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Highly stable lyophilized homogeneous bead-based immunoassays, for on-site detection of bio warfare agents from complex matrices Adva Mechaly, Sharon Marx, Orly Levy, Shmuel Yitzhaki, and Morly Fisher Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00362 • Publication Date (Web): 02 Jun 2016 Downloaded from http://pubs.acs.org on June 3, 2016

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Analytical Chemistry

Highly stable lyophilized homogeneous bead-based immunoassays, for on-site detection of bio warfare agents from complex matrices Adva Mechaly†, Sharon Marx‡, Orly Levy†, Shmuel Yitzhaki†, Morly Fisher† †

Department of Infectious Diseases, IIBR, Ness-Ziona 74100, Israel.

‡ Department of Physical

Chemistry, IIBR, Ness-Ziona 74100, Israel.

ABSRACT: This study shows the development of dry, highly stable immunoassays for the detection of bio warfare (BW) agents in complex matrices. Thermal stability was achieved by the lyophilization of the complete, homogeneous, bead based immunoassay in a special stabilizing buffer, resulting in a ready to use, simple assay, which exhibited long shelf and high temperature endurance (up to one week at 100oC). The developed methodology was successfully implemented for the preservation of TRF (Time Resolved Fluorescence), Alexa-fluorophores and HRP (Horse Radish Peroxidase) based bead assays, enabling multiplexed detection. The multiplexed assay was successfully implemented for the detection of B. anthracis, Botulinum B and Tularemia in complex matrices.

The preservation of proteins in dry solid matrices, utilizing solutions of trehalose, sucrose, mannitol, sorbitol and other sugars, has been demonstrated extensively over the years

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. The stabilization effect of these solutions is believed to arise from a

combination of two basic phenomena: "glass dynamics" and "water substitution". The "glass dynamic" hypothesis states that a good stabilizer forms a rigid, inert matrix around a protein, resulting in limited protein mobility thereby minimizing aggregation, unfolding and other chemical degradation reactions. The "water substitution" concept suggests that during drying, hydrogen bonds, existing between the protein and the surrounding water molecules, are replaced with newly formed protein-sugar interactions, thereby leading to the preservation of protein structure and function. Polymers and additional proteins can also be utilized for protein stabilization as was demonstrated by us in a previous study where the thermal stability of butyryl cholinesterase was enhanced by drying the enzyme in films composed of trehalose in combination with the polymer poly-(vinyl-pyrrolidone) (PVP) and Bovine Serum 1 ACS Paragon Plus Environment


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Albumin (BSA) 8. Those additional components are believed to form hydrogen bonds and electrostatic interactions with the enzyme, thereby enhancing its stability 9-11. In addition to the preservation of different proteins such as enzymes and antibodies, several groups have successfully demonstrated the stabilization and preservation of antibody based complete immunoassays, i.e., plate anchored ELISA 12-14. However, to our knowledge, only one group attempted the preservation of a complete, 15

homogeneous, bead based immunoassay

. This attempt, utilizing a commercial

stabilization buffer with an unknown composition, was only partially successful, enabling the preservation of the assay components separately but not as a homogeneous assay. In this work we demonstrate for the first time the successful preservation and stabilization of complete, homogeneous bead-based immunoassays. The need to stabilize immunoassays, especially those intended for BW agents' detection, stems from their use in remotely deployed labs and their infrequent use in clinical labs. In order to establish a successful stabilization methodology we utilized homogeneous, magnetic bead-based assays based on time resolved fluorescence (TRF) detection. Europium (EuIII) chelates, utilized as the detection molecules, have strong fluorescence signals, long decay times (1 msec), a large stokes shift (>200 nm) and a narrow emission peak, allowing the label to be read once the nonspecific background has already peaked and decayed

16,17

. These properties result in enhanced signal-to-

noise (S/N) ratios, leading to sensitive assays as has been demonstrated previously 18. The use of magnetic beads has many additional advantages including: 1. A high surface-to-volume ratio which enables convenient and easy analyte-antibody binding, 2. Improved antibody-analyte kinetics due to dispersion of the beads in the well, 3. The convenient separation and concentration of the bead coated target molecules from unbound analytes in the mixture, which is advantageous when handling complex samples 19,20.

As proof of concept we developed separate tests for the detection of three different soluble and particulate BW agents: B. anthracis spores, Botulinum B toxin and Francisella tularensis bacteria. These antigens are class A threat agents that can be easily dispersed in water or food 21-24 and can result in widespread illness. The complete homogeneous magnetic-beads based immunoassays were successfully preserved via lyophilization in a suitable, optimized buffer. The developed 2 ACS Paragon Plus Environment

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methodology enabled long term storage of the dried, ready to use assays at room temperature (desiccated), so that the only task remaining for the end-point user is the addition of the evaluated sample and analysis using TRF. In order to establish the wide applicability of the stabilization methodology we further applied it for the preservation of bead based tests with different reporter antibodies such as Alexa and HRP. In both cases, the tests were successfully dried, exhibiting high temperature stability.

EXPERIMENTAL SECTION

Materials and Reagents. Streptavidin micro-beads, 6-7x108 particles/ml (Dynal M-280 Streptavidin. Catalog num. 11206D). Enhancement solution (PerkinElmer, Inc. 1244-105). Polymer: Polyvinylpyrrolidone (PVP) K90 M.W 360,000 (Fluka 81440), Trehalose, (Sigma T-9531), Albumin from bovine serum (BSA) (Sigma A5611), Chelate: Diethylenetriamine pentaacetic acid (DTPA) (Sigma D6518). Normal rabbit serum (NRS) (Biological Industries 04-008-1A). BW agents. B. anthracis strain ∆14185, a nontoxinogenic, nonencapsulated (ToxCap-) derivative of ATCC 14185 25 (Bacillus Genetic Stock center) was obtained from the Israel Institute for Biological Research collection. Spores were produced in sporulation medium SSM, as previously described 26. Clostridium botulinum A, B and E strains were obtained from the IIBR collection (A198, B592 and E450, respectively). Sequence analysis revealed compliance of the neurotoxin genes with serotypes 6A2 (Accession Number M30196), Danish (Accession Number M81186) and NCTC11219 (Accession Number X62683) for toxins A, B and E respectively 27. Toxins were prepared from concentrated supernatants of culture grown for 6 days in anaerobic culture tubes. Toxoids were prepared by dialyzing the toxins against 0.14%-0.2% formaldehyde at 35oC for 2-4 weeks

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. F. tularensis subsp. holarctica

strain LVS (ATCC 29684) was inactivated by exposure of 5x109 cfu/ml to 3 doses of 75,000 µj/cm3 UV radiation. BW agents for specificity profile determination: Bacillus subtilis DSM 675, Bacillus cereus 569, Bacillus megaterium and Bacillus turingensis turingensis were from the Israel Institute for Biological Research collection and were prepared in sporulation medium SSM as described for B. anthracis. Salmonella typhimurium SL3261 and Escherichia coli TG1 (Lucigen) were inactivated using formaldehyde. Yersinia pestis strain EV76 pgm- (Girard’s strain)

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was prepared as described

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. Pure ricin and abrin were prepared as described

previously 29 30. Protective antigen (PA), part of the B. anthracis toxin was purified by Q-Sepharose chromatography

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. Purified, recombinant, F1 (The capsular protein of

Yersinia pestis) and LcrV (V-antigen, part of the type III secretion system of Yersinia pestis) were prepared as described in the literature 32. Antibodies. Rabbit anti Botulinum B antibodies (RbαBotB) were purified from hyper-immune serum with Aminolink (Pierce, Rockford, IL) columns according to the manufacturer's protocol, as described before

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. Antibodies against B. anthracis

spores (RbαBA) were raised against a soluble exosporium fraction previously

34

33

as described

and purified from hyper-immune serum as described for anti-Botulinum

B polyclonal antibodies. Anti Francisella tularensis polyclonal IgG fraction (RαLVS) was obtained by HiTrap Protein G/A (GE Healthcare, Uppsala, Sweden) chromatography of hyper-immune rabbit serum immunized by LVS, according to the manufacturer’s instructions and dialyzed against PBS pH 7.4. Antibody labeling. Biotinylation of IgG purified antibody fractions was carried out using

sulfo-NHS-SS-biotin

[sulfosuccinimidyl-2-(biotinamido)

ethyl-1,3-

dithiopropionate; Pierce 21331] according to the manufacturer's instructions. An average of 4 biotin residues per antibody was determined according to the HABA ([2(4-hydroxyazobenzene] benzoic acid) method (Pierce 28050). Europium labeling was carried out with DELFIA Eu-N1-ITC chelate (PerkinElmer, Inc. 1244-301) or Europium FluoSpheres, carboxylate modified 0.2µm (Invitrogen F20881) according to the manufacturer's instructions. Alexa (fluorophore) labeling was carried out with Alexa Fluor 488, 594 or 647 protein labeling kits (Molecular Probes A-10235, A20185 and A-20173 respectively). HRP labeling was carried out using EZ-LinkTM activated peroxidase antibody labeling kit (Thermo Scientific. 31497). Labeling was performed according to the manufacturer instructions. Europium bead based detection assays. Assays were performed in a final volume of 130 µl in 96-well Greiner transparent micro plates (Greiner bio-one, catalog num. 655101), See Figure 1. Assay mixture contained both Streptavidin magnetic beads and labeled specific antibodies (80 µl) diluted in standard assay buffer (PBS + 2% NRS + 0.05% TW20 + 8 µg/ml DTPA). The final bead concentration (in 80 µl assay mixture) for all BW detection assays was ∼8x106 particles/ml. For each of the developed tests, a cross-titration matrix of capture (Biotinylated antibodies that were 4 ACS Paragon Plus Environment

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termed "Bio-Abs") and reporter antibodies (Europium labeled antibodies that were termed "Eu-Abs") was carried out in order to establish the optimal antibody concentrations in each assay: 2 nM Bio-RbαBA,

8 nM Eu-RbαBA for the B.

anthracis test; 4 nM Bio-RbαBotB, 4 nM of Eu-RbαBotB for Botulinum B test and 4 nM Bio-RbαLVS, 8 nM of Eu-RbαLVS for Francisella tularensis test. Antigen was diluted in PBS, added to the assay mixture (80 µl) and agitated at room temperature for 30 min in an orbital micro shaker (Dynatech, England). The plates were then washed on a microplate washer (Tecan, Hydrospeed, 30054550) equipped with a smart-2MBS 96 well magnetic plate. Captured beads were re-suspended in 50µl of enhancement solution and the resulting signal (Excitation 340nm, emission, 612nm) was determined using a plate reader (Tecan, infinite F200). Multiplexed assays. Fluorophore bead-based assays were performed in a similar fashion to that described for the Europium bead based assays with the following changes: 1. The tests were performed simultaneously in one assay mixture (Magnetic beads and labeled specific antibodies for the three assays were mixed together). 2. Optimized assay performance was obtained by using a buffer consisting of PBS + 2% NRS + 0.05% TW20. 3. For optimal performance, the assays were performed in 96-well black plates (Nunc. Catalog num. 137101). 4. At the end of the incubation and washing steps the beads were re-suspended in PBS + 0.05% tween20 and then read in a Plate Reader (Tecan, infinite F200) in the appropriate wave lengths. For detection from complex matrices, analytes were inoculated in commercially available beverages (1% fat milk, 3% fat milk, Soy milk, infant formula, orange juice and apple juice). The pH of both apple and orange juices was corrected to 6.5 using 10N NaOH. Untreated tap water was used. Environmental extracts were prepared by extracting road sampled swabs in PBS. Signal analysis for bead based assays. Results for all bead based assays were calculated as signal-to-noise (S/N) ratios between antigen (S) - or PBS (N) containing tests. This calculation enabled the normalization of multiple experiments and the determination of a universal threshold for positive samples. In order to evaluate the limit of detection (LOD), the average background reading for each assay was calculated as the mean response of at least six "noise" samples. The LOD was 5 ACS Paragon Plus Environment


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defined as 5 standard deviations (SD) above the average background with a coefficient of variation (CV) < 15%. These values yielded a signal (noise + 5xSD)-tonoise (average noise) ratio which was considered to be the LOD threshold. The threshold LOD values obtained for the various tests ranged from S/N=1.4 to S/N=1.7. For simplicity in data presentation and comparison, a value of S/N≥2 was set as the positive threshold for all assays in accordance with ICH guidelines for validation of analytical procedures 35. Evaporation assays. Assay mixtures (magnetic beads and labeled antibodies) were prepared in an optimized evaporation buffer (standard buffer containing 5% trehalose and 1% BSA) as described for standard Europium bead based detection assays. During the optimization process we evaluated a range of BSA, trehalose and PVP concentrations (0.3-1% for of BSA and PVP and 1.5-5% for trehalose). For the drying process, polymer addition was found to be detrimental (hindered beads reconstitution). The assay mixture was aliquoted (80 µl / well) in 96 well plates and allowed to dry for two days at 37oC. For long term storage at different temperatures the dried plates were sealed in the presence of desiccants (Natrasorb, Multisorb Technologies, Inc.). For detection, dried wells were re-suspended in 80µl PBS and the antigen was added as described. Assay performance was compared to freshly prepared assays. Freeze drying assays. Assay mixtures (magnetic beads and labeled antibodies) were prepared in an optimized lyophilization buffer (standard buffer containing 3.3% trehalose, 0.6% BSA and 0.3% PVP) as described for standard Europium bead based detection assays. During the optimization process we evaluated a range of BSA, trehalose and PVP concentrations (0.3-1% for of BSA and PVP and 1.5-5% for trehalose). In addition we assessed the contribution of different polymers (Polyox WSR-301, Carbopol EZ-1, Klucel EEF, Cellulose GUM 7LF, Viscarin PVA and Matrosol 250LR from Formulator's Polymer Tool Kit, Pragmatics Inc. Elkhart IN 46516) that are known stabilizers and shelf life extenders at 0.5%-1% concentration. Assay mixtures were aliquoted (80 µl/well) in 96 well plates, frozen in liquid nitrogen and lyophilized at 0.01mbar for at least 16 hours in an ALPHA 1-2 LDplus lyophilizer (CHRIST, Andre Unteren Sose 5O, Germany). For long term storage in different temperatures, the dried plates were sealed with an aluminum foil in the presence of desiccants. For detection, dried ELISA wells were re-suspended in 80µl 6 ACS Paragon Plus Environment

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PBS and the antigen was added as described. Assay performance was compared to freshly prepared assays. Electron Microscopy. Scanning electron microscopy (Quanta FEG 200, FEI) analysis was performed under low vacuum conditions with an accelerating voltage of 10kv. The samples were placed on carbon tape (EMC Cat. #77816) on an aluminum mount (EMS Cat. #75520). Statistical analysis. The statistical difference between the S/N ratios of the lyophilzed tests at different temperatures for different antigen concentrations was analyzed using Single Factor ANOVA. A p-value of less than 0.05 was considered to indicate that there was no statistical difference between the examined numerals. Safety considerations. All crude antigens were prepared under BSL3 conditions. Experiments were carried out under BSL2 conditions with attenuated/inactivated strains or low concentration of the indicated antigens.

RESULTS AND DISCUSSION

Development of time resolved fluorescence (TRF) magnetic beads based homogeneous immunoassays. The basic principle of the homogeneous assay used in this study is depicted in Figure 1. We utilized a time resolved fluorescence (TRF) magnetic-beads based assay, that consists of streptavidin coated magnetic beads and antigen specific antibodies, which are either biotinylated or coupled to Europium (EuIII). Assay components were suspended in a standard assay buffer (PBS + 2% NRS + 0.05% Tween 20 + 8 µg/ml DTPA, see materials and methods) and the suspension was incubated with the antigen containing sample, leading to the formation of a "sandwich" immunoassay linked to the magnetic beads (via the biotin-avidin interaction). This complex was then separated using magnetic force and the beads were re-suspended in enhancement solution thereby triggering the dissociation of the EuIII ions from the labeled antibody. Upon excitation, the resulting highly fluorescent chelates emitted a signal which was proportional to the antigen concentration in the sample. Three individual assays were developed for the detection of soluble and particulate BW agents, i.e: B. anthracis spores, Botulinum B toxin and Francisella tularensis bacteria. Dose response curves for the detection of the three BW agents are presented in Figure 2. As can be seen, the resulting homogeneous assays are sensitive (Limit of 7 ACS Paragon Plus Environment


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detection of 4x105 cfu/ml B. anthracis spores, 1 ng/ml Botulinum B and 4x104 cfu/ml Francisella tularensis, based on signal to nose ratio > 2) and demonstrate a broad dynamic range (2 to 3 orders of magnitude). The assays were rapid (40 minutes in total) and simple to operate. Evaporation vs. lyophilization as a whole test stabilizing technique. It was previously shown that drying by means of evaporation in sugar-containing solutions is the method of choice for the preservation of plate anchored ELISA immunoassays

12-

14

. To evaluate the potential application of evaporation as a drying methodology for

magnetic beads based homogeneous immunoassays, assay components (magnetic beads and labeled specific antibodies) were suspended in standard assay buffer and in the same buffer supplemented with various concentrations of sugars and polymers. Assay mixtures were then evaporated in standard ELISA plates (Evaporation of the liquids was done by incubating the uncovered plates at 37oC for 48 hours) and their performance was evaluated compared to freshly made assays in the same buffers. It is important to note that freshly made assay performance was practically identical regardless of buffer composition. However, after evaporation, only buffers supplemented with sugars were able to preserve the assay’s activity, supporting the assumption that the addition of a sugar moiety is essential for preservation of assay performance. In the standard buffer, no activity could be detected after drying, due to aggregation and clump-formation of the beads after re-suspension (This phenomenon could be observed with the naked eye). The preservation of assay performance utilizing the final optimized buffer (standard buffer supplemented with 5% trehalose and 1% BSA) for all the developed assays is presented in figure 3. As can be seen in figure 3 (Green vs. red symbols in all the graphs) while the B. anthracis spore assay was successfully preserved via evaporation (panel A), both Botulinum B (panel B) and Francisella tularensis (panel C) bead based assays suffered irrevocable damage due to the evaporation processes. As a result these assays exhibited a significant reduction in assay sensitivity. It appears that different antibodies exhibit different sensitivities to the evaporation process which is somewhat surprising, since the antibodies implemented in all three tests are polyclonal antibodies which supposedly include different populations. We can therefore assume that the polyclonal fractions contain dominant antibody populations exhibiting different sensitivities to the drying process. 8 ACS Paragon Plus Environment

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Evaporation is a slow process that involves considerable changes in the concentration of all assay components, and might therefore lead to irreversible changes in protein structure and function. An elegant way to dry proteins with minimal functionality loss is lyophilization. In this process, the sample is rapidly frozen (using liquid nitrogen) and the water molecules are then evaporated via sublimation using low pressure. This method is employed routinely for the preservation of antigens or proteins

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. To

evaluate the implementation of lyophilization for the preservation of complete immunological bead based assays, the three assays were lyophilized in a panel of different buffers. As was observed for the evaporation methodology, when the lyophilization was carried out in the standard assay buffer, complete loss of activity was observed for all the assays. However, in contrast to evaporation, it was observed that for optimal performance after lyophilization, the addition of both trehalose and the polymer PVP to the buffer formulation was advantageous. Figure 3 demonstrates the preservation of assay activity after lyophilization of the assay mixture in the optimized lyophilization buffer (blue vs red symbols in all graphs). As can clearly be seen, lyophilization proved to be superior to evaporation for the preservation of assay performance for all of the assays (blue vs. green symbols in all graphs). Some reduction in assay performance can be seen for the tularemia test, probably due to partial denaturation of a small fraction of the antibodies. Microscopic analysis of the dried assays using scanning electron microscope (SEM) revealed that the assays differ substantially in the dry state, Figure 4. The lyophilized assay (B) consists of a powdery material with individual magnetic beads that seem to be coated with the sugar/polymer buffer. In contrast, the evaporated assay (A) appears as a glassy sugary film with bead aggregates that are clearly embedded in the film. We assume that the dispersion of discreet beads in the powder contribute greatly to their successful recovery since after re-suspension the beads are in their initial form. Effect of lyophilization on assay specificity. One of the most important parameters of an immunoassay is its specificity. This is especially true for the detection of BW agents, where accurate identification of the agents has a crucial impact on subsequent treatment protocols and other operational considerations. In order to verify that the lyophilization of the complete homogeneous assay does not impair its specificity profile, the response of a freshly made vs. lyophilized assay was compared against an array of relevant soluble and particulate BW agents. Results are presented in figure 5. 9 ACS Paragon Plus Environment


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In each graph, left to the dotted line, the positive response of the test to its native antigen is presented. For the non-relevant antigens (right of the dotted line in each graph), the response (S/N) was well below the threshold (S/N=2) for each test, indicating that the lyophilization process does not change antibody specificity patterns and the tests do not show any cross reactivity with the non-relevant antigens examined. As expected, the Botulinum B test shows some cross reactivity with Botulinum A, due to the immunogenic response to the Hemagglutinins present on both toxins. Accelerated stability testing. The successful stabilization of the assay was further investigated for its thermal stability, namely, whether it is possible to incubate the tests at high temperatures for long periods of time with no loss of assay activity. To this end, lyophilized assays for the three agents were stored in temperatures ranging between 37oC to 100oC, for a week. After a short cooling period, the dry assays were reconstituted and tested. The results are presented in figure 6. The statistical difference between the S/N ratios determined for each antigen concentration at the different incubation temperatures was calculated using single factor ANOVA (See materials and methods). No statistical difference was observed, indicating that the tests are indeed stable for the indicated time. No apparent loss of activity was also observed at 100oC (for a week), but is this case the plate was so deformed that this experiment was performed only once and is therefore not presented in the graph. Application of the lyophilized assays for colorimetric end point detection. To extend the possible applications of the developed methodology and establish its broad potential, different kinds of reporting molecules, other than Europium were tested. To this end reporter antibodies conjugated to HRP were prepared, bearing in mind that for the end point user it might be advantageous to be able to determine a positive result without the need for a sophisticated reader, using visible color change. HRPconjugates were prepared for all agents. The tests were optimized and the activity of the lyophilized tests was compared to freshly-made ones. The tests were successfully lyophilized and retained their activity after being stored for a week at 85oC (Figure S1). Application of the lyophilized assays in multiplexed detection. Another possible application of the developed stabilizing methodology is multiplexed detection. This 10 ACS Paragon Plus Environment

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will enable the end point user to simultaneously detect one of several antigens in the same sample, thus identifying several different threats in one relevant complex sample. In this scenario several tests are co-lyophilized and stabilized in the same test tube, while for each test, the reporting antibody is labeled with a different marker. The marker can be Quantum dots (QDs) 36-38, or other fluorophores 39,40. Reconstitution of the dry assay, addition of the antigen and subsequent excitation of the sample in the proper excitation wavelength will induce fluorescence only of the label of the antibody linked to the relevant assay, see scheme in figure 7. Multiplexed detection of dry lyophilized B. anthracis, Botulinum B and Francisella tularensis bacteria assays using different Alexafluor tags was studied. Each reporting antibody was labeled with a different Alexafluor tag (Alexa 488, Alexa 647 and Alexa 594 for B. anthracis, Botulinum B and Francisella tularensis specific antibodies respectively) and was excited in different and distinct wavelengths. The three tests (magnetic beads and specific labeled antibodies) were mixed together and lyophilized using the above mentioned buffer. The dried assays were challenged with different concentrations of the BW agents, and the whole plate was excited at the appropriate wave lengths for the three different markers (488, 594 and 647 nm). Figure 8 portrays the clear distinction of each agent from the two other agents, using the unique fluorescent tag applied for each antibody. Each antigen was detected with excellent selectivity and no cross reaction with the other BW agent. The performance of the lyophilized assay was similar to that of a freshly made assay. To further establish the relevance of the methodology, the multiplex assay was applied for the detection of the three agents from relevant complex samples, i.e. different beverages (1% fat and 3% fat milk, tap water, soy milk, infant formula, apple juice, orange juice and environmental samples). Food samples, specifically milk are considered as a possible target for bio-terror attacks especially with regard to B. anthracis spores and Botulinum toxin

41,42

. In humans, three types of anthrax have

been recorded according to the route of infection: cutaneous, gastrointestinal and inhalational 43. Studies carried out in animal models indicated that infectious doses for gastric anthrax vary from 107 to 1010 B. anthracis spores

44

underlining the relevance

of the developed test for detection of anthrax from liquids. Botulinum toxin's natural infection route is the gastric tract with an estimated human 50% lethal dose (HLD50) of 1 ng/kg body weight

45

. Our developed test is indeed applicable for Botulinum 11

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detection from liquids, enabling detection in the relevant toxification range. The multiplexed detection of the three agents was successfully carried out in the different beverages (All beverages were applied "as is" in the test with no dilution) using the lyophilized assay, as depicted in figure 9. Detection of the antigens from the different beverages was similar to that achieved in PBS. No cross reactivity was observed between the three antigens as was demonstrated (see Figure 8). Assay performance was also evaluated after incubation at 85oC for a week. No significant loss in activity was detected as was shown for the Europium based test, indicating that the multiplexed assay is stable at elevated temperatures (Figure S2).

CONCLUSIONS

In this work we show for the first time the drying and stabilization of a complete bead-based homogeneous immunoassay. The methodology was applied successfully for the preservation of three different immunoassay tests for the detection of both soluble and particulate BW agents, i.e, B. anthracis, Botulinum B and Tularemia. Although the reported LODs for the developed tests are at the upper range of some published works for immunodetection of these antigens that each assay depends on the antigen

53

46-52

and/or antibodies

, one has to remember 54

implemented in the

assay, as well as assay duration. In fact, in a recent comparison of eleven commercially available rapid tests for "on site" detection of B. anthracis and F. tularensis

55

it was found that while the lateral flow assays examined in the study

were short (15 min), easy to operate and could be stored with no refrigeration, they all demonstrated lower detection sensitivities for the tested antigens (108 cfu/ml B. anthracis and 107-108 cfu/ml for F. tularensis). Other commercially available kits, evaluated in the aforementioned study (based on immunofiltration), proved to be more sensitive (detection of 106 cfu/ml B. anthracis and 104 cfu/ml for F. tularensis). However, they included several washing steps and some components of the kit needed to be stored at 4oC, rendering the kits unsuitable for "on site" testing. Hence, our developed homogeneous assays are comparable to current immunodetection methods, are faster, highly stable and simpler to operate and will be beneficial for detection of the antigens in different complex samples as was demonstrated (Figure 9). Moreover, we recently attempted the incorporation of FluoSpheres® Europium Luminescent Microspheres as reporting molecules, demonstrating an order of magnitude 12 ACS Paragon Plus Environment

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improvement in the detection limit of the Botulinum B bead-based assay (LOD of 0.2ng/ml Botulinum B). Preliminary results indicate that the developed test could be lyophilized and reconstituted successfully (Figure S3). We believe that the work presented in this study sets the foundation for application of the developed tests for "on site" detection. Utilizing a simple magnet rack and an Eppendorf tube, combined with a hand held device for signal determination (for example GloMax-Multi Jr. by Promega), one could easily implement the developed stabilized tests for low throughput applications. For high throughput applications, one would have to adapt the assay to some sort of a microfluidic device, as was previously demonstrated 56. The wide applicability of the developed methodology was demonstrated for the various assays, combining different reporter molecules utilizing three different reporting mechanisms, i.e, Europium (TRF), HRP (enzyme moiety) and Alexa fluorophores (fluorescence, multiplexed detection). The lyophilized assays, in all assay formats, exhibited high stability at elevated temperatures. The fact that the assays retained their activity even after several days at 100oC, indicates that the assays can be easily transported, handled and stored at end point locations with no refrigeration or freezing..



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Figure Legends Figure 1. Schematic representation of bead-based TRF assay. The assay consisted of A. A suspension of streptavidin coated magnetic beads and antigen specific antibodies, which were either biotinylated or coupled to Europium (EuIII). Assay components were suspended in assay buffer and the suspension was incubated with the antigen containing sample (30 min.), leading to the formation of a "sandwich" immunoassay linked to the magnetic beads (via the biotin-avidin interaction). B. Separation of unbound components using magnetic force and C. Re-suspension of the “sandwich” immunoassay in enhancement solution and signal determination.

Figure 2. Dose response curves of time resolved fluorescence bead-based immunoassays for the detection of B. anthracis (A), Botulinum B (B) and F. tularensis (C). Antigens were diluted in PBS and analyzed with freshly made homogeneous tests. Points are average of at least three independent sets of measurements conducted on different days. Signal-to-noise (S/N) ratios were calculated as described in materials and methods and the limit of detection for each test was determined for S/N ratios ≥ 2 (Black dashed line).

Figure 3. Evaluation of time resolved fluorescence assay performance - evaporation vs. lyophilization. Assay components (magnetic beads and biotinylated or europium labeled specific antibodies) were suspended in optimized drying buffer and assay performance was evaluated after lyophilization (blue) or evaporation (green) in comparison to freshly prepared tests (red). Assay activity was determined with different concentrations of B. anthracis spores: 5x105, 2x106, 6x106 and 1.8x107 cfu/ml (A), Botulinum B: 1, 3, 9, and 27 ng/ml (B) and F. tularensis: 5x104, 5x105, 1.5x106 and 5x106 cfu/ml (C). Points are average of at least three independent sets of measurements. The limit of detection for each test was determined for S/N ratios ≥ 2 (Black dashed line).

Figure 4. Scanning Electron Microscopy analysis of complete bead-based immunoassays after A. evaporation, B. lyophilization. The analysis was performed under low vacuum condition.


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Figure 5. Evaluation of time resolved fluorescence assay specificity after lyophilization. Assays were lyophilized (Filled bars) and assay specificity was evaluated in comparison to freshly prepared tests (Empty bars) for B. anthracis (A), Botulinum B (B) and F. tularensis (C). S/N ratios were calculated as described in materials and methods and the limit of detection for each test was determined for S/N ratios ≥ 2 (Black dashed line).

Figure 6. Thermal stability measurements. Time resolved fluorescence assays were lyophilized in ELISA plates and stored (desiccated) at 37oC (green bars), 60oC (yellow bars), 80oC (orange bars) and 90oC (red bars) for one week. Assay activity was determined with different concentrations of B. anthracis spores: 2x106, 6x106 and 1.8x107 cfu/ml (A), Botulinum B: 3, 9, and 27 ng/ml (B) and F. tularensis: 5x105, 1.5x106 and 5x106 cfu/ml (C). Average S/N values and standard deviation (error bars) were calculated for three independent experiments.

Figure 7. Schematic representation of multiplex bead-based immunoassay.

Figure 8. Multiplexed, lyophilized immunoassays for detection of B. anthracis (A), F. tularemsis (B) and Botulinum B (C). The multiplexed assay (based on Alexa fluorophores reporter antibodies) was lyophilized, and challenged with different concentration of B. anthracis spores (3x106, 9x106 and 2.7x107 cfu/ml), Botulinum B (5, 14, and 45 ng/ml) and F. tularensis (1x106, 3x106 and 9x106 cfu/ml). The plate was then excited with 488 nm (A), 647 nm (B) and 594 nm (C) and the signal was determined for each Alexa fluorophore separately. Average S/N values are presented for each antigen (Positive signal = S/N ≥ 2).

Figure 9. Multiplexed detection of B. anthracis (A), Botulinum B (B) and F. tularensis (C) from different beverages. The multiplexed assay was lyophilized and challenged with different concentration of B. anthracis spores (3x106, 9x106 and 2.7x107 cfu/ml) (A), Botulinum B (5, 14, and 45 ng/ml) (B) and F. tularensis (1x106, 3x106 and 9x106 cfu/ml) (C) diluted in PBS or and other beverages, as indicated in the graphs. The beverages were incorporated "as is" in the test. The pH of the acidic beverages (apple and orange juices) was corrected to 6.5 using 10N NaOH. The plate was then excited with 488 nm, 647 nm and 594 nm and the signal was determined for 19 ACS Paragon Plus Environment


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each Alexa fluorophore separately. Average S/N ratios for each antigen were calculated as described in materials and methods and the limit of detection for each test was determined for S/N ratios ≥ 2 (Black dashed line).


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Figure 1.



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Figure 2.

A.

B.

C.


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Figure 3.

A.

B.


C.


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Figure 4.


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Figure 5.

A. 35

S/N

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12

18

C.

16

30

10

25

8

20

B.

14 12 10

6

8

15

6

4

10

4

5

2

0

0

2 0

BotB

F. tularensis

B. anthracis



Figure 6.

100

1000

1000

37oC

S/N

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100

100

10

10

60oC

10

80oC 90oC

1

1 2.0E+06

6.0E+06

1.8E+07

B. anthracis (cfu/ml)

1 3

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27

Botulinum B (ng/ml)


5.0E+05

1.5E+06

4.5E+06

F. tularensis (cfu/ml)

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Figure 7.



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Figure 8.

A.

B. Anthracis (cfu/m l)

B.

F. tularensis (cfu/m l)

Botulinum B (ng/m l)

C.








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Figure 9.

A.

B.

10 8 6 4

C.

40

12

S/N

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PBS Tap water Environment 1% Fat milk 3% Fat milk Soy milk Infant formula Apple juice Orange juice

35 30 25

16

PBS

14

Tap water

12

Environment 1% Fat milk

10

3% Fat milk

20

Soy milk

8

15

Infant formula

6

Apple juice 10

2

4

Orange juice

2

5

0

0

0

0

3.0E+06

9.0E+06

B. anthracis (cfu/ml)

2.7E+07

PBS Tap water Environmental 1% Fat milk 3% Fat milk Soy milk Infant formula Apple juice Orange juice

0

5

15

45

Botulinum B (ng/ml)


0

1.0E+06

3.0E+06

F. tularensis (cfu/ml)

9.0E+06


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For TOC only


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Highly Stable Lyophilized Homogeneous Bead ... - ACS Publications

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