Article pubs.acs.org/ac
Bispecific Antibody-Mediated Detection of the Staphylococcus aureus Thermonuclease Sarah J. Wagstaffe,† Katja E. Hill,‡ David W. Williams,‡ Beverley J. Randle,‡ David W. Thomas,‡ Phil Stephens,‡ and D. Jason Riley*,† †
Department of Materials, Imperial College London, Exhibition Road, London, SW7 2AZ, United Kingdom Tissue Engineering and Reparative Dentistry, School of Dentistry, Heath Park, Cardiff University, CF14 4XY, United Kingdom
‡
ABSTRACT: We report a novel fluorescence-based immunoassay which enables qualitative detection of the Staphylococcus aureus Thermonuclease (TNase) enzyme, thus providing confirmation of the presence of the S. aureus bacterium in vitro. The biomedical problem of chronic wound healing and the continuing emergence of antibiotic-resistant species is addressed in the development of a detection system capable of the rapid, real-time assessment of bacterial load and diversity. The use of bispecific antibodies (BsAb) provides integration of the molecular detection and signal response components of a standard immunoassay due to steric hindrance-mediated release of prebound fluorescent reporter molecules upon specific binding of TNase to adjacent sites. Rhodamine and fluorescein-labeled hemocyanin from Megathura crenulata (KLH) were prepared as effective immunoconjugates containing a sensitive fluorescent reporter moiety. BsAb that both specifically quenched the fluorescence of the reporter conjugate and bound the TNase target antigen were produced using cell fusion techniques. Assays were then performed to analyze the properties attributable to the steric hindrance-mediated release of the fluorescent reporter molecules upon adjacent TNase binding. This was performed by monitoring the intensity of fluorescence emission of the immunogenic reporter conjugate released into an aqueous environment at 578 and 520 nm, respectively.
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glaucescens,13 but they have since been found to be expressed most commonly by S. aureus. The presence of the TNase enzyme can be considered indicative of S. aureus bacterial infection, thus providing an effective detection target for this study. Naturally occurring antibodies are bivalent and monospecific, containing two identical antigen binding sites, specific to a single antigen. Despite having two recognition sites, examples have been found in nature where only one binding site is functional due to steric hindrance between adjacent antibodies or within asymmetric structures.14,15 These findings led researchers to hypothesize that, in such antibodies, interaction at one high affinity antigen binding site could cause a decrease in affinity at the second site, leading to the release of molecules bound at the latter site. It followed that engineering of antibodies to contain two distinct antigen binding fragments could provide the basis for a generic molecular signal system, where the displacement of a detectable reporter molecule could act as an indicator for specific antigen binding. Bispecific antibodies (BsAb) have two different antigen binding fragments that have specificity toward two distinctly different antigens. Molecular binding to one arm displaces a previously bound reporter from the adjacent site16 as illustrated in Figure 1. This functionality is the result of steric hindrance around the
hronic wounds are those that have failed to follow the normal stages of healing, resulting in persistent and elevated inflammatory cell activity.1 Wound healing processes are complex, requiring the collaborative efforts of many cells and tissues,2−5 and the time scale of recovery can be greatly delayed as a result of both exogenous (pathophysiological) and endogenous factors (e.g., microbial invasion).6 The exact role of microorganisms in healing impairment is the subject of intense debate;7,8 some believe that bacterial load is key while others think that it is the presence of certain bacterial species or that multiple species act synergistically to produce a pathogenic community.9,10 Despite the fact that the precise interactions between bacteria and impaired wound healing are unclear, it has been almost universally acknowledged that a state of chronic inflammation is synonymous with polymicrobial colonization of the wound bed. Staphylococcus aureus is currently the most frequent cause of wound infection among hospitalized patients, with studies suggesting that it is present in 43% of infected leg ulcers.11,12 This, together with the emergence of methicillin-resistant S. aureus (MRSA) in recent decades, led to its selection as a target bacterium for these investigations. Staphylococcus aureus is an opportunistic pathogen that produces many toxins or superantigens which can cause mild infections or life-threatening disease. These toxins are a group of bacterial proteins characterized by their capacity to stimulate an antigenic response, and the S. aureus TNase enzyme is a good example of this. The first bacterial TNases were purified from cell extracts of Streptomyces © 2012 American Chemical Society
Received: December 21, 2011 Accepted: May 25, 2012 Published: May 25, 2012 5876
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software was used alongside the Quantamaster for fluorescence data collection and analysis. The maximal excitation wavelength was utilized for rhodamine (543 nm), and the emission wavelength range was scanned from 553 to 700 nm. The maximal excitation wavelength for fluorescein was utilized at 491 nm, and the emission was scanned from 501 to 700 nm. Reagents. Analyses were performed in PBS (phosphate buffered saline) containing 0.01 mol dm−3 phosphate buffer, 0.0027 mol dm−3 potassium chloride, and 0.137 mol dm−3 sodium chloride, pH 7.4, at 25 °C (Sigma Aldrich). Hemocyanin from Megathura crenulata in lyophilized form (Sigma Aldrich) was reconstituted with 2 cm3 of deionized water to yield an opalescent stock solution at 10 mg cm−3 in 31 mmol dm−3 sodium phosphate buffer, pH 7.4, at 25 °C, containing 0.46 mol dm−3 NaCl, 2% polyvinylpyrrolidone (PVP), and 41 mmol dm−3 sucrose. Bovine serum albumin (BSA) was obtained in powder form and dissolved in sodium bicarbonate buffer 50 mM, pH 8.5, at a concentration of 10 mg cm−3 (Sigma Aldrich). NHS-rhodamine (5-carboxytetramethylrhodaminesuccinimidyl ester) and NHS-LC-fluorescein (6[fluorescein-5(6)-carboxamido]hexanoicacid N-hydroxysuccinimide ester) (Sigma Aldrich) stock solutions (1 mg cm−3) were prepared in dimethylsulfoxide (DMSO, Sigma Aldrich). Sephadex G-25 (Sigma Aldrich) was preswollen before column packing in excess PBS buffer solution (6 × 10−3 mol dm−3) at 3:1 volume to mass of Sephadex G-25. Solutions of blue dextran (5 mg cm−3) and methyl red (5 mg cm−3) dyes in PBS (6 × 10−3 mol dm−3) were made up as void volume markers for gel filtration (Sigma Aldrich). Spectroscopic Analysis of Fluorescent Hapten and Protein Carrier. Fluorescence characterization of haptens and carrier proteins within appropriate concentration ranges was performed at the following excitation and emission wavelengths: KLH (λex 294 nm, λem 304−700 nm); NHS-rhodamine (λex 543 nm, λem 553−700 nm); NHS-LC-fluorescein (λex 491 nm, λ em 501−700 nm). The linear concentration to fluorescence emission range was plotted for KLH, NHSrhodamine, and NHS-LC-fluorescein, and the limit of detection of each species was determined. Immunogenic Conjugate Synthesis and Purification. NHS-rhodamine and NHS-LC-fluorescein were conjugated with the protein carriers KLH and BSA resulting in the formation of a stable amide link. NHS-rhodamine/NHS-LCfluorescein (1 mg cm−3, 1 cm3 DMSO) was slowly added to 2 cm3 of reconstituted KLH (10 mg cm−3) on ice with stirring; the reaction vessel was protected from light and reacted on ice for 2 h. The solutions were stored at −20 °C until required. Unreacted NHS-rhodamine/NHS-LC-fluorescein and KLH, together with reaction byproduct, were removed by gel filtration in Sephadex G-25, utilizing dextran blue and methyl red as high and low molecular weight markers, respectively, in a concomitant column. Sephadex G-25 was preswollen at room temperature overnight before use. Gel filtration collections were diluted and analyzed spectroscopically; the maximum emission at corresponding wavelengths were analyzed and utilized to calculate the relative concentrations of hapten and carrier protein in each fraction. Following purification, samples were stored at 4 °C and discarded after 2 months. Samples were diluted in PBS buffer prior to immediate use in immunoassay investigations. Upon visible precipitation of the protein conjugates, samples were discarded due to concentration variability.
Figure 1. Schematic of a bispecific antibody. Adapted with permission from ref 16. Copyright 2004 Elsevier. Bispecific antibodies function so that molecular binding to one arm displaces the previously bound reporter from the adjacent site. A detected release of the fluorescent KLH conjugate reporter into solution signals the bispecific antibody molecular recognition of bacterial antigen (Staphylococcus aureus Thermonuclease (TNase) enzyme), consistent with steric hindrance between two different antibody binding events.
antibody hinge region. The binding of a second large antigen to the antigen binding site will result in the affinity-dependent displacement of one of the antigens. Cell fusion techniques are employed in BsAb production, to generate a library of antibody structures with varied orientation of binding sites.17 Those antibodies with binding sites facilitating sufficient interaction between the two bound entities to result in steric hindrancemediated release of the lower affinity antigen can then be selected. The relationship between antigen binding and reporter release has been reported to be linear.16 The mean distance between antigen binding sites of IgG subclasses has been measured by neutron and X-ray scattering to be 117−134 Å with variation of 40 Å.18 As a consequence, it is thought that antigens and reporters should be large, to maximize intramolecular steric effects, thus restricting the use of self-signaling antibody events in reporting the presence of low molecular weight analytes. Bispecific antibodies have also been produced that bind to two separate epitopes on the same protein,19 effectively increasing the specificity of the antibody to that protein when compared with monoclonal antibodies. Bispecific antibodies have been developed for numerous potential applications including immunoassays, immunohistochemistry, immunotherapy, and immunoimaging.20−24 Production and functional analysis of two BsAb series specific to the S. aureus TNase generated by cell fusion techniques is described. The first “A” series was directed against S. aureus TNase and rhodamine-Keyhole Limpet Hemocyanin (KLH) immunoconjugate antigen and the second “B” series was against S. aureus TNase and a fluorescein-KLH immunoconjugate. Combinations of antibody binding sites, reactive with various epitopes on either antigen or reporter molecules, were generated to test the concept of self-signaling antibodies. Cell fusion techniques were employed to generate a range of structures where varied orientation of the binding sites was anticipated. This increased the possibility of finding an interaction between antigens and, thus, steric-mediated release of a fluorescent reporter.
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EXPERIMENTAL METHODS Instrumentation. Fluorescence emission spectra were obtained with a PTI Quantamaster Fluorimeter. FeliX32 5877
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Substrate solution (50 μL) (1 mg cm−3 para-nitrophenol phosphate in diethanolamine buffer at 0.1M, pH 9.6) was then added. Plates were left for 15−30 min to develop in the dark before reading the plates at 405 nm using a microplate reader (Thermomax microplate reader, Molecular Devices, Sunnyvale, CA). Functional Analysis of Bispecific Antibodies: Fluorescence Quenching Immunoassays. KLH-rhodamine/ KLH-LC-fluorescein solutions (4 cm3, 0.02 mg cm−3) were excited at 543 and 491 nm, and the fluorescence emission was quantified at 578/520 nm respectively. “A” series and “B” series antibodies (10 μL, 1 mg cm−3) were added to the solutions and incubated with tilt rotation for 30 min. Second fluorescence emission spectra were taken after this incubation, and the percentage decrease in the fluorescence emission was calculated. The solutions of the reporter bound to antibodies of the “A” and “B” series were subjected to further 30 min incubations with tilt rotation with the bacterial antigen TNase (10 μL, 0.02 mg cm−3). A third fluorescence emission spectra was taken after the incubation time, and the percentage increase in the fluorescence emission upon incubation was calculated. Functional Analysis of Bispecific Antibodies: Dynabead Immunoassays. Immunoassay investigations were performed using DYNABEAD technology. Dynabeads M-280 sheep antimouse IgG coated superparamagnetic beads (Invitrogen) were selected as a solid support for immunoassay investigations. The beads were approximately 2.8 μm in diameter with affinity purified sheep antimouse IgG covalently bound to the surface. The sheep antimouse IgGs were specifically directed toward the Fc fragment of mouse IgG, thus anchoring the bispecific antibodies in such an orientation that both antigen binding sites would be accessible during the immunoassay investigations. The beads were supplied as a suspension containing (6−7) × 108 beads cm−3, at a concentration of approximately 10 mg cm−3 in PBS, pH 7.4, with BSA (0.1% w/v) and NaN3 (0.02% v/v). The beads were resuspended thoroughly by tilt rotation for 20 min prior to transferring 29.85 μL of the resuspended beads into a reaction vessel. Bispecific antibodies of the “A” and “B” series (10 μL, 1 mg cm−3) were added to the beads resuspended in 200 μL of PBS and mixed by rotation for 1 h at room temperature. After this time, the magnet was applied to the reaction vessel and the supernatant was removed by gentle pipetting. The beads were washed in PBS (100 μL) to remove residual, unbound antibody. The beads were incubated with KLH-rhodamine and KLH-fluorescein (2 cm3, 0.02 mg cm−3) for the “A” and “B” series, respectively, with rotation for 1 hour following spectroscopic analysis of the incubating solution. After this incubation time, the beads were removed from solution and the supernatant analyzed by fluorescence spectroscopy. The decrease in fluorescence emission intensity at 580 and 520 nm for rhodamine and fluorescein, respectively, was indicative of specific antibody-reporter binding. The solutions were then washed to remove nonspecifically bound reporter and resuspended in PBS (200 μL). TNase (10 μL, 0.02 mg cm−3) was added to the resuspended beads of each series and incubated with tilt rotation for a further hour. Once again, the supernatant was withdrawn upon bead removal and retained for fluorescence emission analysis using excitation at the appropriate wavelength.
Production of TNase-Reactive Monoclonal Antibodies. A commercial TNase preparation was obtained from Toxin Technology Inc., Sarasota, Florida, USA purified from S. aureus strain FRI 1151M.25 Monoclonal antibodies (Mabs) against S. aureus TNase were raised by murine BALB/c immunization with the commercially available TNase preparation. An immune response was induced by four one-weekly immunizations of 10 μg TNase per dose supported by alum adjuvant given intraperitionally. Induction of an immune response was confirmed by indirect enzyme-linked immunosorbent assay (ELISA) with antisera collected at the end of immunization. One month later, the immune response was boosted by 10 μg of TNase in 0.2 mL of PBS given intravenously, and splenocytes were harvested 4 days later. Monoclonal antibodies were produced by methods described previously.26 Production of Bispecific Antibodies. Two bispecific antibody series were generated: the “A” series specific to the S. aureus TNase and KLH-rhodamine and the second “B” series specific to the S. aureus TNase and KLH-LC-fluorescein reporter moieties. TNase-reactive bispecific antibodies were produced by methods described previously.16 Bispecific antibody-secreting fusomas were generated by fusing thioguanineresistant hybridomas, secreting TNase-reactive monoclonal antibodies, with splenocytes from BALB/c immunized with KLH NHS fluorophore. A fluorophore-directed immune response was induced by four one-weekly immunizations of 10 μg KLH NHS fluorophore per dose supported by alum adjuvant given intraperitionally. Induction of an immune response was confirmed by indirect ELISA with antisera collected at the end of immunization. One month later, the immune response was boosted by 10 μg of KLH NHS fluorophore in 0.2 cm3 of PBS given intravenously, and splenocytes were harvested 4 days later. Supernatants from fusoma cultures were screened by indirect ELISA as described below. Selected cultures were expanded by bulk culture. Secreted antibody was enriched by affinity chromatography of culture supernatant, centrifuged to remove cells, on GammaBind Plus Sepharose, packed in HiTrap columns (methods of ref 16; GE Healthcare Biosciences). Determination of Protein Concentrations. Protein concentrations of purified antibodies were determined colorimetrically using a BCA protein assay kit (Pierce, Rockford, USA) according to the manufacturer’s instructions. Standard Indirect Enzyme-Linked Immunosorbent Assay (ELISA) for Primary Screening. Indirect ELISAs were performed to identify culture supernatants and enriched samples containing monoclonal or bispecific antibodies. Briefly, polystyrene microtiter plates (Falcon, Becton Dickinson, Le Pont de Claix, France) were coated with 50 μL per well of the required antigen (TNase, KLH-fluorescein, BSA-fluorescein, KLH-rhodamine, or BSA-rhodamine) at 10 μg cm−3 diluted in coating buffer (0.1 M sodium carbonate/sodium hydrogen carbonate solution, pH 9.6) overnight at 4 °C. Blocking with 100 μL of 10% w/v fetal calf serum in PBS was performed for 2 h. Test and control samples (50 μL) were then loaded in duplicate and incubated for 1 h. Wells were then washed twice with 150 μL of wash buffer (PBS with 0.05% v/v Tween 20). A second layer antibody (50 μL; antimouse IgG alkaline phosphatase conjugate, Sigma) was then added (1 in 1000 dilution in protein buffer; (0.5% (v/v) BSA in 2 mM HEPES, pH 7.4)) and incubated for 30 min with rocking. This second layer was then removed, and the wells were washed 3 times with 150 μL of wash buffer (PBS 0.02% (v/v) Tween 20). 5878
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Figure 2. (a) Maximum fluorescence emission (λex 294 nm, λem 330 nm) of increasing concentrations of KLH (mg cm−3) showing the concentration range within which the fluorescence emission is linear. (b) Maximum fluorescence emission (λex 543 nm, λem 580 nm) versus concentration of NHSrhodamine (mg cm−3). (c) Maximum fluorescence emission (λex 491 nm, λem 520 nm) versus concentration of NHS-LC-fluorescein (mg cm−3). Equations relate fluorescence emission intensity (I) to concentration (C) of KLH and fluorescence species. Error bars showing standard deviation (n = 5) associated with variation in fluorescence measurements and concentration of stock solutions.
Figure 3. (a) Fluorescence emission spectra of diluted KLH-rhodamine conjugate: Peak I: λex 280 nm, λem 320 nm, showing emission maxima of 1.78 × 107 counts; Peak II: λex 543 nm, λem 578 nm, showing emission maxima of 2.31 × 108 counts, corresponding to KLH and rhodamine components, respectively. (b) Fluorescence emission peak spectra of diluted KLH-fluorescein conjugate: Peak I: λex 280 nm, λex 330 nm, showing emission maxima of 1.24 × 105; Peak II: λex 491 nm and λem 520 nm showing emission maxima of 5.15 × 105, corresponding to KLH and fluorescein immunoconjugate components, respectively.
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tration and fluorescence emission within the concentration range of 0.005−0.25 mg cm−3 (Figure 2a). The process was repeated for both haptens, NHS-rhodamine (λex 543 nm, λem 580 nm) and NHS-LC-fluorescein (λex 491 nm, λem 520 nm), and the linear relationships between fluorophore concentration
RESULTS AND DISCUSSION
Fluorescence Quantification of the Carrier Protein and Fluorescent Haptens. Fluorescence emission quantification of a range of KLH concentrations (λex 280 nm, λem 330 nm) showed a linear relationship between protein concen5879
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Table 1. Showing Specificity (Shaded Boxes) of Each Antibody Generated in the “A” (Rhodamine) and “B” (Fluorescein) Seriesa
a
BsAb, bispecific antibody; TNase, thermonuclease; KLH, Keyhole limpet hemocyanin; BSA, bovine serum albumin; r, rhodamine; fl, fluorescein.
and emission maxima were determined (Figure 2b, c). The mathematical relationships allowed an estimate of the degree of fluorophore labeling to be obtained through calculation of the number of moles of fluorophore per mole of protein in the purified immunoconjugate samples. Preparation of Fluorophore-KLH Immunoconjugates. The fluorescence emission spectra of diluted solutions of each immunoconjugate were analyzed: the concentration of rhodamine-KLH immunoconjugate (Figure 3a) was calculated as 1.25 mg cm−3, and each protein was estimated to have approximately 145 rhodamine molecules bound to its surface. The concentration of fluorescein-KLH immunoconjugate (Figure 3b) was calculated to be 1.4 mg cm−3, and 19 fluorescein molecules per protein were estimated. ELISA Screening of Series “A” (Rhodamine) and Series “B” (Fluorescein) Bispecific Antibodies. Bispecific antibodies were screened for reactivity to TNase, rhodamine/ fluorescein (as appropriate), and KLH as seen in Table 1. All antibodies recognized the parental antigen (TNase) as well as the immunization signal molecule (KLH-rhodamine or KLHfluorescein). To distinguish the site of antibody reactivity on
the immunization signal molecule, antibody preparations were also assayed against KLH and fluorophore, carried on BSA. Antibodies reactive with KLH and KLH fluorophore, but not BSA fluorophore, indicated a binding site recognized on the KLH molecule. In contrast, antibodies reactive with KLH fluorophore and BSA fluorophore, but not KLH, indicated recognition of a binding site on the fluorophore molecule. For TNase-rhodamine-KLH supernatants, 61% (19/33) of cultures secreted antibodies consistent with bispecific reactivity to TNase and rhodamine, with a further 14 cultures recognizing TNase and KLH. In contrast, for TNAse-fluorescein-KLH supernatants, 57% (17/30) cultures secreted antibodies consistent with bispecific reactivity to TNase and fluorescein, with a further 14 cultures recognizing TNase and KLH. Two cultures, B11 and B12, secreted antibodies that were only reactive with the parental antigen TNase and the fluoresceinKLH conjugate used for immunization, suggesting the cultures recognized an antigen specific to the fluorescein KLH structure. Two cultures, B66 and B68, recognized antigen sites shared by the all of the protein fluorophore samples examined. 5880
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Figure 4. Figures showing a percentage decrease and subsequent increase in fluorescence emission intensity associated with specific binding and release of KLH-rhodamine antigen from bispecific antibody association: (a) aqueous fluorescence quenching immunoassays; (b) dynabead facilitated immunoassays. Error bars showing standard deviation (n = 5) associated with fluorescence measurements.
resulting in a significant degree of fluorescence quenching: A23, A88, A89, A103, A106, A118, and A119. The results suggest either that these antibodies have the highest affinity for the reporter antigen, and thus more of the antigen gets bound in the time-controlled experiment, or that in the complexes these antibodies form with the antigen; the epitope is located such that fluorescence quenching is more efficient. DYNABEAD immunoassays (Figure 4b) were performed to determine the quenching mechanism. Dynabeads modified with A23, A88, A89, A103, A106, A118, and A119 resulted in a significantly larger decrease in fluorescence emission of the supernatant upon incubation with an antigen, demonstrating that these antibodies had higher affinities for the fluorescence reporter and that quenching was not a result of epitope location. However, A108 was an exception. No significant quenching was observed with the aqueous immunoassays, but when DYNABEAD immunoassays were performed, a more significant decrease in fluorescence was
Functional Analysis of “A” Series Bispecific Antibodies: Fluorescence Quenching Immunoassays and Dynabead Immunoassays. Figure 4a shows the fluorescence quenching analysis of the “A” series: antibodies specific to the KLH-rhodamine reporter and TNase enzyme. The initial decrease in the fluorescence emission at 580 nm is associated with specific binding of free KLH-rhodamine reporter to the respective antibodies; the increase in fluorescence following TNase addition is attributed to steric-hindrance driven release of the reporter into solution. The initial decrease in the emission stems from the quenching of fluorescence due to antibody−antigen complex formation. The immunoglobulin was present in excess of the reporter to allow complete binding of the reporter in solution. Control experiments quantified the degree of nonspecific (protein−protein) binding to be 2.7%, using a fluorescein specific antibody. Binding of KLHrhodamine to all antibodies in the series showed a decrease in fluorescence emission with a number of bispecific antibodies, 5881
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Figure 5. Figures showing a percentage decrease and subsequent increase in fluorescence emission intensity associated with specific binding and release of KLH-fluorescein antigen from bispecific antibody association: (a) aqueous fluorescence quenching immunoassays; (b) dynabead facilitated immunoassays. Error bars showing standard deviation (n = 5) associated with fluorescence measurements.
observed, suggesting fluorescence quenching assays can be influenced by epitope location and energy transfer mechanisms due to antigen orientation. Upon addition of equimolar solutions of TNase, assessment of the relative affinities of the antibodies toward the TNase and fluorescent reporter molecule was facilitated. Stock solutions (1.06 × 107 mg cm−3) were added to the reaction mixtures, and the percentage increase in fluorescence emission at 580 nm are shown in Figures 4a (aqueous assay) and 4b (DYNABEAD assay). Increased fluorescence emission intensity suggests an increase in volume of free reporter in solution, indicative of reporter release from the antibody as a result of steric hindrance/molecular crowding. Nonspecific binding was again estimated (3.2%), utilizing an antibody specific to a different bacterial antigen. Four of the antibodies thought to have higher affinity toward the KLH-rhodamine reporter, namely, A23, A88, A89, and A103, showed the largest increase in fluorescence
intensity in both assays. For these antibodies to show characteristics attributable to steric-hindrance-mediated release of the reporter in the presence of the TNase antigen, they must have a higher affinity for the TNase enzyme than for the fluorescent reporter. Antibodies A118 and A119 both showed a large degree of fluorescence quenching upon binding to the reporter yet no increase in the presence of TNase. Examining the data from both assays, the antibodies were thus considered to have a higher affinity to the fluorescent reporter than the bacterial antigen, thereby making them unsuitable for use in a “self-signaling” antibody-based immunoassay to detect the presence of the TNase enzyme and thus the S. aureus bacterium. Therefore, antibodies A23, A88, A89, and A103 all showed potential as reporters, where specific TNase binding causes the release of the bound fluorescent reporter from the adjacent antigen binding site. It has previously been hypothesized that binding of one antigen to an antibody 5882
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nm for the rhodamine and fluorescein conjugate, respectively, was analyzed with each bispecific antibody of the series, and quenching was observed upon antibody−antigen complex formation. Increase in the fluorescence intensity observed upon TNase binding was demonstrated with antibodies that have a higher affinity for the bacterial antigen than the fluorescent reporter. These antibodies had specific epitopes that facilitated steric hindrance-mediated release of the fluorescent reporter in a self-signaling event. It is clear that the larger reporter conjugates resulted in a significantly increased steric hindrance between adjacent antigen binding sites, as demonstrated by the larger KLH-rhodamine conjugate. Hence, the use of “self-signaling” antibodies in immunoassays is most suited to larger antigens. The “self-signaling” antibody could offer a generic platform for antibody-based biosensors, removing the need for multiple washing steps and secondary detection agents. When integrated to generate a fluorescent signal as demonstrated, a highly sensitive reporter system is generated for analyte detection, potential quantitation, and high throughput screening. Wound exudates contain a broad spectrum of plasma proteins as well as variations in the bacterial flora; thus, significant nonspecific binding could result in false positive results. This study illustrates the high specificity of this assay. In addition, nonspecific protein binding estimated using the Pseudomonas aeruginosa lipopolysaccaride (LPS) antigen as a control gave results showing variations in the fluorescence quenching did not rise beyond 4%. These preliminary investigations suggest a highly sensitive assay with potential application in biological specimens with limited risk of false positive results. Significant progress is thus reported in the qualitative detection of the TNase enzyme and hence the presence of S. aureus species in vitro. The real-time analysis of bacterial species within chronic wounds will allow the evidencebased prescription of targeted antibiotics, which has not been achievable to-date.
binding site could lead to a reduction in the affinity of the adjacent arm for its associated antigen in certain antibody species. This could be a plausible explanation for the lack of affinity and/or steric hindrance-mediated release of the fluorescence reporter in antibodies A118 and A119. Functional Analysis of “B” Series Bispecific Antibodies: Fluorescence Quenching Immunoassays and Dynabead Immunoassays. Figure 5a shows the fluorescence quenching analysis of the “B” series: antibodies specific to the KLH-fluorescein reporter and TNase enzyme. The initial decrease in the fluorescence emission at 520 nm was associated with specific binding of free KLH-fluorescein reporter to the respective antibodies. The increase in fluorescence following TNase addition was attributed to steric-hindrance-mediated release of free reporter into solution. Only a few antibodies (namely, B34, B52, B53, and B59) showed significant fluorescence quenching due to antibody−antigen complex formation. (Nonspecific protein−protein binding was estimated at 1.8%). Many of the antibodies showed an increase in fluorescence upon binding when aqueous immunoassays were performed, a result not reproduced during the DYNABEAD assays, possibly due to complex formation in aqueous solution. All antibodies in the series showed a decrease in fluorescence emission during the DYNABEAD assays, with the same antibodies showing significantly higher affinities for the fluorescein reporter antigen as in the aqueous assays. The results suggest these antibodies had the highest affinity for the reporter antigen and, thus, that more of the antigen gets bound in the time-controlled experiment. However, Figure 5 shows those antibodies, found to have a higher affinity for the fluorescent reporter, do not have a correspondingly higher affinity for the TNase antigen as was seen with the rhodamine reporter system. Stock solutions (1.06 × 107 mg cm−3) were added to the reaction mixtures, with the percentage increase in the fluorescence emission at 520 nm shown in Figures 5a (aqueous assay) and 5b (DYNABEAD assay). Examining the data from both assays, the antibodies were considered to have a higher affinity toward the fluorescent reporter than the bacterial antigen, making them unsuitable for use in a “self-signaling” antibody-based immunoassay to detect the S. aureus TNase. The degree of fluorescent labeling is of considerable importance: the fluorescein-labeled reporter contained an estimated 19 molecules per KLH protein, compared to the rhodamine-labeled reporter (approximately 145 molecules per KLH). The KLH-rhodamine reporter molecule was, therefore, significantly larger than the KLH-fluorescein antigen. Due to the small size of this fluorescein conjugate, it is possible that both antigens could bind to the antibody without producing sufficient steric hindrance/molecular crowding to facilitate free reporter release into solution and a corresponding increase in fluorescence emission intensity.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank the Engineering and Physical Sciences Research Council for funding this research (Grant Number EP/ D505437/2). We also thank Dr. S. Bamford for technical support.
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REFERENCES
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CONCLUSIONS We report a novel immunosensor based on self-signaling antibodies, where specific antigen binding causes the release of a bound reporter from an adjacent antigen binding site to generate a direct measurable response. These bispecific antibodies demonstrate properties consistent with molecular crowding and steric hindrance around the IgG hinge region. Two KLH-fluorophore/TNase specific antibody series were generated, each member of the series specific to an epitope on either the KLH or fluorescent moiety of the reporter conjugate. The decrease in fluorescence emission intensity at 580 nm/520 5883
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dx.doi.org/10.1021/ac203403d | Anal. Chem. 2012, 84, 5876−5884
