A highly-sensitive sandwich ELISA procedure based on one-step antibody immobilization for potential in vitro diagnostics

doi:10.1038/protex.2014.011

Method Article

A highly-sensitive sandwich ELISA procedure based on one-step antibody immobilization for potential in vitro diagnostics

https://doi.org/10.1038/protex.2014.011

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A highly-sensitive sandwich enzyme-linked immunosorbent (ELISA) procedure was developed for detecting human fetuin A (HFA), a specific biomarker for a plethora of various infection and inflammation associated diseases. In this one-step antibody (Ab) immobilization, the capture anti-HFA Ab formed a stable complex with 3-aminopropyltriethoxysilane (APTES), via ionic and hydrophobic interactions. The complex adsorbed on a microtiter plate (MTP) as an ELISA platform for HFA, and exhibited linearity from 4.9 pg mL^-1 to 20 ng mL^-1, a limit of detection of 7 pg mL^-1 and an analytical sensitivity of 10 pg mL^-1, i.e. 51-fold more sensitive than the commercial sandwich ELISA kit. The precision obtained by this procedure for the detection of HFA spiked in human whole blood and plasma was in agreement with the commercial kit. The anti-HFA Ab-bound MTPs stored at 4°C in 0.1 M phosphate-buffered saline, pH 7.4 displayed no significant decrease in original activities after two months.

Chemistry

Biological techniques

ELISA

human fetuin A

3-aminopropyltriethoxysilane (APTES)

one-step antibody immobilization

in vitro diagnostics.

With high-throughput and precision, excellent reproducibility, and high sensitivity, ELISA serves a gold standard for in vitro diagnostics (IVD) in healthcare, industry and other diversified analytical settings. In the last 50 years, over 300,000 peer-reviewed articles related to ELISA have been published, attesting its unmatched analytical performance. There are numerous advanced and improved ELISA formats for IVD, which employ improved immobilization chemistry, signal enhancement strategy, lab-on-a-chip technology, biosensing and new immunoassay formats. Regardless of the platform, the performance of such analytical platforms is dependent upon the selection of an appropriate Ab immobilization strategy^1-3. To date, various immobilization schemes have been considered and tested: physical adsorption, oriented binding, covalent binding, click chemistry, site-directed immobilization and non-covalent binding^4-10. In general, such immobilization protocols involve a complex, multi-step procedure with costly reagents and additional instruments.

This protocol is a highly-simplified one-step Ab immobilization-based sandwich ELISA procedure¹¹ towards the development of rapid, cost-effective and highly-sensitive IVD kits. The dilution of capture Ab in APTES forms a stable APTES-polymer/Ab complex that is sorbed onto the MTP for the detection of HFA. HFA serves as a model analyte because all the sandwich immunoassay components are commercially-available in the form of a kit. The reduced amount of HFA is a specific biomarker for acute infectious and inflammatory diseases¹². In contrast, an elevated level of HFA correlates with adiposity¹³, metabolic disease¹⁴, cardiovascular dysfunction¹⁵, atherosclerosis (related to vascular calcification with increased vascular stiffness)¹⁶, diabetes¹⁷, metabolic syndrome^18,19, arthritis²⁰ and malaria²¹. The determination of HFA is also highly relevant in hepatocellular carcinoma²², breast cancer²³ and neurological diseases including multiple sclerosis²⁴. The results obtained by the new immobilization format (NIF) were superior to the commercial conventional immobilization format (CIF) and our previously-developed covalent immobilization format (CovIF)-based ELISA procedures²⁵. Its applicability for the detection of HFA in PBS and HFA spiked in diluted human whole blood and plasma compared favorably with the conventional and covalent crosslinking-based ELISA procedure²⁵. Excellent storage stability of anti-HFA Ab-bound MTPs at 4°C in 0.1 M PBS, pH 7.4 is also demonstrated, illustrating the potential application of this novel immobilization strategy.

• Human fetuin A ELISA kit (R & D Systems, cat. no. DY1184e) !CAUTION Store reconstituted Ab and antigen at 4°C, if they are to be used within a month. Otherwise, aliquot out and stored at -80°C for up to 6 months. The kit comprises of

• Mouse anti-HFA capture Ab (720 μg mL^-1)

• HFA (20 ng mL^-1)

• Biotin-labeled goat anti-HFA detection Ab

• HRP-conjugated streptavidin !CAUTION Store in the dark 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.

• Sulfuric acid (Aldrich, cat. no. 339741) !CAUTION Avoid skin contact as it is a strongly corrosive agent and an irritant. Use personal protective equipment (PPE) such as chemical safety glasses, chemical-resistant shoes and lab coats, 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.

• 3-Aminopropyltriethoxysilane (APTES) (Sigma Aldrich, cat. no. A3684) !CAUTION Higher concentrations are potentially toxic. It is a skin and eye irritant. Avoid contact with skin and eyes. Handle inside a fume cabinet. CRITICAL Prepare in autoclaved DIW (18Ω), 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 DIW (18Ω), see REAGENT SETUP.

• TMB substrate kit (Thermo Scientific, cat. no. 34021)

• TMB solution (0.4 g L^-1) !CAUTION Skin, eye and lung irritant. In case of skin contact, wash with plenty of water. CRITICAL TMB to peroxide ratio is critical for color development. Maintain the ratio 1:1.

• Peroxide solution (containing 0.02 % (v/v) H₂O₂ in citric acid buffer). !CAUTION Strong oxidizing agent. Harmful if swallowed. Severe risk of damage to eyes. Rinse immediately with plenty of water in case of contact and seek medical attention. Wear suitable protection and work in safety cabinet or fume cupboard.

• Deionized water (18Ω, DIW). (Direct-Q® 3Water Purification System, Millipore)

• 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 version 11.2 from Systat

REAGENT SETUP

PBS Add a BupH phosphate buffered saline pack to 100 mL of autoclaved DIW, dissolve well and make the volume up to 500 mL using autoclaved DIW. Each pack makes 500 mL of PBS at pH 7.2, which can be stored at room temperature (RT) for a week and at 4ºC for up to four weeks.

APTES The procured APTES solution has a purity of 99%. Reconstitute in autoclaved DIW to make 1% (v/v) APTES just before mixing with capture anti-HFA Ab.

• -70°C Freezer (operating range -60 to -80°C) (New Brunswick)

• 2-8°C Refrigerator (Future)

• Direct-Q^®3Water Purification System (Millipore)

• Tecan Infinite M200 Pro microplate reader (Tecan)

• Mini incubator (Labnet Inc.)

• PVC fume cupboard Chemflow range (CSC Ltd.)

Ab immobilization and BSA blocking TIMING 1 h

Mix anti-HFA capture Ab (8 µg mL^-1) with 1% (v/v) APTES in the ratio of 1:1 (v/v). Incubate each of the desired wells of a 96-well MTP with 100 μL of the freshly prepared anti-HFA capture Ab solution, with a final concentration of 4 µg mL^-1 in 0.5% APTES, for 30 min at RT. Wash five times with 300 μL of 0.1M PBS, pH 7.4. Washing can also be performed with an automatic plate washer.
Block the Ab-bound wells with 300 µL of 1% (w/v) BSA for 30 min at 37°C followed by extensive PBS washing (as mentioned in step 1). CRITICAL STEP Use filtered BSA to remove any microbial or other contaminants. ?TROUBLESHOOTING

HFA sandwich ELISA TIMING 2 h 45 min

Add 100 µL of varying HFA concentrations (4.9 pg mL^-1-20 ng mL^-1) to different BSA-blocked wells. Incubate for 1 h at 37°C and wash extensively with PBS (as stated in step 1).
Add 100 µL of biotinylated anti-HFA Ab (200 ng mL^-1) in each of the HFA-captured wells. Incubate for 1 h at 37 °C and wash extensively with PBS (as mentioned in step 1).
Add 100 µL of streptavidin-HRP conjugate (diluted 1:200) to the biotinylated anti-HFA Ab treated wells. Incubate for 20 min at 37°C and wash extensively with PBS (as mentioned in step 1). ?TROUBLESHOOTING
Add 100 µL of TMB-H₂O₂ mixture to each of these wells and incubate at RT to develop color for 20 min. ?TROUBLESHOOTING
Stop the enzyme-substrate reaction by adding 50 µL of 1 N H₂SO₄ to each well.
Determine the absorbance at a primary wavelength of 450 nm taking 540 nm as the reference wavelength in the Tecan Infinite M200 Pro microplate reader. CRITICAL STEP Determine the absorbance within 10 min of stopping the enzyme substrate reaction.

Steps 1-2, Ab immobilization and BSA blocking: 1 h

Steps 3-8, HFA sandwich ELISA: 2 h 45 min

Troubleshooting advice is provided in Table 1.

The NIF shown in Figure 1 detected HFA in the range of 4.9 pg mL^-1 - 20 ng mL^-1 with linearity from 1.6 - 20 ng mL^-1 (Figure 2a). The estimated limit of detection (LOD) and analytical sensitivity^6,25-27 were 7 and 10 pg mL^-1, respectively. Various experimental process controls were investigated to determine the efficiency of BSA blocking; non-specific interaction of BSA with HFA, biotinylated anti-HFA Ab and SA-HRP; and, non-specific interaction of capture anti-HFA Ab with biotinylated anti-HFA Ab. In general, the detection was highly specific for HFA without any interference of immunological reagents (Figure 2b). The intra- and inter-day variability ranged from 1.2 - 8.5 % and 2.1 - 10.2 %, respectively, with a half-effective concentration (EC50) of 2.6 ng mL^-1. The intra- and inter-day variability was calculated from five assay repeats (in triplicate) on a single and five different days, respectively.

A comparison of the NIF versus the commercial CIF and CovIF-based sandwich ELISAs is presented in Table 2. The NIF significantly decreased the overall assay time from 20 h to ~4 h. Its analytical sensitivity is 10 pg mL^-1, i.e., 51-fold lower than the commercial CIF (510 pg mL^-1). It also enabled 28-fold faster Ab immobilization than CIF. Moreover, it has a better LOD and analytical sensitivity in comparison to the HFA sandwich ELISA^6,25 based on heterobifunctional crosslinking of Ab to APTES-functionalized platforms, and surface plasmon resonance (SPR)-based immunoassay formats^1,28. All the ELISA formats were performed under the same conditions with the same assay components to minimize experimental variability. The higher sensitivity of the NIF is mainly due to the higher Ab immobilization density via APTES-assisted physical adsorption, where APTES serves as a diluting and binding agent for anti-HFA capture Ab apart from a surface functionalization agent.

The detection of HFA spiked in diluted human whole blood and plasma (Figure 2c) using the NIF exhibited similar precision as the commercial kit, showing its applicability for the determination of HFA in clinical and bioanalytical settings. The NIF is the most sensitive immunoassay format in comparison to the CIF and CovIF for HFA. The stability of Ab immobilized on MTPs was excellent after two months of storage at 4 ºC in 0.1 M PBS (pH 7.4). No significant decrease in the Ab functional activity was observed after eight weeks (Figure 2d), evincing the leach-proof Ab immobilization. The preparation is very rapid (1 h for Ab immobilization and blocking), therefore, it might obviate the preparation of Ab-bound MTPs in priori considering most antibodies are only good within two weeks of storage.

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The authors declare no competing financial interests.

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A highly-sensitive sandwich ELISA procedure based on one-step antibody immobilization for potential in vitro diagnostics

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