Detection of Staphylococcus aureus toxins using immuno-PCR

ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 5, pp. 526–531. © Pleiades Publishing, Ltd., 2014. Original Russian Text © A.V. Maerle, D.Yu. Ryazantsev, O.A. Dmitrenko, E.E. Petrova, R.L. Komaleva, I.V. Sergeev, D.Yu. Trofimov, S.K. Zavriev, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 5, pp. 571–577.

Detection of Staphylococcus aureus Toxins Using ImmunoPCR A. V. Maerlea, 1, D. Yu. Ryazantsevb, O. A. Dmitrenkoc, E. E. Petrovab, R. L. Komalevab, I. V. Sergeeva, D. Yu. Trofimovd, and S. K. Zavrievb a

NRC Institute of Immunology, Federal Medical and Biological Agency, Kashirskoe sh. 24/2, Moscow, 115478 Russia b Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow c Gamaleya Research Institute of Epidemiology and Microbiology, Moscow d JSC “NPF DNKTekhnologiya”, Moscow Received December 4, 2013, in final form, March 23, 2014

Abstract—A highly sensitive test system, based on the immunoPCR method, was developed for the detec tion of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key ele ment of the developed systems was to obtain supramolecular complexes of bisbiotinylated oligodeoxynucle otides and streptavidin, which were to be used as DNAtags. Specificity studies showed no crossreactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not lower than 10 pg/mL. Keywords: immunoPCR, Staphylococcus aureus, supramolecular complexes DOI: 10.1134/S1068162014050112 1 INTRODUCTION

ImmunoPCR is a highly sensitive method for the detection of antigens of different natures such as spe cific antibodies, cytokines, tumor markers, prions, viral and bacterial antigens, including toxins [1]. This method was devised by Sano et al. [2] in 1992. The main idea was to use a specific DNA fragment (DNAtag) and a polymerase chain reaction (PCR) for the detection of interaction between “antigen–antibody.” Amplifi cation of the DNAtags associated with a specific anti body, followed by detection of the amplification prod ucts allow us to achieve a higher detection sensitivity for the detection of antigens compared with the ELISA. The main areas of development of the method were the selection of the solid phase for immobiliza tion of antibodies or antigens, selection of the optimal DNAtags and the method of the binding of the DNA tag with the detection antibody. Staphylococci (Staphylococcus aureus etc.) pro duce large amounts of toxins, from which a group exhibiting properties of superantigens can be selected. This group includes staphylococcal enterotoxins (SEA, SEB, SEC and remaining until SEV), and toxic shock syndrome toxin (TSST) [3]. Conventional anti gens are absorbed by antigen presenting cells (APC) and undergo processing followed by the appearance of antigenic peptides complexed with molecules of MHC Abbreviations: APC, antigenpresenting cells; Bt, biotin; SEA, staphylococcal enterotoxin A; Str, streptavidin; TSST, toxic shock syndrome toxin. 1 Corresponding author: phone: +7 (926) 1664720; email: [email protected].

on the surface of the APC. These complexes are recog nizable by the Tcell receptors of Tlymphocytes and can lead to the further development of a specific immune response. Only one cell out of 105–106 naive Tcells are stimulated [4]. Superantigens do not undergo processing in the APC cytoplasm, however, they are capable of binding with molecules of Tcell receptors on CD4positive Tlymphocytes and mole cules of major histocompatibility complex of class II on the APC surface directly [5]. Thus the specificity of Tlymphocytes is ignored. Due to such a nonspecific stimulation of Tlymphocytes, a large release of proin flammatory cytokines occurs, since the level of stimu lated Tlymphocytes can reach 20% of the total amount [6]. Superantigens play an important etio pathogenic role in the development of a number of dif ferent severe pathological conditions in humans, such as menstrual and nonmenstrual toxic shock syndrome, staphylococcal pneumonia and endocarditis. The association of superantigens with the development of staphylococcal purple lightning and extreme hyper thermia syndrome has been recently described. Food poisonings caused by S. aureus are syndromes when superantigens of staphylococci (A, B, C, D, E and G) are the main cause of the disease [7, 8]. A majority of severe cases of toxic shock syndrome (TSS) are caused by the superantigen properties of TSST, SEB and SEC. In turn, SEA is the main reason for staphylococcal food poisoning [9]. ELISA and latex agglutination methods are the main laboratory methods for the identification of sta phylococcal toxins. However, the sensitivity of these

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methods is not higher than 30 and 500 pg/mL, respec tively [10–13]. Increased sensitivity of detection of staphylococcal toxins is possible by the use of the immunoPCR method demonstrated by Fischer et al. [14] and Rajkovic et al. [15]. In these studies, the limit of detection of staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) were 100 [14] and 10 pg/mL [14, 15] respectively. This study deals with the optimization of the immunoPCR method that we developed earlier [16] and SEA and TSST detection in samples of superna tants of Staphylococcus aureus cell culture of clinical isolates. RESULTS AND DISCUSSION To date, there is no gold standard in the development of test systems based on immunoPCR. The key points are: the selection of a solid phase for immobilizing the binding antibody, the choice of blocking components and choice of the method for the introduction of the DNAtag, and minimization of background signals. The result of this work is a technique developed based on immunoPCR using supramolecular complexes of sin glestranded DNA with streptavidin as the tag. We used a pair of monoclonal antibodies with dif ferent epitopes of SEA and TSST toxins. The first (binding) antibodies result in antigen capture from the sample and its immobilization on the solid phase, and then the second (detecting) biotinylated antibody binds to another epitope of the corresponding toxin. The DNAtag with biotin via streptavidin binds to the formed immunological complex and can be detected by PCR. Solid phase. As a solid phase can be used ELISA and PCR tubes and plates [1] with improved sorption of protein and plates with preadsorbed streptavidin [17] binding the first (binding) antidodies modified with biotin and magnetic particles [1, 18]. For the solid phase for the immobilization of the first (binding) antibodies we choose a polycarbonate plate for the PCR (Greiner, Germany). The surface of wells of this plate has sufficient capacity for the sorp tion of antibodies. We have chosen the optimal condi tions for modification of wells by binding antibodies: 40 µL of the binding antibody solution (20 µg/mL) in PBS buffer for 16 h at +4°C. The Vshape of the wells allows using it directly in the PCR thermocycler and it eliminates the necessity of cleaving the DNAtag with

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500 bp

70 bp 1

М

2

М

3

4

5

Fig. 1 Electrophoregram of oligo(N)Str conjugate with Bt2oligo(N) and streptavidin molar ratio 1 : 2, 1 : 1 and 2 : 1 (lanes 1, 2 and 3, respectively); Btoligo(N)streptavidin conjugate (lane 4); free Bt2oligo(N) (lane 5). M, molecu lar marker GeneRuler 1 kb Plus DNA Ladder (Fermentas, Lithuania).

the subsequent transfer of the solution from the plate in the PCR tubes. DNAtag. For DNAtags we used supramolecular complexes [(biotin)DNA(biotin)streptavidin]n [16, 19], the conditions for preparation of these complexes were developed in this study. For this purpose single stranded oligodeoxynucleotides (oligo(N)) with lengths of 60 bp with one (Btoligo(N) and two mole cules of biotin (Bt2oligo (N)) were synthesized. As shown in the previous study [16], singlestranded oli gonucleotide used as DNAtag, allows achieving higher sensitivity of the method compared with dou blestranded DNAtags. For the use of conjugates of oligodeoxynucleotides with streptavidin in immunoPCR, the conditions for their production were chosen and optimized. Deter mining the criterion for selection was the minimal detectable SEA concentration in TBS. Conjugates of Bt2oligo(N) with streptavidin with different molar ratios of the components (Fig. 1, lanes 1–3) and con jugate of monobiotinylated oligonucleotide Btoligo (N) with streptavidin (Fig. 1, lane 4) were obtained; the characteristics of these conjugates are shown in Table 1. The formation of the product with electro phoretic mobility, equivalent to an oligonucleotide with a length of about 350 bp in all three conjugates Bt2oligo(N)streptavidin (Nos. 1–3, Table 1) with

Table 1. Characteristics of oligo(N)streptavidin conjugates Conjugate No.

1

Modification of oligonucleotide The molar ratio of oligo(N)streptavidin The minimum detectable concentration of SEA in TBS buffer, pg/mL RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

2

3

3',5'bisbiotinil 1:2 10

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2:1 10

1:1 100

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12.0

ΔCn, normalized value

10.0 8.0

(а)

f(x) = 0.886ln(x) + 7.072 R2 = 0.994

6.0 4.0 2.0 0 0.001

1 0.01 0.1 10 SEA concentration in the sample, ng/mL

100

(b) 10.0


8.0

f(x) = 0.851ln(x) + 6.478 R2 = 0.992

6.0 4.0 2.0 0 0.001

0.01 0.1 10 1 100 TSST concentration in the sample, ng/mL

Fig. 2. Calibration curves obtained by analysis of tenfold dilutions of SEA (a) and TSST (b) in TBS. Linear detec tion range of the toxin for both systems was from 1 to 10 ng/mL. Detection limit was 1 pg/mL.

various molar ratios of the components proves the formation of supramolecular complexes [(biotin)DNA(biotin)streptavidin]n. The formation of supramolecular complexes increases the amount of nucleotide tags associated with a single antibody, which allows achieving higher sensitivity compared to conjugate 4. By comparing the results obtained for the use of conjugates Nos. 1–4 in immunoPCR, conju gate 2 was chosen for further study, since it allowed achieving higher detection sensitivity of SEA in TBS (Table 1). Taking into account that in all three cases with the use of bisbiotinylated oligonucleotide, supramolecular complexes with the same electro phoretic mobility were formed, we can assume that the reduction in detection sensitivity of SEA in TBS using conjugates 1 and 3, compared with an equimolar con jugate 2 is associated with the presence of a certain percentage of free streptavidin in conjugate 1 (excess of streptavidin) and the blocking of the free valences of streptavidin in conjugate 3 (excess of Bt2oligo(N)).

Blocking agents. In order to reduce the level of nonspecific binding of the DNAtag and second anti bodies on the surface of the solid phase, we selected conditions for the blocking of nonspecific adsorption. Bovine serum albumin (BSA), salmon sperm DNA and skimmed milk were used as blocking components. It has been shown that in our system, blocking by BSA solution in the presence or absence of salmon sperm DNA, or dry milk provides low background values. The addition of salmon sperm DNA or dry milk slightly reduces the background value, but at the same time increases the variability of the threshold cycles for duplicates of one sample. Therefore, the BSA solution in TBS was chosen for blocking. Characteristics of the developed test systems. For the development and optimization of the test systems purified SEA and TSST toxin preparations were used. The optimized protocol for the immunePCR test is shown in the experimental section. We reached the limit of detection of purified toxin in TBS buffer for both systems, the limit of detection was 1 pg/mL of the test sample, which exceeds the sensitivity of corre sponding ELISA systems by 300 and 200 times for the detection of SEA and TSST respectively [16]. For quantitative detection of SEA and TSST in unknown samples, linear regression of the mean normalized ΔCn values and purified toxin concentration in the respective linear ranges was performed. The linear range for both systems was in the range from 1 pg/mL to 10 ng/mL. Linear curves represent acceptable regression coefficients, 0.994 and 0.992 (for SEA and TSST, respectively) (Figs. 2a and 2b). It was of interest to investigate the possibility of determining the presence of SEA and TSST in the supernatants of broth cultures of S. aureus clinical iso lates. For this purpose a detection test was performed (“introducedfound” method). A series of samples in which dilution of control samples of toxins in the supernatant was carried in overnight S. aureus cultures which did not contain the toxin, followed by a 10fold dilution in a TBS buffer was performed. The percent age of “detection” for TSST was 110% (3 pg/mL), 91% (30 pg/mL) and 92 % (3 ng/mL). The percentage of “detection” for SEA also was in the range of 90– 110%. Thus, the possibility of not only SEA and TSST detection in supernatants of cultures of clinical iso lates, but also quantitative detection with tenfold dilu tion in the TBS buffer was demonstrated. Detection of SEA and TSST in S. aureus culture supernatants, obtained from clinical samples obtained in 2009–2011 from patients with pneumonia (autopsy material, sputum) or bacteremia (blood). The results of SEA and TSST detection in samples was obtained by 10fold dilutions of supernatants of overnight cul tures of clinical isolates in a TBS buffer are shown in Table 2 and Fig. 3. Table 2 shows the results of determining the pres ence of SEA and TSST in the supernatant of cultures

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Table 2. Comparison of genotyping data and results of detection of staphylococcal toxins by immunoPCR analysis No. 1 2 3 4 5 6 7 8 9 10

Source of isolation*

sea

tst

782 784 706 713 715

a.m. a.m. a.m. a.m. Sputum r.s. r.s. Blood a.m. a.m.

+ + + + + – – – – –

– – – – – + + + – –

DSM FRI 1169 6 765 738

Data of immunoPCR analysis, ΔCn

Genotyping data**

Number of isolate

SEA

TSST

4.0 3.3 5.3 3.9 2.6 –0.9 –0.7 –1.0 –0.8 –1.1

–1.0 –1.1 –1.1 –0.5 –1.0 3.1 1.3 5.9 –0.8 –1.1

* a.m., autopsy material; r.s., reference collection strainTSST producer, ** the presence of superantigens genes accordingly to the pre viously obtained PCR data.


plexes. The synthesis of these complexes allowed achieving higher detection sensitivity of SEA and TSST up to 10 pg/mL in the supernatant of the bacte rial culture. The time for the preparation of the plate (sorption of primary antibodies and blocking of nonspecific adsorption) was 18 hours; the time for the analysis was not more than 5 hours. The possibility to determine bacterial toxins in such complex matrices as food by methods based on 7 6 5


of clinical isolates completely coincided with the pres ence of the respective genes encoding toxins from investigated strains. In sample numbers 6, 7 and 8, obtained from clinical isolates of S. aureus with tst gene, TSST content in the supernatant of overnight cultures was in the range of 140 pg/mL (No. 7) to 26 ng/mL (No. 8). For samples 1, 2, 3, 4 and 5 (sea gene) SEA content ranged from 100 pg/mL (No. 5) to 2.5 ng/mL (No. 3). Data obtained by immunoPCR are repre sented as normalized ΔCn values. It is important to note the absence of false positive results (see Table 2). This fact allows us to conclude that for these samples the developed test system showed 100% specificity. Our study of the adaptation of the immunoPCR method for the detection of staphylococcal toxins was not the first such attempt. Some progress was achieved in the studies of Rajkovic et al. [15] and Fischer et al. [14]. In one study [15] streptavidin and monobiotiny lated dsDNAtag were used and a sensitivity of SEB detection of 10 pg/mL was achieved. Fischer et al. [14] applied a covalent conjugate of a detection antibody and DNAtag with the restriction site which allowed the transfer of the DNAtag from the formed immu nological complex in tubes for PCR by cleavage of DNA fragments with a restriction enzyme [14]. This allowed the authors to achieve the following sensitivity of the detection: SEA, 100 pg/mL and SEB, 10 pg/mL, but the analysis procedure was more complicated. In this study, the optimization of the immunoPCR protocol was achieved by selecting optimal PCR plates that allowed the performance of the entire analysis without transfer of DNAtags, which improved the conditions of sorption of binding antibodies and the development of the optimal composition of the blocking solution. One of the main advantages of this method is the use of supramolecular [Bt2oligo(N)Str]n com

TSST SEA

4 3 2 1 0 –1 –2 1

2

3

4

5

6

7

8

9

10 NC

Fig. 3. Results of TSST and SEA determination, obtained for tenfold dilution of S. aureus overnight bacterial culture supernatants in TBS buffer Nos. 1–10 (see Table. 2); NC—negative control (1 : 10 dilution of supernatant over night S. aureus culture, not containing SEA and TSST in TBS buffer). Vol. 40

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the use of antibodies, especially by the immunoPCR method, should be subjected to a detailed study of each developed system. The positive experience of Liang et al. [22], who demonstrated the absence of an inhibitory effect on the detection of group A strepto coccus in a sample of sonication meat suggests the possibility to adapt our system to detect staphylococ cal toxins in food, but the analytical characteristics may differ from those obtained for culture superna tants. The use of supramolecular complexes has another advantage in comparison with the direct antibody DNA conjugates, since it requires only biotinylation of the detecting antibodies. Based on methodological and economical parameters, this is the most accessible method for modifying antibodies compared with the methods for the production of direct conjugates. Application of this method allows using enzyme linked immunoassay based on biotinylated antibodies for immunoPCR without laborintensive technolo gies. The natural limitation for the development of test systems based on supramolecular complexes is the lack of simultaneous detection of several antigens using appropriate detecting monoclonal antibodies. The developed highly sensitive detection system can be used in studies for the dynamics of SEA and TSST production in the culture medium under various conditions. Further trials of the developed test systems are planned for experiments for the detection of sta phylococcal toxins directly in clinical samples. These trials will allow the detection of SEA and TSST in swabs from suspected infection sites and human bio logical fluids. The use of the supramolecular com plexes [Bt2oligo(N)Str] allows us to develop a highly sensitive and simpletouse test system based on the immunoPCR method. MATERIALS AND METHODS Preparation of DNAtag. Singlestranded DNA tags biotinylated at single (3') and double (5'and 3') ends of the chain with a size 60 nt were prepared by direct oligonucleotide synthesis. Synthesis was per formed on an automated DNA synthesizer ASM800 (Biosset, Russia). The corresponding solid phase was used for the insertion of biotin on the 3'end of oligo nucleotides: 3'Protected Biotin Serinol CPG (Glen Research, United States). Biotin at the 5'end was inserted via phosphoramidite Protected Biotin Serinol Phosphoramidite (Glen Research, United States). Purification of oligonucleotides was carried out by HPLC and the purity of the product was confirmed by MALDITOF mass spectrometry (mass spectrometer microflex LT (Bruker Daltonik, Germany)). Oligonu cleotide concentration was determined by spectro photometer NanoDrop 2000c (Thermo Scientific, United States).

Preparation of supramolecular complexes [Bt2 oligo(N)Str]n. Supramolecular complexes was pre pared by mixing a 10 µM Bt2oligo(N) solution and a 10 µM streptavidin solution in different ratios and the incubation of these solutions at 4°С for 30 min [16,19]. Agarose gel electrophoresis was used for the analysis of the complexes obtained. The complexes were stored at –70°C in 50% glycerol. Antibodies and toxins. The purified SEA and TSST toxins were provided by Y.V. Vertiev (N.F. Gamalei Scientific Research Institute of Epidemiology and Microbiology, Ministry of Health of the Russian Fed eration, Moscow). Monoclonal antibodies against TSST (TSST5/ TSST14bt) and SEA (SEA14/SEA10bt) were earlier produced by the authors [21]. TSST5 and SEA14 antibodies were used as the first (binding) antibodies. Biotinylated TSST14bt and SEA10bt antibodies were used as the second (detecting) anti bodies. Strains of S. aureus. We used clinical isolates obtained in 2009–2011 from patients with pneumonia (autopsy material, sputum) or bacteraemia (blood) and also reference strains producing TSST, obtained from the laboratory of Professor Bergdoll (United States) and Professor Ch. Lammler (Germany). All preclinical isolates were tested for the presence of the following toxin genes with superantigen activity: sea, seb, sec, seh, seg, tst, lukSPV and lukFPV [8]. For immunoPCR, clinical isolates of S. aureus were cul tured in the liquid medium, Brain heart infusion (Conda, Spain), for 18 h at 37°С without shaking and then centrifuged at 10000 rpm for 15 min. The obtained supernatants were analyzed for the detection of toxin content by immunoPCR. Detection of the number of cells in the overnight cultures was per formed by the inoculation of tenfold dilutions on a solid agar medium followed by counting the number of colonies. The amount of S. aureus bacteria for all cul tures was determined in the range of 1 × 107–1 × 108 cells per 1 mL. ImmunoPCR. The basic protocol is based on the study of Niemeyer et al. [20]. A Greiner polycarbonate PCR plate was used for the immobilization of the binding antibody, 20 µg/mL antibodies in 40 µL PBS (137 mM NaCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4) was added in the well of the plate, incubated at 4°C for 12 h and followed by being washed 3 times with TBS (20 mM TrisHCl, 150 mM NaCl, pH 7.5). Blocking of the remaining adsorption sites was performed with 1% BSA in TBS, using 150 µL/well for 2 h at room temperature. Then it was washed (standard washing) three times with TETBS (0.1 mM EDTA and 0.1% Tween 20 in TBS). Incubation of a 40 µL toxin sample solution in TBS was performed for 1 h at room temperature, followed by standard TETBS washing. A biotinylated detecting antibody in TBS (40 µL with a concentration of 0.5 µg/mL) was added and incubated


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for 1 h followed by a standard washing. A solution of a complex of biotinylated oligonucleotide with strepta vidin in TBS (concentration 5 pmol/L, 40 µL/well) was added and incubated for 45 min at room tempera ture, then washed 9 times with 150 µL TETBS and 3 times with 150 µL TBS. Tubes were then completely dried of buffer residues. Then 40 µL of amplification mixture for “realtime” PCR was added [16] and amplification and analysis of the results were per formed. PCR was performed according to the following program: 94°С 5 min (1 cycle); 94°C 5 seconds, 60°C 15 seconds (40 cycles) using detecting thermocycler DT96 (JSC NPF DNKTekhnologiya, Russia), the detection of signal was performed at 60°С using detec tion channel FAM. PCR results were analyzed using the software supplied with the detection thermocycler, calculating values of the threshold cycles (Cp). Each experiment was performed with at least two replicates. The results were analyzed as described pre viously [16]. For all samples of the total number of amplification cycles, threshold cycle values were sub tracted, and then mean values and standard deviations for the values were calculated. The limit of detection (LOD) was calculated using the formula LOD=[ aver age(40 – Cp) for negative control without antigen + 3* s.d. (40 – Cp) for negative control without antigen]. Normalized values (ΔCn) were calculated using the formula [ΔCn(i) = (average(40 – Cp)i – LOD]. Sam ples with normalized value less than “0” were consid ered negative, and with the value higher or equal “0” were considered as positive. Only normalized values were compared between different experiments. ACKNOWLEDGMENTS

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This work was financially supported by the Minis try of Industry and Trade of the Russian Federation (SC No. 12411.0810200.13.V13).

17. Lind, K. and Kubista, M., J. Immunol. Methods, 2005, vol. 304, pp. 107–116.

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