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Method Article
A highly-sensitive graphene-based immunoassay procedure for the detection of human lipocalin-2
Sandeep Kumar Vashist
Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
We report a highly-sensitive graphene nanoplatelets (GNPs)-based sandwich enzyme-linked immunosorbent immunoassay (ELISA) procedure for the detection of human lipocalin-2 (LCN2). It involves the functionalization of 96-well microtiter plate (MTP) with GNPs (dispersed in 3-aminopropyltriethoxysilane (APTES)). APTES generates the free amino groups on MTP and GNPs, which are used for the leach-proof covalent binding of anti-LCN2 capture antibody by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) based heterobifunctional crosslinking. The developed immunoassay detects LCN2 in the range of 0.6-5120 pg mL-1 with limit of detection and analytical sensitivity of 0.7 and 1 pg mL-1, respectively. It is the most sensitive immunoassay for LCN2, which has 80-fold higher analytical sensitivity and 3-fold reduced immunoassay duration than the commercial sandwich ELISA kit. The anti-LCN2 antibody-bound MTPs, stored at 4°C in 0.1M phosphate buffered saline (PBS), were highly stable as they did not show any decrease in functional activity for 8 weeks.
Keywords
Sandwich ELISA, human lipocalin-2, graphene nanoplatelets, EDC, APTES
Figure 1
Figure 2
The highly sensitive detection of LCN21, also known as neutrophil gelatinase-associated lipocalin (NGAL), is desired in clinical diagnosis as it is an important prognostic and diagnostic biomarker for several medical conditions. It is a small 25 kDa glycoprotein, which is found in neutrophils and certain epithelia such as renal tubules. The LCN2 level in healthy humans is 0.7 ng mL-1, which increases in cases of ischemic2 or nephrotoxic injury3, cardiovascular surgery, kidney transplants4-6, coronary heart disease7-8, fatty liver disease, obesity9, bacterial infections10-11 and cancer12-15. The down-regulation of LCN2 occurs in glioblastoma16, ovarian cancer14, microglial cells17 and in response to excessive exposure to high electromagnetic fields18.
The developed GNPs-based sandwich ELISA procedure19 has higher sensitivity and reduced immunoassay duration in comparison to commercial sandwich ELISA kits and our previously developed immunoassay procedures20-24. It involves the APTES functionalization of GNPs and MTP, and EDC-based heterobifunctional crosslinking of anti-LCN2 capture antibodies (Fig. 1). The high sensitivity of the developed procedure is mainly due to the increased surface area provided by GNPs, which leads to higher immobilization density of anti-LCN2 capture antibodies. The developed immunoassay detects the entire pathophysiological range of LCN2 in the real sample matrices i.e. diluted human serum, plasma and whole blood. It has a very high precision as it correlates well with the conventional ELISA. Therefore, it is ideal for mass-use clinical and industrial settings as the anti-LCN2 antibody-bound MTPs, stored at 4°C in 0.1 M phosphate buffered saline (PBS), did not lose their functional activity even after 8 weeks. Moreover, it is a generic sandwich ELISA procedure that can also be employed in in vitro diagnostics (IVD) for the detection of other disease biomarkers and analytes.
• Human Lipocalin-2/NGAL DuoSet ELISA kit (R & D Systems, cat. no. DY1184e). All the assay components in the kit were reconstituted in phosphate buffered saline (PBS, 0.1 M, pH 7.2) with 1% (v/v) bovine serum albumin (BSA), while PBST (PBS with 0.05% Tween 20) was used for washing steps. The buffers were made with ultrapure water (UPW). The kit comprises of
• Rat anti-human LCN2 capture antibody (360 μg mL-1) !CAUTION Store reconstituted antibody at 2-8°C for up to 60 days or at -20°C to -70°C for up to 6 months. Dilute to a working concentration of 2 μg mL-1 in 0.1 M PBS, pH 7.2.
• Recombinant human LCN2 (90 ng mL-1). !CAUTION Store reconstituted LCN2 at 2-8°C for up to 60 days or at -70°C for up to 6 months. Dilute to the dynamic concentration range of the assay in 0.1 M PBS, pH 7.2) with 1% BSA. Make the dilutions of human LCN2 in BSA-preblocked sample vials to minimize the analyte loss due to non-specific binding on sample tube surfaces25.
• Biotinylated goat anti-human LCN2 detection antibody (18 μg mL-1). !CAUTION Store reconstituted antibody at 2-8°C for up to 60 days or at -20°C to -70°C for up to 6 months. Dilute to a working concentration of 100 ng mL-1 in 0.1 M PBS, pH 7.2) with 1% BSA.
• Streptavidin conjugated to HRP. !CAUTION Store at 2-8°C in dark for up to 6 months after initial use as streptavidin is light-sensitive.
• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, cat. no. 37525) !CRITICAL To avoid microbial contamination in BSA, filter prior to use.
• Sulphuric acid (Aldrich, cat. no. 339741) !CAUTION Avoid skin contact as it is a strong corrosive agent and an irritant. Use personal protective equipment (PPE), i.e. chemical-resistant shoes, lab coats and chemical safety glasses, for handling. Handle only in a fume cabinet. In case of skin contact, wash immediately with acid neutralizers and seek medical advice as soon as possible.
• KOH pellets (99.99%), semiconductor grade (Sigma Aldrich, cat. no. 306568) !CAUTION Use PPE for handling and handle only in a fume cabinet as it can cause severe burns. Avoid contact with skin and eyes. CRITICAL The KOH concentration must be 1% (w/v) in autoclaved UPW as higher concentrations lead to surface defects and strongly impacts the antibody immobilization.
• APTES (Sigma Aldrich, Ireland, cat. no. A3684) !CAUTION Higher concentrations are highly toxic. Avoid contact with skin and eyes as it is a skin and eye irritant. Handle inside a fume cabinet. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• GNPs (diameter 5 µm) (Cheap Tubes, Grade 2) !CAUTION Avoid inhalation. CRITICAL Mix 1 mg of GNPs with 1 mL of 0.25% APTES and disperse by keeping in an ultrasonic bath for 1 h before use.
• 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (Thermo Scientific, cat. no. 22981) !CAUTION Equilibrate to room temperature (RT) as it is hygroscopic and may lose activity upon water absorption. Handle inside a fume cabinet as it is an irritant. CRITICAL Store at -20°C. Reconstitute in 0.1 M MES, pH 4.7, see REAGENT SETUP.
• BupH Phosphate Buffered Saline Packs (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) (Thermo Scientific, cat. no. 18372) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• BupH MES Buffered Saline Packs (0.1 M MES [2-(N- morpholino)ethane sulfonic acid], 0.9 % (w/v) sodium chloride, pH 4.7) (Thermo Scientific, cat. no. 28390) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• TMB substrate kit (Thermo Scientific, cat. no. 34021). It comprises of
• TMB solution (0.4 g L-1) !CAUTION Skin, eye and lung irritant. In case of skin contact, wash with plenty of water. CRITICAL STEP Maintain the TMB:peroxide ratio as 1:1 as it is critical for the development of color.
• Peroxide solution (containing 0.02 % (v/v) H2O2 in citric acid buffer) (Thermo Scientific). !CAUTION It is a strong oxidizing agent, which is harmful if swallowed and carries a severe risk of damage to eyes. In case of contact, rinse immediately with plenty of water and seek medical attention as soon as possible. Wear PPE and work inside the safety cabinet or fume cabinet.
• Nunc microwell 96-well polystyrene plates, flat bottom (non-treated), sterile (Sigma Aldrich, cat. no. P7491)
• Eppendorf microtubes (1.5 mL; Sigma Aldrich, cat. no. Z 606340)
• SigmaPlot software bundle version 11.2 from Systat for curve plotting and assay analysis
• -80°C freezer (operating range -60 to -80°C) (Thermo Scientific, UK)
• 2-8°C Refrigerator (Future, UK)
• Direct-Q3 Water Purification System (Millipore, USA)
• Tecan Infinite M200 Pro microplate reader (Tecan, Austria GmBH)
• Mini Incubator (Labnet Inc., UK)
• Ultrasonic cleaner (Branson Ultrasonics, USA)
REAGENT SETUP
PBS Add a BupH Phosphate Buffered Saline Pack to 100 mL of autoclaved UPW, dissolve well and make the volume up to 500 mL using autoclaved UPW. Each pack makes 500 mL of PBS at pH 7.2, which can be stored at RT for a week and at 4ºC for up to four weeks.
MES Add a BupH MES Buffered Saline Pack to 100 mL of autoclaved UPW, dissolve well and make the volume up to 500 mL using autoclaved UPW. Each pack makes 500 mL of MES at pH 4.7, which can be stored at RT for two weeks.
APTES The procured APTES solution has a purity of 99%. Reconstitute in autoclaved UPW to make 2% (v/v) APTES just before functionalization.
GNPs Mix 1 mg of GNPs with 1 mL of 0.25% APTES and disperse by keeping in an ultrasonic bath for 1 h before use.
EDC The commercial pack contains 25 g EDC. Reconstitute in 0.1 M MES buffer, pH 4.7 to make 8 mg mL-1. Aliquots can be stored effectively for six months at -20°C.
H2SO4 The concentrated solution is 37 N. Add 5.4 mL of 37 N H2SO4 to 94.6 mL UPW to make a 2 N solution. Reaction of acid and water is exothermic, therefore, acid should always be added to water. It can be stored indefinitely.
Surface activation and GNPs-functionalization (TIMING 1 h 5 min)
- Put 100 μL of 1% (w/v) KOH into the wells of 96-well MTP followed by incubation for 5 min at 37°C and subsequent washing with 300 μL of UPW five times. Washing can also be performed with an automatic plate washer. CRITICAL STEP KOH treatment is optimal at 5 min but it should not be longer than 10 min, which can cause surface aberrations that impact the bioanalytical performance of developed immunoassay. ?TROUBLESHOOTING
Incubate the KOH-treated MTP with 100 μL of GNPs (1 mg mL-1) in 0.25% (v/v) APTES for 1 h at RT inside the fume cabinet. Wash the GNPs-functionalized wells extensively with UPW (as described in step 1). CRITICAL STEP One mg of GNPs should be mixed with 1 mL of 0.25% APTES and dispersed by keeping in an ultrasonic bath for 1 h before use. ?TROUBLESHOOTING
Antibody immobilization and BSA blocking (TIMING 1 h 30 min)
- Incubate 990 μL of anti-LCN2 capture antibody (2 µg mL-1) with 10 μL of EDC (4 mg mL-1) for 15 min at 37°C. CRITICAL STEP The optimized concentration of EDC is critical for the crosslinking of antibodies.?TROUBLESHOOTING
- Put 100 µL of EDC-activated anti-LCN2 antibody to the GNPs-functionalized wells followed by incubation for 1 h at 37°C and subsequent washing with 400 µL of PBST five times. ?TROUBLESHOOTING
Block the antibody-bound wells with 400 µL of 1% (w/v) BSA for 30 min at 37°C followed by extensive PBST washing (as mentioned in step 4). CRITICAL STEP Use diagnostic grade BSA that does not interfere with the immunoassay components. Also filter the prepared BSA in order to remove any contaminanting substances. ?TROUBLESHOOTING
LCN2 sandwich ELISA (TIMING 2 h 40 min)
- Add 100 µL of varying LCN2 concentrations (0.6-5120 pg mL-1) to different BSA-blocked wells. Incubate for 1 h at 37°C and wash extensively with PBST (as stated in step 4).
- Add 100 µL of biotinylated anti-LCN2 antibody in each of the LCN2-captured wells. Incubate for 1 h at 37°C and wash extensively with PBS (as mentioned in step 4). ?TROUBLESHOOTING
- Add 100 µL of SA-HRP conjugate to the biotinylated anti-LCN2 antibody treated wells. Incubate for 20 min at 37°C and wash extensively with PBST (as mentioned in step 4). ?TROUBLESHOOTING
- Add 100 µL of TMB-H2O2 mixture to the wells of MTP and incubate at RT for 20 min to develop color.
- Stop the enzyme-substrate reaction by adding 50 µL of 2 N H2SO4 to each well.
- Measure the absorbance at a primary wavelength of 450 nm and secondary wavelength of 540 nm in the Tecan MTP reader. CRITICAL STEP Determine the absorbance after 5 min of stopping the enzyme substrate reaction so that the reaction is effectively stopped. But the reading should be taken before 10 min.
Steps 1-2, Surface activation and GNPs-functionalization: 1 h 5 min
Steps 3-5, Antibody immobilization and BSA blocking: 1 h 30 min
Steps 6-11, LCN2 sandwich ELISA: 2 h 40 min
Troubleshooting advice is provided in Table 1.
The developed GNPs-based sandwich ELISA19 detected 0.6-5120 pg mL-1 of LCN2 (Fig. 2A) with linearity in the range of 80-2560 pg mL-1. The limit of detection (LOD) and analytical sensitivity of LCN2 immunoassay were 0.7 pg mL-1 and 1 pg mL-1, respectively. Various experimental controls were employed that demonstrated the absence of non-specific interactions between the immunoassay components (Fig. 2B). The inter- and intra-day variability were in the range of 1-10.2 % and 2-13.7 %, respectively, while the half-effective concentration (EC50) was 435 pg mL-1. The intraday variability was determined by six assay repeats in a day, while the interday variability was calculated from six assay repeats on six different days. The developed immunoassay has high precision as it correlates well with the commercial sandwich ELISA. It enables LCN2 detection in atual sample matrices (Fig. 2C), as demonstrated by the analysis of human LCN2-spiked samples that were prepared by mixing various human LCN2 concentrations in 1:1000 diluted human plasma, serum and whole blood.
The developed procedure significantly decreased the overall assay duration by more than 3-fold i.e. from 20 h \(commercial sandwich ELISA) to 5.5 h. It has 80-fold better analytical sensitivity \(1 pg mL<sup>-1</sup>) than that of commercial LCN2 ELISA \(80 pg mL<sup>-1</sup>). Moreover, it has better bioanalytical performance \(lower LOD and better analytical sensitivity) than our previously developed LCN2 immunoassay<sup>20</sup>. All these immunoassays were performed under identical conditions using the same components to obviate process variability. The higher sensitivity of the developed ELISA is mainly due to the increased surface area provided by GNPs functionalization, which leads to high antibody immobilization density. The antibody immobilization strategy further enables the leach-proof covalent binding of capture anti-LCN2 antibodies by EDC-based crosslinking of its carboxyl groups to the free amino groups on silanized GNPs-functionalized MTP.
The anti-LCN2 capture antibody-bound MTPs can be stored at 4°C in 0.1 M PBS for up to 8 weeks without any significant loss of their functional activity \(**Fig. 2D**), which is desired for clinical and industrial analysis. The developed procedure is multisubstrate-compatible<sup>19</sup> and can be used to perform sandwich ELISA on various substrates using our previously developed modified MTP format<sup>25</sup>. The multisubstrate-compatibility was due to the generic nature of silanization and GNPs functionalization steps. The developed procedure has got tremendous potential to improve the bioanalytical performance of sandwich ELISA-based IVD kits.- Kjeldsen, L., Johnsen, A.H., Sengelov, H. & Borregaard, N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J. Biol. Chem. 268, 10425-10432 (1993).
- Mishra, J. et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J. Am. Soc. Nephrol. 14, 2534-2543 (2003).
- Mishra, J. et al. Neutrophil gelatinase-associated lipocalin: a novel early urinary biomarker for cisplatin nephrotoxicity. Am. J. Nephrol. 24, 307-315 (2004).
- Malyszko, J., Malyszko, J.S., Pawlak, K. & Mysliwiec, M. NGAL (neutrophil gelatinase-associated lipocalin) is a new sensitive marker of kidney function in renal-allograft recipients. Transplant International 20, P602 (2007).
- Mishra, J. et al. Kidney NGAL is a novel early marker of acute injury following transplantation. Pediatr. Nephrol. 21, 856-863 (2006).
- Parikh, C.R. et al. Urine NGAL and IL-18 are predictive biomarkers for delayed graft function following kidney transplantation. Am. J. Transplant. 6, 1639-1645 (2006).
- Bachorzewska-Gajewska, H. et al. Neutrophil gelatinase-associated lipocalin (NGAL) correlations with cystatin C, serum creatinine and eGFR in patients with normal serum creatinine undergoing coronary angiography. Nephrol. Dial. Transplant 22, 295-296 (2007).
- Malyszko, J. et al. Serum neutrophil gelatinase-associated lipocalin as a marker of renal function in hypertensive and normotensive patients with coronary artery disease. Nephrology 13, 153-156 (2008).
- Wang, Y. et al. Lipocalin-2 is an inflammatory marker closely associated with obesity, insulin resistance, and hyperglycemia in humans. Clin. Chem. 53, 34-41 (2007).
- Flo, T.H. et al. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432, 917-921 (2004).
- Xu, S.Y., Pauksen, K. & Venge, P. Serum measurements of human neutrophil lipocalin (HNL) discriminate between acute bacterial and viral infections. Scand. J. Clin. Lab. Invest. 55, 125-131 (1995).
- Fernandez, C.A. et al. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients. Clin. Cancer Res. 11, 5390-5395 (2005).
- Laurell, H. et al. Identification of biomarkers of human pancreatic adenocarcinomas by expression profiling and validation with gene expression analysis in endoscopic ultrasound-guided fine needle aspiration samples. World J. Gastroenterol. 12, 3344-3351 (2006).
- Lim, R. et al. Neutrophil gelatinase-associated lipocalin (NGAL) an early-screening biomarker for ovarian cancer: NGAL is associated with epidermal growth factor-induced epithelio-mesenchymal transition. Int. J. Cancer 120, 2426-2434 (2007).
- Zhang, H. et al. Upregulation of neutrophil gelatinase-associated lipocalin in oesophageal squamous cell carcinoma: significant correlation with cell differentiation and tumour invasion. J. Clin. Pathol. 60, 555-561 (2007).
- Zheng, L.T. et al. Down-regulation of lipocalin 2 contributes to chemoresistance in glioblastoma cells. J. Neurochem. 111, 1238-1251 (2009).
- Lee, S. et al. A dual role of lipocalin 2 in the apoptosis and deramification of activated microglia. J. Immunol. 179, 3231-3241 (2007).
- Roushandeh, A.M. et al. Down-regulation of lipocalin 2 expression in mouse testis after electromagnetic field exposure. Iran J. Med. Sci. 34, 265-270 (2009).
- Vashist, S.K. Graphene-based immunoassay for human lipocalin-2. Anal. Biochem. 446, 96-101 (2014).
- Vashist, S.K., Saraswat, M., Holthöfer, H. Development of a rapid sandwich enzyme linked immunosorbent procedure for the highly-sensitive detection of human lipocalin-2/NGAL. Procedia Chem. 6, 141-148 (2012).
- Dixit, C.K., Vashist, S.K., MacCraith, B.D. & O’Kennedy, R. Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays. Nat. Protoc. 6, 439-445 (2011).
- Dixit, C.K. et al. Development of a high sensitivity rapid sandwich ELISA procedure and its comparison with the conventional approach. Anal. Chem. 82, 7049-7052 (2010).
- Vashist, S.K., Dixit, C.K., MacCraith, B.D. & O’Kennedy, R. Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay. Analyst 136, 4431-4436 (2011).
- Vashist, S.K. A sub-picogram sensitive rapid chemiluminescent immunoassay for the detection of human fetuin A. Biosens. Bioelectron. 40, 297-302 (2012).
- Dixit, C.K., Vashist, S.K., MacCraith, B.D. & O’Kennedy, R. Evaluation of apparent non-specific protein loss due to adsorption on sample tube surfaces and/or altered immunogenicity. Analyst 136, 1406-1411 (2011).
- Vashist, S.K., O’Sullivan, S.A., O’Neill, F., Holthofer, H., O’Reilly, B. & Dixit, C.K. A multiwell plate for biological assays. WIPO Publication number WO2010/044083 (2010).
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Associated Publications
Graphene-based immunoassay for human lipocalin-2
by Sandeep Kumar Vashist
Analytical Biochemistry (30 December, 2013)
Method Article
A highly-sensitive graphene-based immunoassay procedure for the detection of human lipocalin-2
Sandeep Kumar Vashist
Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 31 Dec, 2013
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This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
We report a highly-sensitive graphene nanoplatelets (GNPs)-based sandwich enzyme-linked immunosorbent immunoassay (ELISA) procedure for the detection of human lipocalin-2 (LCN2). It involves the functionalization of 96-well microtiter plate (MTP) with GNPs (dispersed in 3-aminopropyltriethoxysilane (APTES)). APTES generates the free amino groups on MTP and GNPs, which are used for the leach-proof covalent binding of anti-LCN2 capture antibody by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) based heterobifunctional crosslinking. The developed immunoassay detects LCN2 in the range of 0.6-5120 pg mL-1 with limit of detection and analytical sensitivity of 0.7 and 1 pg mL-1, respectively. It is the most sensitive immunoassay for LCN2, which has 80-fold higher analytical sensitivity and 3-fold reduced immunoassay duration than the commercial sandwich ELISA kit. The anti-LCN2 antibody-bound MTPs, stored at 4°C in 0.1M phosphate buffered saline (PBS), were highly stable as they did not show any decrease in functional activity for 8 weeks.
Figure 1
Figure 2
The highly sensitive detection of LCN21, also known as neutrophil gelatinase-associated lipocalin (NGAL), is desired in clinical diagnosis as it is an important prognostic and diagnostic biomarker for several medical conditions. It is a small 25 kDa glycoprotein, which is found in neutrophils and certain epithelia such as renal tubules. The LCN2 level in healthy humans is 0.7 ng mL-1, which increases in cases of ischemic2 or nephrotoxic injury3, cardiovascular surgery, kidney transplants4-6, coronary heart disease7-8, fatty liver disease, obesity9, bacterial infections10-11 and cancer12-15. The down-regulation of LCN2 occurs in glioblastoma16, ovarian cancer14, microglial cells17 and in response to excessive exposure to high electromagnetic fields18.
The developed GNPs-based sandwich ELISA procedure19 has higher sensitivity and reduced immunoassay duration in comparison to commercial sandwich ELISA kits and our previously developed immunoassay procedures20-24. It involves the APTES functionalization of GNPs and MTP, and EDC-based heterobifunctional crosslinking of anti-LCN2 capture antibodies (Fig. 1). The high sensitivity of the developed procedure is mainly due to the increased surface area provided by GNPs, which leads to higher immobilization density of anti-LCN2 capture antibodies. The developed immunoassay detects the entire pathophysiological range of LCN2 in the real sample matrices i.e. diluted human serum, plasma and whole blood. It has a very high precision as it correlates well with the conventional ELISA. Therefore, it is ideal for mass-use clinical and industrial settings as the anti-LCN2 antibody-bound MTPs, stored at 4°C in 0.1 M phosphate buffered saline (PBS), did not lose their functional activity even after 8 weeks. Moreover, it is a generic sandwich ELISA procedure that can also be employed in in vitro diagnostics (IVD) for the detection of other disease biomarkers and analytes.
• Human Lipocalin-2/NGAL DuoSet ELISA kit (R & D Systems, cat. no. DY1184e). All the assay components in the kit were reconstituted in phosphate buffered saline (PBS, 0.1 M, pH 7.2) with 1% (v/v) bovine serum albumin (BSA), while PBST (PBS with 0.05% Tween 20) was used for washing steps. The buffers were made with ultrapure water (UPW). The kit comprises of
• Rat anti-human LCN2 capture antibody (360 μg mL-1) !CAUTION Store reconstituted antibody at 2-8°C for up to 60 days or at -20°C to -70°C for up to 6 months. Dilute to a working concentration of 2 μg mL-1 in 0.1 M PBS, pH 7.2.
• Recombinant human LCN2 (90 ng mL-1). !CAUTION Store reconstituted LCN2 at 2-8°C for up to 60 days or at -70°C for up to 6 months. Dilute to the dynamic concentration range of the assay in 0.1 M PBS, pH 7.2) with 1% BSA. Make the dilutions of human LCN2 in BSA-preblocked sample vials to minimize the analyte loss due to non-specific binding on sample tube surfaces25.
• Biotinylated goat anti-human LCN2 detection antibody (18 μg mL-1). !CAUTION Store reconstituted antibody at 2-8°C for up to 60 days or at -20°C to -70°C for up to 6 months. Dilute to a working concentration of 100 ng mL-1 in 0.1 M PBS, pH 7.2) with 1% BSA.
• Streptavidin conjugated to HRP. !CAUTION Store at 2-8°C in dark for up to 6 months after initial use as streptavidin is light-sensitive.
• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, cat. no. 37525) !CRITICAL To avoid microbial contamination in BSA, filter prior to use.
• Sulphuric acid (Aldrich, cat. no. 339741) !CAUTION Avoid skin contact as it is a strong corrosive agent and an irritant. Use personal protective equipment (PPE), i.e. chemical-resistant shoes, lab coats and chemical safety glasses, for handling. Handle only in a fume cabinet. In case of skin contact, wash immediately with acid neutralizers and seek medical advice as soon as possible.
• KOH pellets (99.99%), semiconductor grade (Sigma Aldrich, cat. no. 306568) !CAUTION Use PPE for handling and handle only in a fume cabinet as it can cause severe burns. Avoid contact with skin and eyes. CRITICAL The KOH concentration must be 1% (w/v) in autoclaved UPW as higher concentrations lead to surface defects and strongly impacts the antibody immobilization.
• APTES (Sigma Aldrich, Ireland, cat. no. A3684) !CAUTION Higher concentrations are highly toxic. Avoid contact with skin and eyes as it is a skin and eye irritant. Handle inside a fume cabinet. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• GNPs (diameter 5 µm) (Cheap Tubes, Grade 2) !CAUTION Avoid inhalation. CRITICAL Mix 1 mg of GNPs with 1 mL of 0.25% APTES and disperse by keeping in an ultrasonic bath for 1 h before use.
• 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (Thermo Scientific, cat. no. 22981) !CAUTION Equilibrate to room temperature (RT) as it is hygroscopic and may lose activity upon water absorption. Handle inside a fume cabinet as it is an irritant. CRITICAL Store at -20°C. Reconstitute in 0.1 M MES, pH 4.7, see REAGENT SETUP.
• BupH Phosphate Buffered Saline Packs (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) (Thermo Scientific, cat. no. 18372) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• BupH MES Buffered Saline Packs (0.1 M MES [2-(N- morpholino)ethane sulfonic acid], 0.9 % (w/v) sodium chloride, pH 4.7) (Thermo Scientific, cat. no. 28390) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved UPW, see REAGENT SETUP.
• TMB substrate kit (Thermo Scientific, cat. no. 34021). It comprises of
• TMB solution (0.4 g L-1) !CAUTION Skin, eye and lung irritant. In case of skin contact, wash with plenty of water. CRITICAL STEP Maintain the TMB:peroxide ratio as 1:1 as it is critical for the development of color.
• Peroxide solution (containing 0.02 % (v/v) H2O2 in citric acid buffer) (Thermo Scientific). !CAUTION It is a strong oxidizing agent, which is harmful if swallowed and carries a severe risk of damage to eyes. In case of contact, rinse immediately with plenty of water and seek medical attention as soon as possible. Wear PPE and work inside the safety cabinet or fume cabinet.
• Nunc microwell 96-well polystyrene plates, flat bottom (non-treated), sterile (Sigma Aldrich, cat. no. P7491)
• Eppendorf microtubes (1.5 mL; Sigma Aldrich, cat. no. Z 606340)
• SigmaPlot software bundle version 11.2 from Systat for curve plotting and assay analysis
• -80°C freezer (operating range -60 to -80°C) (Thermo Scientific, UK)
• 2-8°C Refrigerator (Future, UK)
• Direct-Q3 Water Purification System (Millipore, USA)
• Tecan Infinite M200 Pro microplate reader (Tecan, Austria GmBH)
• Mini Incubator (Labnet Inc., UK)
• Ultrasonic cleaner (Branson Ultrasonics, USA)
REAGENT SETUP
PBS Add a BupH Phosphate Buffered Saline Pack to 100 mL of autoclaved UPW, dissolve well and make the volume up to 500 mL using autoclaved UPW. Each pack makes 500 mL of PBS at pH 7.2, which can be stored at RT for a week and at 4ºC for up to four weeks.
MES Add a BupH MES Buffered Saline Pack to 100 mL of autoclaved UPW, dissolve well and make the volume up to 500 mL using autoclaved UPW. Each pack makes 500 mL of MES at pH 4.7, which can be stored at RT for two weeks.
APTES The procured APTES solution has a purity of 99%. Reconstitute in autoclaved UPW to make 2% (v/v) APTES just before functionalization.
GNPs Mix 1 mg of GNPs with 1 mL of 0.25% APTES and disperse by keeping in an ultrasonic bath for 1 h before use.
EDC The commercial pack contains 25 g EDC. Reconstitute in 0.1 M MES buffer, pH 4.7 to make 8 mg mL-1. Aliquots can be stored effectively for six months at -20°C.
H2SO4 The concentrated solution is 37 N. Add 5.4 mL of 37 N H2SO4 to 94.6 mL UPW to make a 2 N solution. Reaction of acid and water is exothermic, therefore, acid should always be added to water. It can be stored indefinitely.
Surface activation and GNPs-functionalization (TIMING 1 h 5 min)
- Put 100 μL of 1% (w/v) KOH into the wells of 96-well MTP followed by incubation for 5 min at 37°C and subsequent washing with 300 μL of UPW five times. Washing can also be performed with an automatic plate washer. CRITICAL STEP KOH treatment is optimal at 5 min but it should not be longer than 10 min, which can cause surface aberrations that impact the bioanalytical performance of developed immunoassay. ?TROUBLESHOOTING
Incubate the KOH-treated MTP with 100 μL of GNPs (1 mg mL-1) in 0.25% (v/v) APTES for 1 h at RT inside the fume cabinet. Wash the GNPs-functionalized wells extensively with UPW (as described in step 1). CRITICAL STEP One mg of GNPs should be mixed with 1 mL of 0.25% APTES and dispersed by keeping in an ultrasonic bath for 1 h before use. ?TROUBLESHOOTING
Antibody immobilization and BSA blocking (TIMING 1 h 30 min)
- Incubate 990 μL of anti-LCN2 capture antibody (2 µg mL-1) with 10 μL of EDC (4 mg mL-1) for 15 min at 37°C. CRITICAL STEP The optimized concentration of EDC is critical for the crosslinking of antibodies.?TROUBLESHOOTING
- Put 100 µL of EDC-activated anti-LCN2 antibody to the GNPs-functionalized wells followed by incubation for 1 h at 37°C and subsequent washing with 400 µL of PBST five times. ?TROUBLESHOOTING
Block the antibody-bound wells with 400 µL of 1% (w/v) BSA for 30 min at 37°C followed by extensive PBST washing (as mentioned in step 4). CRITICAL STEP Use diagnostic grade BSA that does not interfere with the immunoassay components. Also filter the prepared BSA in order to remove any contaminanting substances. ?TROUBLESHOOTING
LCN2 sandwich ELISA (TIMING 2 h 40 min)
- Add 100 µL of varying LCN2 concentrations (0.6-5120 pg mL-1) to different BSA-blocked wells. Incubate for 1 h at 37°C and wash extensively with PBST (as stated in step 4).
- Add 100 µL of biotinylated anti-LCN2 antibody in each of the LCN2-captured wells. Incubate for 1 h at 37°C and wash extensively with PBS (as mentioned in step 4). ?TROUBLESHOOTING
- Add 100 µL of SA-HRP conjugate to the biotinylated anti-LCN2 antibody treated wells. Incubate for 20 min at 37°C and wash extensively with PBST (as mentioned in step 4). ?TROUBLESHOOTING
- Add 100 µL of TMB-H2O2 mixture to the wells of MTP and incubate at RT for 20 min to develop color.
- Stop the enzyme-substrate reaction by adding 50 µL of 2 N H2SO4 to each well.
- Measure the absorbance at a primary wavelength of 450 nm and secondary wavelength of 540 nm in the Tecan MTP reader. CRITICAL STEP Determine the absorbance after 5 min of stopping the enzyme substrate reaction so that the reaction is effectively stopped. But the reading should be taken before 10 min.
Steps 1-2, Surface activation and GNPs-functionalization: 1 h 5 min
Steps 3-5, Antibody immobilization and BSA blocking: 1 h 30 min
Steps 6-11, LCN2 sandwich ELISA: 2 h 40 min
Troubleshooting advice is provided in Table 1.
The developed GNPs-based sandwich ELISA19 detected 0.6-5120 pg mL-1 of LCN2 (Fig. 2A) with linearity in the range of 80-2560 pg mL-1. The limit of detection (LOD) and analytical sensitivity of LCN2 immunoassay were 0.7 pg mL-1 and 1 pg mL-1, respectively. Various experimental controls were employed that demonstrated the absence of non-specific interactions between the immunoassay components (Fig. 2B). The inter- and intra-day variability were in the range of 1-10.2 % and 2-13.7 %, respectively, while the half-effective concentration (EC50) was 435 pg mL-1. The intraday variability was determined by six assay repeats in a day, while the interday variability was calculated from six assay repeats on six different days. The developed immunoassay has high precision as it correlates well with the commercial sandwich ELISA. It enables LCN2 detection in atual sample matrices (Fig. 2C), as demonstrated by the analysis of human LCN2-spiked samples that were prepared by mixing various human LCN2 concentrations in 1:1000 diluted human plasma, serum and whole blood.
The developed procedure significantly decreased the overall assay duration by more than 3-fold i.e. from 20 h \(commercial sandwich ELISA) to 5.5 h. It has 80-fold better analytical sensitivity \(1 pg mL<sup>-1</sup>) than that of commercial LCN2 ELISA \(80 pg mL<sup>-1</sup>). Moreover, it has better bioanalytical performance \(lower LOD and better analytical sensitivity) than our previously developed LCN2 immunoassay<sup>20</sup>. All these immunoassays were performed under identical conditions using the same components to obviate process variability. The higher sensitivity of the developed ELISA is mainly due to the increased surface area provided by GNPs functionalization, which leads to high antibody immobilization density. The antibody immobilization strategy further enables the leach-proof covalent binding of capture anti-LCN2 antibodies by EDC-based crosslinking of its carboxyl groups to the free amino groups on silanized GNPs-functionalized MTP.
The anti-LCN2 capture antibody-bound MTPs can be stored at 4°C in 0.1 M PBS for up to 8 weeks without any significant loss of their functional activity \(**Fig. 2D**), which is desired for clinical and industrial analysis. The developed procedure is multisubstrate-compatible<sup>19</sup> and can be used to perform sandwich ELISA on various substrates using our previously developed modified MTP format<sup>25</sup>. The multisubstrate-compatibility was due to the generic nature of silanization and GNPs functionalization steps. The developed procedure has got tremendous potential to improve the bioanalytical performance of sandwich ELISA-based IVD kits.- Kjeldsen, L., Johnsen, A.H., Sengelov, H. & Borregaard, N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J. Biol. Chem. 268, 10425-10432 (1993).
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Associated Publications
Graphene-based immunoassay for human lipocalin-2
by Sandeep Kumar Vashist
Analytical Biochemistry (30 December, 2013)