Appl Microbiol Biotechnol (2014) 98:6095–6103 DOI 10.1007/s00253-014-5777-5

METHODS AND PROTOCOLS

An application of capsid-specific artificial ankyrin repeat protein produced in E. coli for immunochromatographic assay as a surrogate for antibody Sawitree Nangola & Weeraya Thongkum & Somphot Saoin & Aftab A. Ansari & Chatchai Tayapiwatana

Received: 31 January 2014 / Revised: 11 April 2014 / Accepted: 12 April 2014 / Published online: 8 May 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Immunochromatographic strip test is a unique type of rapid test that has been developed for use as part of a diagnostic kit for the rapid detection of antibodies and/or other proteins of interest. For the detection of target proteins, most of the commercial tests are assembled based on the conjugation of colloidal gold particles to monoclonal antibodies embedded within the conjugate pad of a strip test. In this study, we tested the novel concept of using an artificial non-antibody structure for generating a colloidal gold conjugate (CGC). We exploited the property of an ankyrin repeat protein that specifically binds to the HIV-1 capsid protein termed AnkGAG1D4. This construct was applied as a model structure to create Ank1D4-CGC and used as a new type of visible detector system and termed it ankyrin-based immunochromatographic strip (ABIS) test. The ABIS test was shown to be highly sensitive with a lower limit of detection of the target protein at 0.1 μg/ml. Moreover, the ABIS test was not only highly sensitive but also shared a level of S. Nangola Division of Clinical Immunology and Transfusion Sciences, School of Allied Health Sciences, University of Phayao, Phayao 56000, Thailand A. A. Ansari Department of Pathology, Emory University School of Medicine, Atlanta, GA 30332, USA W. Thongkum : S. Saoin Division of Clinical Immunology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 52000, Thailand C. Tayapiwatana (*) Division of Clinical Immunology and Biomedical Technology Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 52000, Thailand e-mail: [email protected]

specificity within the same range of the commercial test kit. The results of the studies presented herein therefore demonstrate the novel application of an artificial nonimmunoglobulin structure (ankyrin repeat protein) as the new line of a visible detector using a rapid diagnostic test with characteristics that have the potential to be superior to those that utilize antibody-based tests. Keywords AnkGAG1D4 . HIV . Capsid protein . Immunochromatographic assay . Artificial ankyrin repeat protein

Introduction The formulation of an artificial repeat ankyrin protein of 33 amino acids that forms two antiparallel alpha helices followed by a beta hairpin loop that facilitates conjugation to the next repeat structure leading to an elongated molecule provides a unique reagent that forms the basis of this communication (Binz et al. 2003; Boersma and Plückthun 2011). This basic molecular architecture has been utilized for the construction of a novel class of scaffold protein library that has led to the generation of several target-specific binding reagents that directly evolved from two other constructed libraries that included the designed ankyrin repeat proteins (DARPins) library (Binz et al. 2003) and the artificial ankyrin repeat protein library (Nangola et al. 2012). This novel scaffold has several unique characteristics. Firstly, it shares the characteristics of binding specificity and affinity comparable to antibodies. Thus, the randomized amino acids that are located on the binding concave and the high number of internal repeats in concert promote specific interactions, resulting in a higher binding affinity (Binz et al. 2004). Secondly, since it is stably expressed at high levels in

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Escherichia coli and is conveniently purified using a simple nickel column via either N- or C-terminal, “His” tag and its low-cost production indicates its superiority over the use of conventional antibodies. Its unique compact structure does not require disulfide bonding and leaves open the option of expressing it within the bacterial cytoplasm so that it can be easily produced in a soluble form at a large-scale level (Boersma and Plückthun 2011). Thirdly, since it is a nondisulfide-containing structure, unique cysteine at specific sites can be introduced to enable the chemical coupling with several molecules of choices such as drugs, fluorescein dyes, or polyethylene glycol (PEG) (Simon et al. 2012). Therefore, the derivation of most target-specific DARPins have expanded their application to several fields such as tumor targeting and therapy (Simon et al. 2013), viral retargeting for improving the efficiency of adenoviruses (Ads) as gene transfer vectors (Dreier et al. 2013), antiviral agents by both extracellular (Mann et al. 2013) and intracellular functions (Nangola et al. 2012), diagnostics, and a number of additional biomedical applications (Huber et al. 2007). In the studies reported herein, we have utilized the repeat synthetic ankyrin protein as an efficient visual detector by applying it as a probe for an immunochromatographic method. In 2012, we reported on the successful retrieval of a capsid-specific ankyrin, AnkGAG1D4 among others, from our libraries of artificially constructed ankyrins (Nangola et al. 2012). These molecules have been characterized for their binding specificity and affinity for the HIV-1 capsid domain. Therefore, the conjugation of this AnkGAG1D4 with colloidal gold particles for use as a visual detector was reasoned to provide a unique reagent that can be utilized for the detection of the HIV-1 capsid protein in the immunochromatographic strip test (ICT) or rapid test. The prototype test kit name ankyrin-based immunochromatographic strip (ABIS) test was developed and optimized to yield a novel rapid test platform with high sensitivity and specificity. This kit utilizes a tiny amount of sample, and the result can be simply read by the naked eye. The consistency of this test was assessed by evaluating the sensitivity and specificity resulting from standard capsid-spiked samples. Results from our preliminary studies indicate that our ABIS test is suitable for further development as a novel platform as a diagnostic tool.

Materials and methods Large-scale preparation and purification of capsid-specific ankyrin AnkGAG1D4 (PDB: 4HLL_A), an artificial capsid-specific ankyrin repeat protein, was fundamentally retrieved from

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our artificial ankyrin repeat protein library by standard phage display technique as described elsewhere (Nangola et al. 2012). The gene encoding AnkGAG1D4 was subcloned into pQE30 cytoplasmic expression vector for large-scale protein preparation in bacteria M15[pREP4] strain (Qiagen). Largescale purification of ankyrins was achieved as described previously (Nangola et al. 2012). Briefly, M15[pREP4] cells containing pQE30-1D4 were cultured in a shaking incubator at 37 °C in 2 l of LB broth containing 100 μg/ml ampicillin, 25 μg/ml kanamycin, and 1 % D-glucose. When the optical density (OD) 600 reached a value of 0.8, 1 mM IPTG was added to the broth, and the cultures were incubated for additional 4 h at 30 °C with shaking. Cells were collected by centrifugation at 3,000×g in a refrigerated centrifuge at 4 °C for 30 min and resuspended with phosphate-buffered saline (PBS) containing a cocktail of protease inhibitors followed by repeated freeze/thaw cycles. Cell lysates were collected by high-speed centrifugation at 10,000×g, 4 °C for 30 min, and then loaded onto a HisTrap™ column (GE Healthcare) for protein purification. Purity and concentration of protein were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Bradford Protein Assay Kit (Bio-Rad), respectively.

Production of polyclonal anti-ankyrin antibodies New Zealand rabbits were immunized with 200 μg of purified AnkGAG1D4 emulsified in complete Freunds’ adjuvant for the first injection and incomplete Freunds’ adjuvant for three booster doses of 200 μg at monthly intervals. Rabbit sera were collected 1 week after the last injection, and the immunoglobulin fraction was precipitated by standard ammonium sulfate precipitation. In brief, an equal amount of saturated ammonium persulfate solution was added dropwise into the pooled serum with continuing agitation on ice until a precipitate was formed. The mixture was centrifuged at 12,000×g at 4 °C for 1 h. The precipitate containing the immunoglobulin fraction was dissolved with PBS and then dialyzed against PBS solution and the amount of protein determined. An aliquot of the Ig protein was evaluated for the presence of ankyrin-specific antibodies by a simple indirect ELISA test. Briefly, individual wells of a 96-well microtiter plate were coated with purified AnkGAG1D4 by incubating the plate at 4 °C for 16–18 h. Coated wells were blocked with blocking solution (2 % skimmed milk in TBS), and varying dilutions of the samples to be tested were dispensed into appropriate wells. The wells were then washed, and the bound antibodies were detected by the addition of a predetermined optimum concentration of HRP-conjugated swine anti-rabbit IgG. The wells were washed, and TMB substrate was added followed by the addition of 1 N HCl to stop all reactions. Absorbance unit was measured at 450 nm.

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Preparation of ankyrin colloidal gold conjugate The conjugation of colloidal gold to AnkGAG1D4 protein was performed essentially as previously described (Pattarawarapan et al. 2007). Purified AnkGAG1D4 protein was dialyzed against 5 mM borax buffer, and the protein concentration was quantitated using the BCA protein assay. Appropriate concentration of AnkGAG1D4 was mixed with a 15 nm colloidal gold solution at room temperature for 30 min. A solution of 5 % bovine serum albumin (BSA) dissolved in 5 mM NaCl was then added to eliminate uncoupled gold particles. AnkGAG1D4-colloidal gold-conjugated 1D4-CGC was concentrated by centrifugation at 10,000×g for 30 min at 4 °C. The pellet containing 1D4-CGC was resuspended with colloidal gold diluent, and the concentration was determined by measuring the optical density at 520 nm. Production of recombinant H6MACA and H6CA proteins in baculovirus-infected cells The viral target used as a standard target protein consisted of the His-tagged recombinant polyprotein, H6MACA, corresponding to the matrix (p17) and capsid (p24) domains of the HIV-1 Gag precursor and H6CA, corresponding to the mature capsid protein. These two proteins were prepared as previously described (Nangola et al. 2012). In brief, the baculovirus transfer vectors encoding His-tagged MACA domains of Gag (pBlueBac4.5-H6MA-CA) and His-tagged CA (pBlueBac4.5-H6CA) were generated and further cotransfected with Bac-N-Blue™ DNA into Sf9 cells (Invitrogen) following the conditions recommended by the manufacturer (Invitrogen). The recombinant viruses obtained, BV-H6MACA and BV-H6CA, were isolated using the blue plaque selection method and were amplified. Aliquots of the Sf9 cells were infected with BV-H6MACA and BV-H6CA and were harvested at 48 h postinfection (pi), lysed by freezing and thawing. The cell lysates were clarified by centrifugation at 15,000×g for 30 min at 4 °C. His-tagged Gag proteins were purified from clarified Sf9 cell lysates by affinity chromatography on HisTrap column, using ÄKTA Prime™ plus (GE Healthcare Bio-Sciences). Protein concentration was determined using the Bradford protein assay (Thermo Fisher Scientific, Inc.). Purity of His-tagged Gag proteins was assessed by SDS-PAGE analysis using a 15 % acrylamide gel and Coomassie blue staining. Production of monoclonal antibody against capsid protein M88, a monoclonal antibody against capsid protein, was produced in our laboratory as described briefly. Balb/c mice were immunized every 2 weeks with 100 μg H6MACA in 500 μl sterile PBS. The mice were sacrificed, and then spleens were removed, and spleen cells were utilized for the

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preparation of hybridomas using standard hybridoma technology. Supernatant of single clones was tested by indirect ELISA using H6MACA-coated plate and western immunoblotting using H6MACA. The M88 is a clone which gave a positive signal by both the techniques. Results obtained by western immunoblotting revealed two reactive bands of approximately 40 and 24 kDa corresponding to H6MACA and a partially cleaved form of CA (data not shown). These data strongly indicated that clone M88 was the candidate monoclonal antibody with specificity for the capsid protein. Binding activity of 1D4-CGC defined by an indirect sandwich dot blot The indirect dot blot was performed as follows. Nitrocellulose membranes were coated with either recombinant H6MACA (Nangola et al. 2012), BSA, or rabbit anti-ankyrin. Nonspecific binding signal was blocked by incubating the strips in blocking buffer (5 % BSA in PBS). A solution of 1D4-CGC solution with an OD 520 nm=1 was added to the membrane and incubated on a shaker at room temperature for 30 min. The sandwich dot blot was performed as follows. Nitrocellulose membranes were spotted with either purified monoclonal anti-CA, clone M88 (unpublished data), or rabbit anti-ankyrin. The membranes were incubated with blocking buffer to limit non-specific binding. Membranes were reacted with 100 μg/ml of H6MACA recombinant protein for 1 h. Excess amount of H6MACA was removed by subsequent washing five times with washing buffer (0.05 % Tween 20 in PBS) followed by the addition of diluted 1D4-CGC solution to the membrane incubated on a shaker at room temperature for 30 min. ABIS test The use of artificial ankyrin repeat protein as a novel reagent that serves as a visible detector was termed the ABIS test. This first prototype test was applied to detect HIV-1 capsid protein in spiked samples. The ABIS test relied on a sandwich assay format as shown in Fig. 1. A solution of 1D4-CGC with an OD 520 nm value of 60 was absorbed onto a conjugate pad. In the detection part, the membrane was coated with M88, and rabbit anti-ankyrin polyclonal antibodies were identified as the test line and control line, respectively, according to the standard manufacturing process. In principle, the ABIS test involves dipping the strip in 200 μl of sample allowing the sample to flow by capillary force through the conjugate pad. In the presence of the target molecule, H6MACA or H6CA, 1D4-CGC becomes dissolved and forms a complex with its ligand. The complex then gradually migrates towards the test line and is trapped by the immobilized M88 monoclonal antibody and forms a band that can be visualized. The remaining trace amount of 1D4-CGC migrates towards the control

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line that consists of rabbit anti-ankyrin antibody and can also be then visualized. Thus, if the sample is positive, the assay results in two red-purple lines. However, in the absent of the CA molecule, only a single line at the control area will appear. ABIS test performance In efforts to evaluate the ABIS test, strips were dipped into standard H6MACA diluted in PBS at various concentrations ranging from 0.1 to 100 μg/ml. Visible results were observed by the naked eye within 5 min. The lowest concentration giving the red-purple color at the test line was highlighted as the lower limit of detection (LOD). Each dilution was evaluated in triplicate. Both H6MACA and H6CA were utilized to evaluate the efficiency of ABIS test. In addition, strips were placed in a microtiter plate containing standard H6CA diluted in PBS at the same level as H6MACA. Results were observed, and the LOD was assessed by the same strategy. Effect of salt and pH on ABIS test H6CA was spiked into pH-adjusted phosphate buffer saline ranging from pH 5 to 10 and used as a test samples for evaluating the effect of pH. Moreover, phosphate buffer pH 7.4 containing NaCl at 0.1, 0.5, and 1.0 M was added with H6CA at various concentrations. ABIS test strips were immersed into samples and imaged within 5 min. A preliminary assessment of the ABIS test To evaluate the performance of the ABIS test under the range of conditions in which it is likely to be used in practice, plasma samples spiked with both H6MACA- and H6CA-containing capsids were utilized. Additionally, 25 samples of normal plasma and 25 blinded samples containing various concentrations of capsid protein ranging from 0.1 to 100 μg/ml were used as unknown negative and positive samples, respectively. The diagnostic sensitivity and specificity of the test were considered using a 2×2 table (Banoo et al. 2010).

Result Production and evaluation of polyclonal antibody against ankyrin AnkGAG1D4 Sera were collected following the last boost of the rabbits, and an aliquot was tested for the presence of specific antibodies against AnkGAG1D4 protein by both ELISA and western immunoblotting. Positive results were obtained when this aliquot of diluted serum was added to AnkGAG1D4-coated wells. Data displayed in Fig. 2a shows the representative net

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results (experimental − negative control values). Consequently, a single reactive band consistent with the molecular weight of ankyrin protein was observed by western immunoblotting (Fig. 2b). These results demonstrate that high titers of polyclonal anti-AnkGAG1D4 antibodies are present in the serum. To prepare a large-scale amount of the capture antibody to be utilized for spraying at the control line, polyclonal anti-AnkGAG1D4 was further isolated by the standard salting-out method. After precipitation, the gamma globulin fraction was enriched from 40 % as found in normal serum to 97 % in the precipitated gamma globulin fraction. Thus, a high concentration of polyclonal antibody was successfully isolated from serum. This precipitated polyclonal anti-AnkGAG1D4 was further applied at control line of the ABIS test kit. Binding reactivity of 1D4-CGC The conjugation efficiency and the binding reactivity of 1D4CGC were directly verified with rabbit anti-ankyrin and H6MA-CA, respectively. Red-purple dots appeared at both areas, but not at the BSA-coated control area (Fig. 3a). In addition, the intensity of red-purple dots was correlated with the amount of coated H6MACA. Moreover, the high intensity displayed by the positive dot was observed at the anti-ankyrin area. These results indicate the success of the 1D4-CGC preparation and support the view that the conjugation process did not alter the binding activity of ankyrin. Consequently, a sandwich dot blot was performed to assess the binding activity of 1D4-CGC with the soluble antigen mimicking the sandwich format of the ABIS test method (Fig. 3b). Various intensities of red-purple dots appeared at two different concentrations of M88 monoclonal antibody only in the presence of standard H6MACA. Consequently, these dots reflect the fact that the binding sites of both clone M88 and AnkGAG1D4 located at the heterogeneous positions did not appear to alter the reaction. Therefore, these results served as a guide for us to design an appropriate system of our ABIS test by using clone M88 at the test line. Performance of the ABIS test The performance of ABIS test as stated above relies on the format of the sandwich assay, where the capsid proteins are sandwiched between 1D4-CGC and the immobilized clone M88 on the nitrocellulose membrane (Fig. 1). The protocol is as follows. The sample solution is allowed to flow through the conjugate pad while dipping the ABIS test in the sample. This procedure facilitates the conjugate to become dissolved and is subsequently pulled by capillary action to flow along the nitrocellulose membrane and migrate towards the test line and control line. In the presence of the capsid protein, it binds to the clone M88 capture line together with 1D4-CGC. As a result, a red-purple color line is formed and appears on the test

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Fig. 1 Schematic of ankyrinbased immunochromatographic strip (ABIS) test for capsid detection. The ABIS test is composed of 1D4-CGC that has been previously absorbed on the conjugate pad. The monoclonal antibody clone M88 and rabbit anti-ankyrin antibodies are coated on the nitrocellulose strip at the test line and control line, respectively (a). The ABIS test involves dipping the strip into the sample to be tested. As shown, if the sample contains the capsid (c), it forms two lines, whereas in the absence of the capsid (b), it only forms a single line

line. Any excess unbound conjugate produces a purple color on the control line, this being an indication of the technical validity of the test. After a 5-min incubation period, the color intensity of the test line is initially firstly assessed visually with the naked eye. The lower LOD of the ABIS test was achieved by testing for the capsid antigen, H6MACA, serially diluted in PBS. The lowest concentration of standard capsid protein resulting in the development of the red-purple color at the test line was defined as LOD. As expected, the red-purple line appeared Fig. 2 Polyclonal antiAnkGAG1D4 production. Rabbitimmunized serum was collected to test the antibody titer by ELISA (a) and specificity by western immunoblotting (b). Protein marker and AnkGAG1D4 were loaded at lanes M and 1, respectively

readily at low-capsid protein concentrations. Thus, two positive lines could be readily observed while testing our novel ABIS platform utilizing as little as 1 μg/ml of antigen in the sample (Fig. 4a). In addition, to evaluate the interfering effect of serum proteins, ABIS strips were immersed into H6MACA serially diluted in HIV-negative plasma. As expected, the color intensity was directly proportional to the capsid concentration in the spiked plasma (Fig. 4b) without false-positive or false-negative results. At the highest concentration of protein target tested (Fig. 4c, d), the weaker signals were observed

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Fig. 3 An assessment of 1D4CGC by dot blot. The efficiency of colloidal gold conjugation and the binding activity of 1D4-CGC were evaluated by direct dot blot (a) and sandwich dot blot (b)

comparing to the signal obtained at a lower concentration. This phenomenon happened because of the limited number of 1D4-CGC coated onto the conjugated pad. Therefore, at the high concentration, there are capsid-1D4-CGC complexes and free capsid protein that migrate to the capture line, and the inhibition effect occurs resulting in the weaker signal. Furthermore, LOD of this test system showed the sensitivity of the detection with a positive test line at 0.5 μg/ml. This phenomenon resulted from the slower migration rate of plasma through the membrane compared to that of the PBS sample. This result indicates that the ABIS test might be applied to use as a potential diagnostic test for clinical use. Furthermore, to assess the performance of the ABIS strip in detecting proteins of interest in a mature form, PBS and normal plasma were spiked with recombinant H6CA and used as test samples. Interestingly, positive signals could be observed in samples containing various concentrations of protein with an intensity that correlated with the amount of protein (Fig. 4c). The LOD was as low as 0.1 μg/ml in plasma sample (Fig. 4d). This submicrogram level of mature capsid protein was able to be detectable because of the higher molar ratio compared with the full-length protein. Influence of pH and salt concentrations on the detection of HIV-1 capsid protein To address the effect of pH and salt concentration on the accuracy of the test results, ABIS test strips were immersed in H6CA-spiked PBS with a pH ranging from 5 to 10. Positive signals appeared at all tested conditions (Fig. 5a), reflecting that 1D4-CGC was stable at a broad range of pH. With regards to the effect of high-salt concentration inducing gold colloidal precipitation, we additionally assessed the range of salt concentration in the efficiency of the ABIS test. Tested samples, H6CA-spiked phosphate buffer with a range of 0.1 to 1 M of

NaCl, were prepared and used to address this objective. No false-positive or non-specific binding and false-negative results were observed as shown in Fig. 5b. These findings suggest that all colloidal gold particles were stabilized by both ankyrin repeat protein and albumin used during the conjugation process and did not interfere with accuracy. Sensitivity and specificity of ABIS test With regards to the main application of the ABIS test for use as a diagnostic kit in the future, the basic performance characteristics of a test designed to distinguish positive from negative samples is clearly required. Results of the ABIS test with samples experimentally incubated with recombinant capsid protein are shown in Fig. 6. It is to be noted that samples giving a red-purple color at the test line were interpreted as a positive result. In contrast, no color at test line was graded as a negative result. Sensitivity and specificity were calculated from the results using a 2×2 table, as shown in Table 1. Our ABIS test showed high sensitivity (96 %) and specificity (100 %) comparable to several diagnostic kits indicated the efficiency of applying 1D4-CGC as a visible detector with an acceptable error and, moreover, the probability of applying the ABIS test as a diagnostic tool in the future.

Discussion Ankyrin repeat proteins are member of non-immunoglobulin structures which are built by stacking of a series of modules to create a unique structure. It appears as an attractive choice for generating a novel class of binding proteins since it shares the same binding ability as antibodies but consists of several superior biochemical properties (Binz et al. 2003). The disulfide-independent structure is the most important factor

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Fig. 4 Performance of ABIS test. Strips were dip into H6MACAspiked PBS (a) and normal plasma (b) and H6CA-spiked PBS (c) and normal plasma (d) ranging from 0.1 to 100 μg/ml

that facilitates its expression within the bacterial cytoplasm and to further insert cysteines for site-specific coupling with several agents. Several target-specific molecules have been generated by standard in vitro evolution including phage display and ribosome display (Binz et al. 2004). These high-affinity structures have been modified for coupling with several agents such as biotin, fluorescein dye, and PEG using both conventional chemical coupling and the insertion of non-natural amino acids for site-specific labeling (Simon et al. 2012). Fig. 5 Effect of salt and pH on ABIS result. Phosphate buffer containing 0.1, 0.5, and 1.0 M NaCl was spiked with H6CA and used as a tested sample for evaluating the effect of salt concentration (a). ABIS tests were dipped into pH-adjusted phosphate buffer saline containing 1 μg/ml of H6CA and buffer control (b). Results were imaged within 5 min

In this study, we have identified a new method for the coupling of this type of molecular architecture with colloidal gold nanoparticles and have utilized these to assemble an antigen detection test named “ABIS test.” AnkGAG1D4 was used as a model protein target for demonstration of the proof of concept. This protein was tagged with colloidal gold particles and dispensed onto the conjugate pad of the ABIS test. The nature of the interaction between the colloidal gold particles and protein is poorly understood as described elsewhere

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Fig. 6 Assessment of sensitivity and specificity of ABIS test in plasma samples. The efficiency of ABIS test was evaluated by tested with blinded samples divided into two groups, 25 positive samples (a) and 25 negative

samples (b), corresponding to capsid-spiked plasma and capsid-negative plasma, respectively. One and two red-purple lines indicate negative and positive results, respectively

(Oliver 2010). However, the consensus opinion is that hydrophobic interactions are the major mechanism involved in the conjugation process, while electrostatic interactions were reduced by adjusting the pH of the gold solution to approximately 0.5 pH units higher than the pI of the protein being conjugated. Interestingly, the external surface of the internal repeat of ankyrin structure displays a hydrophobic property which is suitable to link with gold particles without disturbing the binding motif located within the internal surface (Binz et al. 2003; Nangola et al. 2012). Moreover, concerning to the effect of pH and salt concentration on the stability of protein-gold conjugation, the adsorption of ankyrin structure onto colloidal gold with an optimal condition can stabilize colloidal gold particles from aggregation in a range of pH and salt concentration. Stabilizing agents such as albumin and fibrinogen added in the conjugation process were adsorbed on the remaining space on the surface of colloid gold and stabilized the proteingold particles (Park 1989). The first prototype ABIS test was generated to detect the HIV-1 capsid protein in samples relying on the specificity of AnkGAG1D4. Dot blot results demonstrated the success of the conjugation process with no detectable interfering effect on target binding properties. The ABIS test results showed that it can detect ultra-low amounts of the standard capsid protein suspended in either PBS or normal plasma at nanogram levels which is within a slightly higher range compared with the available high-sensitivity commercial ELISA-based test kits (Saville et al. 2001). However, the test can be further optimized by adjusting several factors to enhance the lowest detection range such as concentration and dispensing rate of both 1D4-CGC and M88 sprayed onto the conjugated pad and

test line, respectively. These factors include the type of membrane and sample pad which are likely to influence the flow rate of sample through all compartments (Hua et al. 2012). Interestingly, this test can detect capsid both as a precursor polyprotein and/or in its mature form, suggesting that 1D4CGC is able to access its binding site. The sensitivity and specificity of this test is within an acceptable range as required for evaluation of diagnostic tests to identify infectious disease (Banoo et al. 2010). Of note, the ABIS test was evaluated using standard capsid-spiked samples which are different from the clinical samples. However, this test needs to be evaluated with HIV-infected samples before use in clinical diagnostics. In HIV-infected sera, most fractions of capsid molecule form immune complex with antibodies resulting in the negative interfering of test and normally ensuing false-negative results (Nishanian et al. 1990). Therefore, to apply this test for clinical use in the future, we need to find out an immune complex dissociation (ICD) procedure such as using low pH and/or thermal treatments to dissociate p24 antigen/anti-p24 antibody complexes before performing the antigen assay (Pokriefka et al. 1993; Goldschmidt et al. 1998). In conclusion, the assay described herein documents the ability to conjugate colloidal gold particles with ankyrin protein for use as a low-cost visible detector in the ABIS test. Additionally, to apply this protein architecture targeting a new target, a process can be conveniently set up by screening the artificial ankyrin repeat protein library for binding to the novel target molecule in question by phage display technique. Gene encoding the target-specific protein can practical isolate and insert into various type of vectors, especially a cytoplasmic expression vector for production of soluble protein in E. coli. Regarding to a disulfide bridge-independent structure, this protein can be expressed in all compartments of cells, especially in the cytoplasm of E. coli, resulting in a high level of protein production in a short time and low cost of protein production. In 1 l of cultivation, up to 100 mg of purified protein can be prepared (data not shown) and sufficed to generate a large batch of ABIS test strips. Moreover, the first prototype test exhibited herein outlines the novel type of the immunochromatographic strip test that can potentially be applied for clinical use, especially in resource-limited countries in the future.

Table 1 The sensitivity and specificity of ABIS test Number of sample/result

Positive samples Negative samples

ABIS result Positive

Negative

24 None

1 25

Appl Microbiol Biotechnol (2014) 98:6095–6103 Acknowledgments The work in Thailand was supported by the National Research University project under the Thailand’s Office of the Commission on Higher Education, the Research Chair Grant of National Sciences and the Technology Development Agency at the Faculty of Associated Medical Sciences, Chiang Mai University, School of Allied Health Sciences, University of Phayao, Thailand Research Fund (TRF), National Research Council of Thailand (NRTC), Health Systems Research Institute (HSRI), and i+Med Laboratories Company Limited. Conflict of interest None.

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An application of capsid-specific artificial ankyrin repeat protein produced in E. coli for immunochromatographic assay as a surrogate for antibody.

Immunochromatographic strip test is a unique type of rapid test that has been developed for use as part of a diagnostic kit for the rapid detection of...
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