ISSN 00036838, Applied Biochemistry and Microbiology, 2014, Vol. 50, No. 6, pp. 683–688. © Pleiades Publishing, Inc., 2014. Original Russian Text © P.V. Khramtsov, M.S. Bochkova, M.B. Rayev, 2014, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2014, Vol. 50, No. 6, pp. 612–618.
Application of Diagnosticum Based on Functionalized Carbon Nanoparticles for Monitoring of Immunoglobulins Affinity Purification P. V. Khramtsov, M. S. Bochkova, and M. B. Rayev Institute of Ecology and Genetics of Microorganisms, Russian Academy of Sciences, Perm, 614081 Russia email:
[email protected] Received November 16, 2013
Abstract—A diagnostic reagent based on carbon nanoparticles covalently functionalized with streptococcus G protein was applied to construct a system for monitoring and optimization of the technology of affinity purification of rabbit polyclonal antibodies to alphafetoprotein. The developed system allows a shorttime (45 min) semiquantitative assessment of the immunoglobulins (IgG) of most higher animals and human beings in blood serum samples and eluate. IgG detection sensitivity using the carbonGprotein diagnosti cum was 80 ng/mL. Approaches to stabilizing the components of the analysis system, which ensures the pres ervation of their functional properties during long storage, were developed. The storage life of the diagnosti cum was more than 20 years, and that of immunosorbents was more than year and a half. A technique of long term immunosorbent storage was developed. Application of the developed testsystem do not require regis tration equipment. DOI: 10.1134/S0003683814060064
INTRODUCTION At present, the need is growing in laboratory, clini cal, and biotechnological practices for simplified test ing methods that make it possible to gain accurate results of the analysis in a very short time. The princi ples and methods used in these tests are different: agglutination of colored particles, immunochro matography, immunofiltration, and dotblot. In most cases, these tests are noninstrumental and can be used even when there is no registration equipment or highly qualified specialists. The Laboratory of Ecological Immunology, UB RAS, developed a number of such test systems. The diagnostic reagent is based on carbon nanoparticles covalently conjugated with affine compounds. Carbon particles perform the function of a colored label, and affine compounds provide specificity of conjugate interaction with the analyte. Carbon diagnosticums were used to create qualitative and semiquantitative rapid analysis systems of antibodies to the heatstable toxin of Yersinia pseudotuberculosis, tetanus toxoid, αfetoprotein (AFP) and human chorionic gonadot ropin (HCG) [1–3]. The Gprotein of Streptococcus, streptavidin, and monoclonal antibodies to gestation hormones were used as the affinity component of the diagnosticums. The sensitivity of these systems lies in a range from 1.5 to 160 ng/mL [2, 4]. The detection sensitivity of IgG with carbonGprotein diagnosti cum was 80 ng/mL [5].
The potential applications of carbon conjugates are not limited by clinical diagnosis. Affinity chromatography is an effective, widely used tool for obtaining immunoglobulin preparations with a high purification degree, which are used for var ious purposes: therapeutic, diagnostic, and research. For affinity chromatography, sorbents and protocols, both commercial and those developed independently with account of special requirements and features, are used. The sources of antibodies are also diverse—animal hyperimmune serum, ascite fluid, and culture medium. The individual features of organisms—pro ducers of antibodies—define the need for adjusting the parameters of the chromatographic system: the saturation rate of the sorbent with antiligands, the sample volume applied, the volumes of elution buffers, etc. When using commercial kits and standard tech niques, it is possible to calculate mathematically parameters based on known data (capacity of the sor bent, examples of elution profile) from suppliers and protocols with sufficient accuracy. When using commercial sorbents for solutions of nonstandard tasks or during independent design of a chromatographic system (for example, for the isola tion of antibodies to rare or synthetic antigens), it is difficult to accurately predict the properties of the affinity sorbent and its behavior in the experiment. A more attractive solution is selfassessment of the dynamics of a chromatographic process using detec tion systems of immunoglobulins in the eluate.
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These systems include analytical methods to quan titatively or semiquantitatively evaluate the content of immunoglobulins of the required specificity in the ini tial material and in eluate samples. This information makes it possible to adjust the chromatography proto col (change of the elution rate, the original sample vol ume, the volume of the sorbent, etc.) and facilitates planning subsequent experiments. Methods for the detection of antibodies in chro matographic samples are numerous, and instrumental approaches based on the use of sensing devices are widespread. These include immunoenzyme assay (IEA) [6–9], nephelometry [10], and biosensors [9]. In most cases, IEA is used because it is a routine labo ratory analysis method, and equipment for the method is the most available. A noninstrumental method based on the immunodiffusion reaction in agar gel is a traditional method [6, 11–14]. The designed system was tested during the isolation of rabbit polyclonal antibodies to human AFP. The aim of this study was to develop noninstru mental solidphase dotimmunoassay system for the control of chromatographic immunoglobulin separa tion using carbon nanoparticles functionalized with streptococcus Gprotein. MATERIALS AND METHODS Materials and Equipment. We used a 1.5 × 12 cm chromatographic column (BioRad, United States), a peristaltic pump P1, flow UV spectrometer 2138 Uvi cord S (Pharmacia LKB, Sweden), a Soniprep 150 Plus ultrasonic disintegrator (MSE, Great Britain), a Zetasizer NanoZS analyzer for measuring particle size (Malvern, England), a NOVAsem 600 scanning elec tron microscope (FEI, United States), an ultrafiltra tion cell Amicon Stirred Cell 8050 (Millipore, Ger many), and a 5804 R centrifuge (Eppendorf, Ger many). Reagents and Buffers. The reagents were cyanogen bromideactivated Sepharose 4B, Sepharose CL6B (Pharmacia Fine Chemicals, Sweden), AFP (Bialexa, Russia), white polystyrene plates for serial dilutions Linbro (United States), agar Difco (United States), nitrocellulose membrane with a pore size of 0.45 μm (BioRad, United States), bovine serum albumin (BSA), human IgG, streptococcus Gprotein (Sigma, United States), glutaraldehyde (AppliChem, Ger many), toluene (Ekros, Russia), glycerol and tween20 (Panreac, Spain). The solutions for immunoassay and synthesis of the diagnosticum were 0.02 M carbonatebicarbonate buffer, pH 9.6 (CBB); 0.15 M NaCl buffered with 0.015 M Naphosphates, pH 7.25 (NaClBP); and NaClBP containing 0.05% tween20 (NaClBPT). The solutions for chromatography were 0.15 M NaCl; 0.1 M glycineHCl pH 2.6; 0.5 and 0.15 M NaCl buffered with 0.03 M Naphosphates, pH 7.25 (0.15 M
and 0.5 M PBS). All solutions were prepared using deionized water. Synthesis of Diagnosticum. Carbonblack was used as a carbon source, which was obtained through con densation of a burning flame of toluene. It was sub jected to multistage washing in organic solvents and filtration to remove the underoxidized products. The obtained amorphous carbon was a mat black powder, insoluble in available solvents. Scanning electron microscopy established that carbon particles were spherical or close to spherical in shape, and the linear dimensions were in the range of 40–70 nm. At the next step of synthesis, 1.0 g of amorphous carbon was suspended in 19 mL of 2% BSA in NaClBP for 1 day under vigorous stirring on a magnetic stirrer. The carbon concentration as calculated to dry sub stance was 5%. The resulting suspension was subjected to an ultrasonic disintegration at 22 kHz. Total sonica tion time was 1 h. In order to remove any remaining large particles the sonicated suspension was centri fuged for 5 min at 1620 g [15]. The supernatant was incubated for 40 min at room temperature with an equal volume of 25% glutaralde hyde solution. The mixture was then centrifuged for 5 min at 1620 g and chromatography was carried out using a Sepharose CL6B containing column to remove unbound BSA and glutaraldehyde. The frac tions from the void volume fraction were combined and concentrated to the initial volume of the carbon suspension in an ultrafiltration cell [15]. The glutaraldehydeactivated suspension, with a volume of 4.5 mL, was incubated with 0.5 mL of Gprotein with a concentration of 10 mg/mL for 100 min with gentle stirring. The resulting suspension was centrifuged for 5 min at 1620 g. The unbound Gprotein was removed by gelchromatography on a Sepharose CL6B column. The void volume fractions containing the conjugate were combined, and BSA and glycerol were added to a final concentration of 1 and 20%, respectively [15]. The particle size of the conjugate was determined by measuring the reverse dynamic light scattering at an angle of 173°C with the Malvern Zetasizer NanoZS analyzer (Great Britain). For measurement the conju gate was diluted in water to a concentration of carbon particles of 0.01%. The diameter of 90% of the parti cles was in the range of 70–200 nm, and particles with a diameter of 90–100 nm dominated. The synthesized conjugates were stored at 2–8°C. This temperature regime ensured the stability of the functional properties of the diagnosticum for more than 20 years [16]. Preparation of Hyperimmune Rabbit Serum and Determination of Titers of AFP Antibodies. The immu nization of rabbits was carried out according to a stan dard method [17] using the Freund’s Complete Adju vant (CFA) and aluminum hydroxide.
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The process was carried out according to the fol lowing scheme: 1st day—first immunization intramuscularly with 200 μg of AFP with three injections of CFA; 29th day—repeated immunization intramuscu larly with 200 μg of AFP with three injections with alu minum hydroxide; 53rd day—the last blood sampling. Before application to the column, all hyperim mune rabbit blood sera were combined. The titer of antibodies in the combined sera was determined by immunodiffusion in agar gel according to the Ouchterlony method [18]. The results were evaluated by the presence of precipitation lines 72 h after initia tion of the analysis. Chromatographic Separation of Polyclonal Antibod ies to AFP. To isolate rabbit polyclonal antibodies to AFP, an affinity sorbent based on Sepharose 4B was synthesized by the method of the manufacturer using a commercial preparation of AFP. The protein in the elu ate was recorded using a flow UV spectrophotometer. The sorbent in the column, having a volume of 11 mL, was washed with 100 mL of 0.5 M PBS. Twenty mL of combined whole rabbit hyperimmune serum was applied at a rate of 1 mL/min with three fold recirculation. Upon completion of each of the 3 cycles of application, a sample with a volume of 300 μL was collected from the eluate for the analysis of the target antibody content. The sorbent was washed consistently with 0.15 M PBS and 0.15 M NaCl, and the antibodies were eluted in 0.1 M glycineHCl buffer with a pH of 2.6. Semiquantitative Determination of Antibodies to AFP in Eluate by SolidPhase DotImmunoassay. Five μL dots of AFP at a concentration of 0.1 mg/mL in CBB was placed in the bottom of polystyrene plate wells. Human IgG and BSA were adsorbed as a posi tive and negative control at concentrations of 0.05 and 0.1 mg/mL, respectively. After 30 min of incubation in a wet chamber at 37°C, the plates were washed by fill ing the wells three times with NaClBPT and immedi ately removing it. Sensitized polystyrene plates are an immunosorbent suitable for further detection of anti bodies in the eluate. Sensitized plates can be stored for long time at room temperature pretreated with 30% aqueous sucrose solution. Under these conditions they retain their propertis for over one and a half years [19]. Three hundred μL of twofold serial dilutions of eluate samples in NaClBPT, ranging from 1 : 2 dilution and ending with 1 : 524 000 dilution, were added in the sensitized plate wells. After incubation for 30 min, the wells were washed three times with NaClBPT, and 150 μL of carbonGprotein conjugate diluted to working titer was added. After 15 min of incubation with gentle stirring, the plates were washed and dried. The optimum period of incubation of the sample and conjugate was selected beforehand. The analysis APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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Fig. 1 Semiquantitative determination of antibody titer to AFP in hyperimmune rabbit serum by Ouchterlony immu nodiffusion method. AFP at a concentration of 125 µg/mL was applied into the central well. The total rabbit hyperim mune serum in a specified dilution was introduced in wells at edges; K is the well with the serum of an intact rabbit (neg ative control).
results were visually evaluated according to the last vis ible dot in serial dilutions. Combined hyperimmune rabbit serum was simi larly analyzed prior to its application to the immun osorbent. RESULTS AND DISCUSSION Analysis of Antibodies to AFP in Combined Hyper immune Rabbit Blood Serum. Two methods were used to determine the titer of polyclonal AFP antibodies in combined rabbit hyperimmune sera. The first method was the dotimmunoassay developed for testing serum on AFP preparationsensitized polystyrene plate using carbon diagnosticum. The second method was agar gel immunodiffusion by Ouchterlony, a traditional nonin strumental method of semiquantitative serological analysis. In the experiment, the sensitivity and speci ficity of the methods were compared. In the Ouchterlony immunodiffusion reaction, the titer of antibodies to AFP in the studied serum was 1/8. The absence of precipitation lines opposite the well with whole serum of intact rabbit was indicative of the absence of a nonspecific interaction of its components with AFP (Fig. 1). In the dotimmunoassay testing of blood serum using carbon diagnosticum on a polystyrene support, the titer of antibodies to AFP in the sample was 1/8000. The correctness of the system and its specificity were confirmed by analysis of control samples (Fig. 2). Comparing the obtained results, it can be con cluded that both systems demonstrated equally high specificity, but the sensitivity of the analysis system constructed on the basis of functionalized carbon par ticles was 3 orders of magnitude higher as compared to the immunodiffusion reaction. Control of Chromatographic Purification of Poly clonal Rabbit Antibodies to AFP. Polyclonal antibodies
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Fig. 2. Semiquantitative determination of antibody titer to AFP in hyperimmune rabbit serum by solidphase dotimmunoassay using (a) carbon diagnosticum with Gprotein and (b) scheme of applying antiligands to plate wells. (a) dilutions of the sample are indicated near the wells; K is the well in which the whole serum of an intact rabbit (negative external control) was applied, and the arrow marks the last visible dot in a series of dilutions. (b) IgG is the zone of the well in which human IgG adsorbed at a con centration of 0.05 mg/mL (internal positive control); AFP is the zone in the well in which AFP adsorbed at a concentration of 0.1 mg/mL; BSA is the zone in the well in which BSA adsorbed at a concentration of 0.1 mg/mL.
to AFP were isolated from rabbit hyperimmune serum using affinity chromatography on Sepharose 4B con jugated with AFP. Control samples of the eluate, col lected after each application cycle, were subjected to testing in the constructed dotimmunoassay system for determination of the dynamics of the target anti body titer changes. The work was broken down into a series of experi ments on the chromatographic separation of AFP antibodies from a certain fraction of combined sera. In the first series of separation, a serum sample with a volume of 10 mL was applied to the column. The analysis of control samples of the eluate after three recyclings detected no antibodies of the required spec ificity in any of them. Thus, as early as during the first cycle of applying the preparation onto the column, all
of the containing target antibodies bound with the sor bent. On this basis it was concluded that, with an ini tial sample volume of hyperimmune rabbit serum equal to 10 mL, there is no need for it to be reapplied to the column. Based on these results, the volume of the prepara tion applied onto the column was increased twice, i.e., to 20 mL. The analysis of control samples demon strated a steady decline in antibody titer in the eluate with each subsequent cycle of applying the sample onto the column (1/16 after first cycle, 1/8 after the second, and 1/2 after the third). However, after the first cycle, the titer of antibodies in the eluate was still two orders lower as compared to the initial sample (titer 1/8000) (Fig. 3). That is, even at a twofold increase in the volume of the applied preparation, the
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(c) Fig. 3. Semiquantitative determination of antibody titer to AFP in eluate samples using the constructed solidphase dotimmu noassay system based on carbon diagnosticum after (a) first, (b) second, and (c) third cycles of applying the preparation onto the column. Arrows indicate the last visible dot in serial dilutions of the eluate sample; dilutions of the sample are indicated close to the wells. K is the well wherein the whole serum of an intact rabbit was applied (external negative control). Scheme of antiligand application onto the wells is as in Fig. 2b.
capacity of the affinity sorbent (upon maintaining other parameters of application unchanged) was found to be sufficient for adsorption of the vast majority of target antibodies contained in the initial sample. Fur ther adsorption of the sample during subsequent cycles proceeded much slower. Hence, the constructed system is a convenient tool for the optimization and monitoring of the chromato graphic purification of antibodies. In this case, its use could make it possible to dispense with additional cycles of application of the preparation as inessential to obtaining the desired product and to draw conclu sions about the possibility of increasing the initial sam ple volume. Carbon diagnosticum based on Gprotein can sim ilarly be used successfully to detect antibodies of any APPLIED BIOCHEMISTRY AND MICROBIOLOGY
specificity isolated from the majority of higher animals used in technological processes (goats, rabbits, mice, guinea pigs, etc.) and humans. Another positive fea ture of the developed control system is the ability of the carbon conjugate to retain functional properties when stored for over 20 years. The constructed control system based on carbon diagnosticums is enough efficient and can be used under certain conditions to monitor chromatographic process in real time (for example, at large volumes of sorbent, low elution rates, etc.).The short analysis time is an additional advantage in retrospective analy sis, especially when a large number of series of chro matographic purification of immunoglobulins are per formed in a short time.
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ACKNOWLEDGMENTS This study was supported by a grant for young sci entists and graduate students of the Ural Branch, Rus sian Academy of Sciences (No.134NP571). REFERENCES 1. Rayev, M., Ambrosov, I., and Briko, N., in Streptococci and the Host. Advances in Experimental Medicine and Biology, Thea Horaud at al., Eds., New York: Plenum Press, 1997, vol. 418, pp. 327–329. 2. Rayev, M.B., Nanobiotekhnologii v neinstrumental’noi immunoanalitike (Nanobiotechnology in NonInstru mental Immunoanalytics), Demakov, V.A., Ed., Yekat erinburg: UrO RAN, 2012. 3. Rayev, M.B., Timchenko, N.F., Bochkova, M.S., Nedashkovskaya, E.P., and Andryukov, B.G., Dokl. Biochem. Biophys., 2013, vol. 451, pp. 221–224. 4. Rayev, M.B., Bochkova, M.S., Timganova, V.P., Khramtsov, P.V., and Tyulenev, A.V., Vestn. Ural. Med. Akad. Nauki, 2011, vol. 38, no. 4/1, pp. 146–147. 5. Bochkova, M.S., Timganova, V.P., and Rayev, M.B., Dokl. Biochem. Biophys., 2013, vol. 449, pp. 63–65. 6. Staak, C., Salchow, F., Clausen, P.H., and Luge, E., J. Immunol. Meth., 1996, vol. 194, no. 2, pp. 141–146. 7. Proll, G., Kumpf, M., Mehlmann, M., Tschmelak, J., Griffith, H., Abuknesha, R., and Gauglitz, G., J. Immunol. Meth., 2004, vol. 292, nos. 1–2, pp. 35–42.
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Translated by M. Novikova
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