Combining magnetic nanoparticle with biotinylated nanobodies for rapid and sensitive detection of influenza H3N2
© Zhu et al.; licensee Springer. 2014
Received: 5 May 2014
Accepted: 7 September 2014
Published: 26 September 2014
Our objective is to develop a rapid and sensitive assay based on magnetic beads to detect the concentration of influenza H3N2. The possibility of using variable domain heavy-chain antibodies (nanobody) as diagnostic tools for influenza H3N2 was investigated. A healthy camel was immunized with inactivated influenza H3N2. A nanobody library of 8 × 108 clones was constructed and phage displayed. After three successive biopanning steps, H3N2-specific nanobodies were successfully isolated, expressed in Escherichia coli, and purified. Sequence analysis of the nanobodies revealed that we possessed four classes of nanobodies against H3N2. Two nanobodies were further used to prepare our rapid diagnostic kit. Biotinylated nanobody was effectively immobilized onto the surface of streptavidin magnetic beads. The modified magnetic beads with nanobody capture specifically influenza H3N2 and can still be recognized by nanobodies conjugated to horseradish peroxidase (HRP) conjugates. Under optimized conditions, the present immunoassay exhibited a relatively high sensitive detection with a limit of 50 ng/mL. In conclusion, by combining magnetic beads with specific nanobodies, this assay provides a promising influenza detection assay to develop a potential rapid, sensitive, and low-cost diagnostic tool to screen for influenza infections.
Influenza A and B viruses cause a pandemic threat to human health throughout the world . Sporadic transmission of influenza viruses from birds to humans could lead to unpredictable pandemic outbreaks. Influenza is an infectious disease of the respiratory tract that can infect millions of people and kills hundreds of thousands of them . Humans, infected with influenza A, manifest typically an acute upper respiratory tract illness characterized by fever, cough, and sore throat. Disease severity depends mainly on the virulence of the influenza virus strain and immune competence of the patients . Influenza viruses are members of the Orthomyxoviridae family, and they are further classed as A, B, and C viruses . Until now, 17 influenza A hemagglutinin (HA) subtypes have been described. However, only a limited number of influenza A viruses (IAV), such as H1, H2, H3, H5, H6, H7, and H9, have been implicated with human infection . The high mutational rate of the virus and frequency of interspecies transmission leading to novel virus subtypes will reduce the current vaccine efficacy and make influenza infection highly unpredictable [6, 7].
Due to a broad vaccine deficiency, effective, early, and sensitive detection of all subtypes of influenza A viruses is of significant importance to reduce the mortality of influenza infection . Simple equipment for the fast and low-cost detection of influenza viruses can attract great attention, because such a tool could reveal the threat before spreading the disease . Diagnostic methods to detect the influenza virus have been reported in several studies. Isolation and cultivation of viruses in cell culture has been regarded as the golden standard for virus detection . However, this method is laborious and time-consuming, and it will take several days to identify the virus. Besides cultivation assays, enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies against influenza viral antigens is a good alternative method for the detection of influenza . PCR, another method used for influenza detection, is more specific, more sensitive, and less time-consuming, compared with traditional methods . However, all of the above methods require sophisticated equipment and are not practical in clinical settings. Therefore, the development of a simple, rapid, and sensitive assay to detect influenza remains a challenge.
Antibody-mediated immunoassays are promising tools for the detection of influenza based on their specificity, accuracy, and stability. Camelidae such as camels, llamas, and alpacas have a humoral immune response that has evolved into heavy-chain-only antibodies. Unlike conventional IgGs, the antigen-binding fragment of these heavy-chain antibodies consists of one single domain referred to as VHH or nanobody, with a molecular weight of approximately 15 kD. A nanobody is one of the smallest known antigen-binding antibody fragment. The CDR3 (the third antigen-binding loop) of nanobodies plays a key role in recognizing complicated structures such as pockets and clefts that are usually inaccessible for conventional antibodies . The reduced size, improved solubility, and good stability of the camelid heavy-chain fragments form the basis of a new generation of antibodies for diagnostic applications [14, 15]. In our previous study, we have isolated nanobodies against human prealbumin (PA) and successfully applied them into a sensitive flow injection chemiluminescence immunoassay with a detection limit of 0.01 μg/L . In addition, a nanobody with specificity to small caffeine molecules has been used in an ELISA to measure caffeine concentration in beverages . Thus, nanobodies provide great alternatives to conventional antibodies for diagnosis of diseases.
A healthy young camel was immunized primarily with pure inactivated influenza A grade 2 (H3N2) virus (1 mL, 100 μg) mixed with an equal volume of Freund’s incomplete adjuvant. This virus was purchased from Microbix Biosystems Inc. (Ontario, Canada) and was inactivated by gamma radiation. To stimulate antigen-specific B cells to raise heavy-chain antibodies, antigen was injected once a week. After seven injections, peripheral blood lymphocytes (PBLs) from 100 mL of blood of the immunized dromedary were isolated by density gradient using Ficoll-Paque™ PLUS (GE Healthcare, Beijing, China) and used to construct the library. All camel experiments were performed according to guidelines approved by Southeast University.
Construction of the immune single-domain antibody library
Primers used in this study
The amplified second PCR products were digested with Pst I and Not I restriction enzymes (NEB, Ipswich, MA, USA), then inserted into the phagemid pMECS . Ligation products were transformed into Escherichia coli TG1 cells by electroporation. The transformants were plated onto 2 × YT containing 1% glucose and 100 μg/mL ampicillin and cultured at 37°C for 16 h. Plating an aliquot of the library and counting the colony number determined the library size. Many clones were selected randomly and used in a colony PCR to estimate the percentage of clones with a proper insert size within our library.
Selection of H3N2-specific nanobodies
A representative fraction of the VHH library was cultured and infected with VCSM13 helper phages to express the VHH at the tip of phage particles . Pure inactivated influenza A grade 2 (H3N2) (20 μg) in coating buffer (0.1 M NaHCO3, pH 8.2) was used as antigen to coat onto microtiter plates (Nunc Immuno Maxsorp, Roskilde, Denmark) at 4°C overnight. The control was 0.1 M NaHCO3 (pH 8.2). After blocking with 0.1% casein in phosphate-buffered saline (PBS) for 2 h and incubation with phage-displayed sdAbs in PBS for 1 h at room temperature, the specific phages were eluted with 100 mM triethylamine, transferred to a fresh tube, and immediately neutralized with 1.0 M Tris-HCl (pH 7.4) and used to infect TG1 cells. This process represented one round of panning. Then, part of the TG1 cells was plated at various dilutions, whereas the remaining of the culture was super-infected with helper phages VCSM13. The generated phage particles were used in the next round of panning. During two to four rounds of panning, the H3N2-specific phages were enriched gradually.
Periplasmic extract ELISA
To detect the H3N2-specific clones, 95 clones were selected randomly for periplasmic extract ELISA (PE-ELISA). After disrupting the cells by osmotic shock and a centrifugation step (i.e., the periplasmic extract), the nanobodies resided in the supernatant, which was incubated with antigen coated in microtiter plates. This technique is referred to as periplasmic extract ELISA or PE-ELISA. Each clone was cultured in 1 mL Terrific Broth (1 L TB: 12 g peptone, 24 g yeast extract, 4 mL glycerol, 170 mM KH2PO4, and 0.72 M K2HPO4) with 100 μg/mL ampicillin, then the expression of VHH by 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was induced. Cells were collected and resuspended into 200 μL TES (0.5 M sucrose, 0.2 M Tris-HCl pH 8.0, 0.5 mM EDTA) for 2 h at 4°C, and 300 μL cold TES/4 was added for 2 h. The supernatant was transferred into the wells of the microtiter plates, in which we have coated inactivated influenza A grade 2 (H3N2) (2 μg/mL). After 1 h, we added mouse anti-HA tag antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 1 h and then an anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich, Saint Louis, MO, USA) for 1 h. After washing with PBST (PBS with 0.05% Tween 20) and addition of the chromogenic solution containing bisphosphate (pNPP) (Sigma-Aldrich, Saint Louis, MO, USA), we read the absorbance at 405 nm with an ELISA reader (Bio-Rad iMark™, Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Expression and purification of selected nanobodies
The selected VHH genes in pMECS were transformed into E. coli WK6 electrocompetent cells to express the nanobodies. The cells were grown in TB supplemented with 0.1% glucose, ampicillin (100 μg/mL), and 2 mM MgCl2, until absorbance at 600 nm reached between 0.6 and 0.9. The expression of nanobodies was subsequently induced with 1 mM IPTG for 16 h at 28°C. After pelleting the cells, we extracted the periplasmic proteins by osmotic shock. Soluble sdAbs containing His-tags were purified from the cell lysate by immobilized metal affinity chromatography (IMAC) using a His-Select column (Sigma-Aldrich, Saint Louis, MO, USA). After washing with PBS, we eluted the His-tagged proteins with a gradient of increasing concentration of imidazole (pH 7.0) and subsequent dialysis of the fractions of interest into PBS.
ELISA for nanobody specificity detection
To detect the specificity of nanobodies which we have purified, these nanobodies were tested to combine with several kinds of avian influenza virus by ELISA. Each inactivated influenza virus (5 μg/mL) was coated onto microtiter plates overnight at 4°C in coating buffer. After blocking with 1% bovine serum albumin (BSA) at room temperature for 2 h, 10 μg/mL of nanobodies was added and the plates were incubated at room temperature for 1 h. The following steps were the same as described above for the periplasmic extract ELISA.
We then decided to detect the specificity of these two nanobodies against the H3N2 virus. Hence, 5 μg/mL of recombinant hemagglutinin (HA) protein (Sino Biological Inc., Beijing, China), H3N2 virus, and BSA (control) were coated independently in 96-well microtiter plates, and residual protein binding sites were then blocked with 1% skimmed milk in PBS. After incubation with nanobodies at 10 μg/mL at room temperature for 1 h, anti-His tag mouse monoclonal antibody (Abbkine, Inc., Redlands, CA, USA) was added for 1 h and then anti-mouse IgG-alkaline phosphatase for 1 h. Finally, after adding the substrate, absorbance at 405 nm was read.
Conjugation with biotin in vivo
We subcloned the VHH gene fragment to a pBAD vector after digestion by Nco I and BstE II restriction endonucleases and co-transformed the ligated material into WK6 cells with pBirA plasmid . The expression of VHH-BAD fusion protein was induced with 1 mM IPTG followed by the addition of 50 μM d-biotin (Bio Basic Inc., Beijing, China) and incubated for 30 min. Cells were collected and the periplasmic proteins extracted by osmotic shock. Soluble nanobodies, which were coupled to biotin, were purified by Streptavidin Mutein Matrix (Roche, Basel, Germany). After washing with PBS, the nanobodies conjugated with biotin were eluted by 6 mM d-biotin and dialyzed into PBS.
Coupling with HRP
To provide a detection function to the nanobodies, we coupled HRP (Sigma-Aldrich, Saint Louis, MO, USA) to our nanobodies. We added 100 μL fresh NaIO4 (0.1 M) into 200 μL HRP (5 mg/mL) for 30 min at 4°C, and 200 μL ethylene glycol (2.5%) was added for 30 min at room temperature. One milliliter of H3N2-specific nanobodies (1 mg/L) was added to incubate overnight at 4°C. Twenty microliters of sodium borohydride (5 mg/mL) was mixed into it for 3 h at 4°C the next day. HRP-conjugated nanobodies were precipitated by saturated ammonium sulfate (SAS). The same volume of SAS was added for 1 h at 4°C and then centrifuged at 10,000g for 30 min. Sediments were resuspended in PBS, and the free nanobodies (i.e., non-conjugated to HRP) were removed by ultrafiltration. Then, HRP-labeled nanobodies (Nbs) were dialyzed into PBS. In order to check the efficiency of the enzyme-labeled nanobody, we coated the H3N2 antigen (5 μg/mL, 100 μL) and used the nanobodies coupling with HRP (10 μg/mL, 100 mL) as detector in our ELISA experiment. After adding 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich, Saint Louis, MO, USA) to react for 10 min and stopping the reaction with 2 M H2SO4, the absorbance was measured at 450 nm.
Double nanobody sandwich method for antigen detection
We used 50 μL Dynabeads M-280 streptavidin (Invitrogen, Carlsbad, CA, USA) for each reaction to capture the Nb3 conjugated with biotin (2 μg/mL) for 30 min. It was washed with PBST for 10 times and 0.1% BSA for 2 times. After blocking with 0.1% BSA for 2 h, serial dilutions of influenza A grade 2 antigen (500 μL) were added for 1 h. The Nb1 coupled with HRP (1 μg/mL) used as a detector was incubated for 45 min followed by washing for 15 times. All of these procedures were performed at room temperature. Finally, we removed the free Nb1-HRP by washing for 25 times using PBST. Then, the visualization was carried out by adding 3,3′,5,5′-tetramethylbenzidine as substrate. After stopping the reaction using 2 M H2SO4, the readings were measured at 450 nm.
Temperature sensitivity test
We detected the residual binding capacity of the nanobodies after being incubated at 37°C for different amounts of time. Antigen (10 μg/mL) was coated at 4°C overnight. Each sample has its own blank control, which was coated with NaHCO3 (0.1 M, pH 8.2). After blocking with 1% skimmed milk for 2 h, 20 μg/mL Nbs were added to incubate for 1 h. The following steps were the same as described for the periplasmic extract ELISA and ELISA for evaluating the nanobody specificity. The signal with nanobodies that were not incubated at 37°C was considered as 100%.
Results and discussion
Construction of immunized phage display nanobody library
For library construction, two restriction enzymes, Pst I and Not I, were introduced into the 5′ and 3′ ends of the final VHH PCR fragments, respectively. In total, 4.8 μg of purified VHH PCR product and 16 μg of linearized pMECS vector were used for the following ligation. A total of 30 electroporations were performed to transform the ligation mixture into bacterial cells and to obtain a high-quality library with great diversity. The size of the constructed library was calculated from the number of independent colonies on plates and shown to reach 8 × 108 colonies (Figure 2C). This size of the library is about 400 times larger than the one reported previously . It should be a good starting point to retrieve antigen-specific nanobodies. Colony PCR analysis on 24 randomly picked colonies revealed that the percentage of colonies with plasmids having an insert of a proper size for a VHH reached 95% (Figure 2D). These clones have unique sequences after sequencing their VHH fragment. All together, these results demonstrated that we have successfully constructed a phage display nanobody library against H3N2.
Bio-panning of phage display library against H3N2
Expression of soluble nanobodies and specificity test
Expression and purification of biotinylated nanobodies
Magnetic particle-based immunoassay is a promising tool for clinical diagnosis . In this method, antibody will be conjugated onto magnetic beads and used as a carrier to capture the analytes from the solution. The magnetic beads are easy to manipulate by using an external magnetic field for only a very short period of time. However, the chemical conjugation of antibodies to magnetic particles is usually not directional. Therefore, the covalent conjugation will lead to a decreased binding efficacy of the antibodies. In order to overcome this problem, we decided to label our nanobodies with biotin based on an in vivo labeling. Streptavidin-coated magnetic beads will incubate with biotin-labeled nanobodies to efficiently capture the antigens in a directional orientation. In this case, the nanobody conjugated on magnetic particles will be fully functional.
Magnetic nanoparticle-based sandwich immunoassay for rapid detection of influenza H3N2
We developed a rapid and sensitive immunoassay for influenza H3N2 detection based on single-domain antigen-binding fragments of camelid-like heavy-chain antibodies. In this study, an immune phage-displayed nanobody library is constructed, from which four classes of nanobodies with good affinity and specificity against H3N2 are successfully isolated and expressed. In addition, Nb3 coupled to magnetic beads and HRP-conjugated Nb1 have been used for rapid and sensitive detection of influenza H3N2. This assay holds a great promise in providing a potentially universal method for detecting all subtypes of influenza viruses.
This work was supported by grants from Jiangsu Nanobody Engineering and Research Center of China (2014-02), Program for New Century Excellent Talents in University (NCET-20130127), National Natural Science Foundation of China (grant numbers 31271365 and 31471216), and Natural Science Foundation of Jiangsu Province (grant number BK2011599). This work was also supported by Key topics for State Key Laboratory of Materials-Oriented Chemical Engineering (ZK20134), and National Natural Science Foundation (grant number 31471692). We are deeply indebted to Prof. Serge Muyldermans and Ph.D Ema Romão of Vrije Universiteit Brussel, Belgium for their help.
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