate for HRP. The absorbance of solution in the wells was measured at 450 nm after terminating the reaction with 0.02 N H2SO4 (Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014). 3. Results 3.1 Displacement assay
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In order to determine the binding specificities of the aptamers, i.e. whether they bind at the same or different sites on tetanus toxoid, displacement studies were carried out by dot blot assay. For this, labelled and unlabelled aptamers were added to tetanus toxoid in a reaction mixture and incubated for 2 h at 25 °C. The amount of labelled aptamer was constant and the amount of unlabelled aptamer was varied. If both aptamers bind at the same site on tetanus toxoid, as the amount of unlabelled aptamer is increased, it will displace the labelled aptamer and the fluorescence intensity of the complex retained on the membrane will be reduced. If the aptamers bind to different sites, no significant change in fluorescence intensity would be observed upon increase in concentration of the unlabelled aptamer. In order to validate the displacement assay, labelled TT-24 was allowed to bind to tetanus toxoid and challenged with unlabelled TT-24. As equilibrium is set up between the labelled aptamer and tetanus toxoid leading to the formation of proteinRNA complex, increasing the amount of the unlabelled aptamer will lead to the displacement of the labelled aptamer from the surface of the protein surface. This will result in reduction in the fluorescence intensity of the protein-RNA complex retained on the membrane. As the amount of unlabelled RNA (TT-24) was increased, the intensity of the retained complex decreased on the membrane in a concentrationdependent manner (Figure 1A). Thus, this positive control validates the use of displacement assay in this work. Next, constant amounts of different labelled aptamers (TT-13, TT-14, TT-20 and TT23) were mixed with increasing amounts of unlabelled TT-24 and incubated with tetanus toxoid. The fluorescence intensity of the protein-RNA complex retained on the 9
membrane was determined by densitometry. The fluorescence intensity of the proteinRNA complex with labelled TT-17 was found to decrease with increasing amounts of unlabelled TT-24 (Figure 1A). Thus, TT-24 was able to displace TT-17 from the protein surface, indicating that these two aptamers probably bind to the same site on the toxoid. Similarly, the fluorescence intensity of the protein-RNA complex with labelled TT-20 was found to decrease with increasing amounts of unlabelled TT-24 (Figure 1A), although not to the same extent as with labelled TT-17, showing that TT20 and TT-24 recognize overlapping regions on the surface of the toxoid. A similar result was seen with labelled TT-13-protein complex. With increasing amount of unlabelled TT-24, the fluorescence intensity of the protein-RNA complex was found to decrease (Figure 1A), although the reduction was much less than with TT-17. Thus, TT-13 and TT-24 may partially share binding site on the surface of the protein. No significant difference in fluorescence intensity of the protein-RNA complex was observed with labelled TT-23 when challenged with increasing amounts of unlabelled TT-24 (Figure 1A). Thus, TT-24 was not able to displace TT-23 from the protein surface, indicating that these two aptamers bind to different regions of the toxoid. Similar comparative measurements were carried out with other aptamer pairs. With increasing amount of unlabelled TT-23, significant reduction in fluorescence intensity of protein-RNA complex in case of labelled TT-13 and TT-17 was observed (Figure 1B), demonstrating the partial displacement of TT-13 and TT-17 by TT-23 from the surface of the toxoid. On the other hand, unlabelled TT-23 was not able to displace labelled TT-20 (Figure 1B). Thus, it is likely that TT-13, TT-17 and TT-23 may share binding sites on tetanus toxoid. It is probable that TT-20 binds to a different site on 10
the protein. On being tested with increasing amounts of unlabelled TT-13, only partial reduction in fluorescence intensity of the protein-RNA complex was seen with labelled TT-17 and TT-20 (Figure 1C). This confirms that TT-13, TT-17 and TT-20 partially share the ‘aptatope’ on the toxoid. Similar overlapping recognition of the toxoid surface was seen with partial displacement of labelled TT-20 by unlabelled TT17 too (Figure 1D). 3.2 Aptamer-linked immobilized sorbent assay (ALISA) Five monoclonal aptamer sequences were selected which have high affinity and selectivity towards tetanus toxoid (Jain et al., 2013). In displacement studies, it was found that two pairs of aptamers were not able to displace each other while the remaining pairs could displace each other from the surface of the protein (tetanus toxoid) (Figure 2A). Thus, the aptamer pairs belonging to the first category (with ≥80 % retention of fluorescence intensity) were not able to displace each other and presumably bound to different regions on the protein. These could be probable candidates to carry out sandwich ALISA. The other aptamers would be suitable candidates for detection of tetanus toxoid, either alone (direct ALISA), or when paired with tetanus toxoid-specific monoclonal antibody, if the latter does not bind to the same epitope as the aptamer. Aptamers TT-20 and TT-23 were shown to bind to different regions on tetanus toxoid (Figure 1B). Hence these were chosen as capture and detection pair for sandwich ALISA. Unlabelled aptamer TT-23 was used as the capture aptamer and labelled aptamer TT-20 was used as the detection aptamer. The capture aptamer was immobilized on three different plates (Table 2), followed by addition of different 11
amounts of the analyte (tetanus toxoid) and a constant amount of the labelled detection aptamer. Detectable and reproducible results could be obtained only with Corning® plate (catalogue no. CLS3912) (Figure 2B) and this plate was used for further studies. The specificity of the test was confirmed by running a control. In this case, the unlabelled (capture) aptamer was not coated on the wells. Tetanus toxoid (5 ng) was added to the well after blocking the unreacted sites in the well with glycine followed by BSA, as described in the Methods section, and was quantified using labelled (detection) aptamer. Negligible fluorescence intensity was observed in this case as compared to coating the well with the capture aptamer (Figure 2C). No toxoid was detected in the washings in the latter case showing that all the protein added could be ‘captured’ by the aptamer. Thus, the increase in fluorescence intensity observed with increasing amounts of tetanus toxoid (Figure 2B) was due to the detection of the toxoid by the sandwich pair and not due to non-specific adsorption of the protein on the well. In order to optimize the amount of capture aptamer, different amounts of the unlabelled aptamer TT-23 were used to coat the wells. With 0.25 ng/l of the aptamer, the resultant fluorescence intensity was found to be quite low (Figure 3A). With 0.5 or 1 ng/l of the capture aptamer, the fluorescence intensity was found to be in the linear range. No significant difference in fluorescence intensity could be observed between the two cases, indicating that 0.5 ng/l of the capture aptamer is sufficient to bind to the highest amount of the toxoid (100 ng). Surprisingly, with 2 ng/l of the capture aptamer, the fluorescence intensity was found to be saturated at higher amounts of the toxoid (Figure 3A). In order to determine the sensitivity of the assay, the experiment 12
was repeated with lower amounts of tetanus toxoid. At lower amounts, both 1 ng/l and 2 ng/l of the capture aptamer showed linear curves over the whole range, i.e. till 5 ng toxoid (Figure 3B). Thus, the signal intensity is linear at lower amounts of the analyte but reaches saturation at higher amounts with higher amounts of the capture aptamer. Next, we compared the use of mouse anti-tetanus toxoid monoclonal antibody with unlabelled TT-23 as the capture reagent. Labelled TT-20 was used as the detection aptamer in both cases. At a lower concentration of the antibody (2 ng/l), the fluorescence intensity of detection was lower while the signal output was higher with a higher concentration of the antibody (4 ng/l) (Figure 4A). Thus, the signal intensity of response did not show any difference irrespective of whether the monoclonal antibody or the aptamer was used as the capture reagent. As the aptamer pair TT-23 and TT-24 was shown to bind to different regions on the toxoid (Figures 1A and 2A), the above experiment was repeated with unlabelled TT24 (2 ng/l) as the capture aptamer and labelled TT-23 as the detection aptamer. This pair was also shown to exhibit reproducible and linear curve with increasing amounts of the analyte (1-5 ng) (Figure 4B). At higher amounts of the toxoid (5-20 ng), the curve for fluorescence intensity was found to reach saturation (Figure 4B), similar to the other pair (TT-20 and TT-23) (Figure 3A). Thus, the method described here is reproducible and not limited to a single pair of aptamers being used for detection of the analyte. 3.3 Direct ALISA
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We repeated the conventional protocol of capture ELISA (Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014) by replacing the antibody with the aptamer for detection. Increasing amounts of tetanus toxoid (1-5 ng) were coated on the walls of the wells. Detection was carried out with labelled TT-23 and TT-24 (0.7 ng/l). Curves obtained with both of the aptamers were found to be linear (Figure 5A) at lower amounts of the analyte and were found to saturate at higher amounts (Figure 5B). Using mouse anti-tetanus toxoid monoclonal antibody as the detection agent (Determan et al., 2006; Jain et al., 2013; Jetani et al., 2014) confirmed that there was no reduction in sensitivity of detection upon using aptamers (Figure 5A). 3.4 Exposure of adjuvanted tetanus toxoid to stress conditions We have shown earlier that when exposed to stress conditions, adjuvanted tetanus toxoid is partially desorbed from the matrix (Jetani et al., 2014). The residual adsorbed toxoid is partially inactive, as determined by measurement of antigenicity by ELISA. In order to determine whether the method reported here has broad applicability, we subjected adjuvanted tetanus toxoid to three stress conditions, viz. thermal, mechanical and freeze-thawing, and determined the residual antigenicity of the adsorbed toxoid by ALISA using unlabelled TT-23 as the capture aptamer and labelled TT-20 as the detection aptamer. In all cases, loss of antigenicity of the adsorbed toxoid was observed (Table 3). Comparison with antigenicity values reported by ELISA [13] showed that there was no significant difference between the measurements using antibodies or aptamers as ligands (Table 3). As exposure to accelerated stress conditions leads to changes in the native conformation of tetanus toxoid (Solanki et al., 2011; 2012), our results show that the selected aptamers are 14
capable of discriminating between antigenically active and inactive forms of tetanus toxoid. Thus, the method developed here can measure changes in antigenicity of tetanus toxoid occurring during the production, storage and transport steps and has comparable sensitivity to conventional ELISA. Thus, it may replace the antibody in the analytical measurement. 4. Discussion Potency of vaccines is confirmed by in vivo assays. These assays are approved by the European Pharmacopoeia and World Health Organization (WHO). In these assays, multiple dilutions of a reference and test preparations are required which leads to the need of a large number of animals (Hendriksen et al., 1987; Hendriksen, 2006; 2007; Schiffelers et al., 2007; Council of Europe, 2013; 2014). The collaborative studies done by National Institute for Biological Standards and Control (NIBSC) and the European Pharmacopoeia have led to the validation of serological methods used for testing the potency of tetanus vaccine (Stickings et al., 2011). These studies validated a correlation between the results obtained by animal challenge and in vitro analysis methods including ELISA and the toxin neutralization test (TNT) assays for checking the potency of testing of vaccines. In order to decrease the use of animals, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) are promoting alternative methods that will help in reduce, refine (less pain and distress) and replace animal use in testing the vaccines. The primary objective of this work was to develop a method which will replace animal testing, 15
minimize the use of antibodies from biological sources and thus reduce batch-to-batch variation in the toxoid production process. For detection of hVEGF (human vascular endothelial growth factor) ELISA method has LOD (limit of detection) of 10 pg but in case of aptamer, the LOD is 100 fg (Yoshida et al., 2009). The aptamer-based assay was able to detect 1 ng of tetanus toxoid, which was the same as with ELISA. Lower concentrations of capture aptamers (0.25 ng/l) were able to detect the maximum amount of analyte although the fluorescence readout was low. This problem could be solved with using marginally higher concentrations (0.5 ng/l) of the aptamers. A diagnostic tool with a pair of aptamers (and no antibody) was recently reported for the detection of Escherichia coli O157:H7 (Wu et al., 2015). Detection was however based on a coupled amplification step. Some of the aptamer pairs, which were found to bind to different sites on the surface of the toxoid, were used for sandwich ALISA. Some of the aptamers were used for direct ALISA for detection of tetanus toxoid. Two pairs of aptamers [(TT-20 and TT-23) and (TT-23 and TT-24)] were confirmed to bind at different positions on the surface of tetanus toxoid. They were further used for development of sandwich ALISA. They were able to detect 1 ng tetanus toxoid. Sensitivity of ELISA for tetanus toxoid was also confirmed. 1 ng of tetanus toxoid could be determined by ELISA. A major drawback of the use of aptamers is their nuclease-instability during in vivo administration (Cerchia and de Franciscis, 2010; Keefe et al., 2010). In the present case, this is not a limiting factor as the application envisaged is in vitro analysis of batch-to-batch variation of the toxoid during production. 5. Conclusion 16
Aptamers are selected by an in vitro evolution process such that they exhibit very high specificity and selectivity for their target molecule. They possess several advantages over antibodies which make them suitable for use in diagnostic and clinical applications. Their in vitro synthesis and amenability to chemical modification for improved stability make them attractive ligands for any binding/inhibitions studies where antibodies could potentially be employed (Jayasena, 1999; Keefe et al., 2010; Lollo et al., 2014; McConnell et al., 2014). Reproducibility of antibody generation protocols in cell cultures or polysera, especially for large-scale production, introduces additional variables. The main purpose of this study was to establish a wholly antibody-less aptamer-linked immobilized sorbent assay (ALISA) for detection of tetanus toxoid. We have shown earlier that the selected aptamers bind specifically to tetanus toxoid and do not show affinity for other proteins with similar properties (Jain et al., 2013). Our developed method matches the sensitivity of the present gold standard method for detection of tetanus toxoid. Complete replacement of antibodies with aptamers makes this protocol less susceptible to adverse environmental conditions and reduces the loss of reproducibility commonly associated with the use of antibodies.
6. Acknowledgements Tetanus toxoid was obtained as a gift from Shantha Biotechnics Ltd., Hyderabad, India. This work was partially supported by Indian Council of Medical Research (ICMR). AKB, KAP, RKC and NKJ acknowledge the award of senior research fellowships from DST-INSPIRE programme, Department of Biotechnology, ICMR 17
and Council for Scientific and Industrial Research, respectively. The funding agency had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
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Figure 1. Displacement assay was carried out to determine the overlap of binding site of the aptamers on the surface of the toxoid. Densitometric analysis of the retained fluorescence intensity of the RNA-protein complex was carried out using ImageQuantTL software (GE Healthcare). Results are shown for increasing amounts of unlabelled (A) TT-24, (B) TT-23, (C) TT-13 and (D) TT-17 aptamers, in the presence of constant amount (0.7 ng/l) of fluorescein labelled TT-13 (cross), TT-17 (triangle), TT-20 (circle), TT-23 (square) and TT-24 (star). In each case, duplicate pairs are avoided. The fluorescence intensity of the RNA-protein complex in the absence of any unlabelled aptamer has been arbitrarily assigned a value of 100 % in each case. Values shown are mean±s.e.m. of three independent experiments.
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Figure 2. Standardization of conditions for carrying out ALISA. (A) Summary of binding results obtained in Figure 1. The table shows the retention of fluorescence intensity (in %) when increasing amounts of unlabelled (UL) aptamer is added to a constant amount of labelled (L) aptamer complexed with tetanus toxoid. Pairs which showed ≥80 % retention of fluorescence intensity on the PDVF membrane were assumed to bind to distinct sites on the surface of the protein and were considered to be suitable for sandwich ALISA. (B) ALISA was carried out with three different Corning® 96-well plates. Features of the plates are described in Table 2. Values shown are mean±s.e.m. of three independent experiments. (C) Specificity of the assay was confirmed by incubating tetanus toxoid on the blocked plate in the presence (sample) or absence (control) of labelled capture aptamer (TT-23). The fluorescence intensity of the well in the presence of the capture aptamer has been arbitrarily assigned a value of 100 % in each case. Values shown are mean±s.e.m. of three independent experiments. 24
Figure 3. Standardization of ALISA conditions was carried out at (A) higher and (B) lower amounts of tetanus toxoid as the analyte. TT-23 was used as the capture aptamer and labelled TT-20 was used as the detection aptamer. Values shown are mean±s.e.m. of three independent experiments.
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Figure 4. Validation of sandwich ALISA. (A) The experiments were repeated using mouse anti-tetanus toxoid monoclonal antibody (solid lines) as the capture agent. Results with TT-23 as the capture agent (dashed lines) are shown for comparison. In both cases, labelled TT-20 was used as the detection agent. (B) The experiment was also repeated using unlabelled TT-24 (2 ng/l) as the capture agent and labelled TT23 (0.7 ng/l) as the detection agent. Values shown are mean±s.e.m. of three independent experiments.
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Figure 5. Comparison of aptamers with antibody for detection of tetanus toxoid by direct measurement. (A) Lower and (B) higher amounts of tetanus toxoid were immobilized on microtitre plate (Sigma catalogue no. CLS3590) wells. Capture ELISA was carried out using mouse monoclonal antibody (diamond, dashed line) and detection of absorbance was carried out at 450 nm (secondary axis). Capture ALISA was carried out using 1 ng/l of labelled TT-23 (triangle, solid line) or TT-24 (circle, solid line). Values shown are mean±s.e.m. of three independent experiments.
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Table 1. RNA aptamers selected against tetanus-toxoid (Jain et al., 2013) and used in this work. Values represent mean±s.e.m. of three independent experiments. Designation TT-13 TT-17 TT-20 TT-23 TT-24
Length of sequence 97 bp 97 bp 97 bp 97 bp 97 bp
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Kd, nM 142±22 271±11 224±25 223±22 181±40
Table 2. Properties of Corning® 96-well plates used in this work. Catalog No. Properties* (Sigma) High binding surface binds medium (>10kD) and large biomolecules CLS3590 that possess ionic groups and/or hydrophobic regions Not treated (or medium binding) polystyrene surface is hydrophobic in nature and binds biomolecules through passive interactions. White CLS3912 microplates enhance luminescent signals and have low background luminescence and fluorescence. High binding surface is capable of binding medium (>10kD) and large biomolecules that possess ionic groups and/or hydrophobic regions. CLS3925 Black microplates have low background fluorescence and minimize light scattering. * As per information available at the company website
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Table 3. Comparison of ALISA in detecting changes in antigenicity of tetanus toxoid. Adjuvanted tetanus toxoid was subjected to thermal, mechanical and freeze-thawing stress as already described. Antigenicity of the toxoid retained on the matrix was determined using ALISA. In both cases, 100 % antigenicity refers to the antigenicity of the adsorbed tetanus toxoid which was incubated at 4 °C for the same time period as the stress condition. Values shown are mean±s.e.m. of three independent experiments.
Stress Thermal Mechanical Freeze-thawing
Residual antigenicity, % Antibody Aptamer (Jetani et al., 2014) 63.40 ± 1.30 71.18 ± 4.16 50.73 ± 2.09 46.69 ± 3.30 24.54 ± 3.07 31.46 ± 6.10
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p-value 0.148 0.294 0.368