A rapid one-step kinetics-based immunoassay procedure for the highly-sensitive detection of C-reactive protein

doi:10.1038/protex.2014.017

Method Article

A rapid one-step kinetics-based immunoassay procedure for the highly-sensitive detection of C-reactive protein

https://doi.org/10.1038/protex.2014.017

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A rapid one-step kinetics-based sandwich enzyme-linked immunosorbent (ELISA) procedure has been developed for highly-sensitive detection of C-reactive protein (CRP) in less than 30 min. With minimal process steps, the procedure is highly simplified and cost-effective. The analysis only involves sequentially the formation of a sandwich immune complex on capture anti-CRP antibody (Ab)-bound Dynabeads, followed by two magnet-assisted washings and an enzymatic reaction. The developed immunoassay (IA) detected CRP in the dynamic range of 0.3-81 ng mL^-1 with a limit of detection and analytical sensitivity of 0.4 ng mL^-1 and 0.7 ng mL^-1. Its analytical precision for analysis of CRP spiked in diluted human serum, whole blood, and ethylenediaminetetraacetic acid (EDTA) plasma samples of patients was validated by conventional ELISA, unravelling its immense potential for in vitro diagnostics (IVD).

Chemistry

Biological techniques

One-step kinetics

immunoassay

C-reactive protein

Dynabeads

in vitro diagnostics

As the gold standard for the detection of CRP in clinical diagnostics, ELISA exhibits high-throughput, excellent reproducibility, high precision and remarkable sensitivity. Last five decades have accumulated over 300,000 peer-reviewed articles related to ELISA. In particular, the last decade has also witnessed considerable advances in diversified CRP assay formats^1-22, lateral flow²⁰, immunoturbidimetry^1,11, surface plasmon resonance¹⁴, piezoresistive cantilever-based IA¹⁰, chemiluminescent IA³, impedimetry²³, electrochemistry¹⁷, reflectometric interference spectroscopy¹⁹, microfluidics⁷ and homogenous bead-based IA²⁴. In general, most of these assay formats are based on a complex procedure comprising of many process steps with a lengthy analysis time^25-28. Therefore, there is uttermost importance for the development of rapid and cost-effective IA procedures with high precision and minimal process steps to guide healthcare professionals to decide on the desired intervention at an early stage.

CRP, a pentameric protein with a molecular weight of 118 kDa, is a member of a class of acute-phase reactants indicating activation of innate and adaptive immunity^13,29,30. It plays an important role in host defense by binding to phosphocholine and related microbial molecules. As an early indicator of infectious or inflammatory conditions^31,32, CRP is usually elevated in patients with neonatal sepsis^33-35, meningitis, pancreatitis, pneumonia and pelvic inflammatory disease and occult bacteremia. The significantly elevated serum CRP levels are associated with malignant diseases, bacterial infections and correlate with increased 30-day mortality rates in hospitalized patients³⁶. The monomeric CRP binds to the surface of damaged cells and platelets, thereby activating the complement cascade that plays an important role in inflammation. The American Heart Association/Center for Disease Control has considered CRP as the best inflammatory marker for clinical diagnosis³⁷. The precise and rapid determination of human C-reactive protein (CRP) is essential for diagnosis and management of neonatal sepsis^33-35,38,39, cardiovascular diseases^40-44, infectious/inflammatory conditions³¹ and diabetes^45-47. The ability for repeated CRP measurements with high precision in an acute setting provides clinicians with the valuable information to assess disease diagnosis and circumvent any unnecessary administration of antibiotics. The normal CRP levels in human serum are usually below 10 µg mL^-1 ³⁰ but up to 350-400 µg mL^-1 in several disease states. The CRP levels in the ranges of 10-40 µg mL^-1, 40-200 µg mL^-1, and >200 µg mL^-1 are the indicators of mild or chronic inflammation and viral infections; acute inflammation and bacterial infections; and severe bacterial infections and burns, respectively³⁰. The CRP levels beyond the cut-off point of 5 µg mL^-1 are indicative of neonatal sepsis, which is diagnosed based on the determination of two CRP concentration ranges normal (0.2-480 µg mL^-1) and high sensitivity (0.08-80 µg mL^-1)³⁰. The high sensitivity CRP assay is performed first, but the normal CRP assay is also performed if the CRP levels are >80 µg mL^-1.

The existing analytical techniques for the determination of CRP have limitations in terms of prolonged IA duration and lower analytical sensitivity, as reviewed recently by Algarra et al.²². The clinical laboratory-based CRP assays, based on latex agglutination or nephelometry, and phosphocholine and O-phosphorylethanolamine based immunoassays can detect CRP only in the detection range of µg mL^-1. On the other hand, the recently developed immunoassay formats, based on electrochemical techniques, nanoparticles, nanocomposites, chemiluminescence, total internal reflection and micromosiac immunoassays, have higher sensitivities in the range of ng mL^-1 to µg mL^-1. Similarly, the surface plasmon resonance based real-time and label-free immunoassay formats have sensitivity between ng mL^-1 to g mL^-1. Therefore, there is a critical need for a highly simplified, cost-effective, precise and highly sensitive IA format for the rapid detection of CRP.

We have developed a rapid one-step kinetics-based sandwich ELISA procedure⁴⁸ (Figure 1) to detect CRP in human whole blood and serum in less than 30 min. It has critically reduced the IA duration by more than 12-fold and the analysis cost by 2.5-fold in comparison to the conventional procedure. This novel ELISA format required also significantly reduced number of process steps and only two washing steps in comparison to the conventional counterpart (Table S1). Moreover, the high analytical precision of the developed procedure implies its tremendous potential for rapid analyte detection in clinical and bioanalytical settings.

• Human CRP Duoset kit (R & D Systems, UK, cat. no. DY1707E) !CAUTION Store reconstituted Ab and antigen at 2-8 °C, if they are used within a month. Otherwise, make aliquots and store at -20 °C to -70 °C for up to 6 months. The kit comprises of

• Mouse anti-human CRP capture Ab (360 μg mL^-1)

• Recombinant human CRP (90 ng mL^-1)

• Biotinylated mouse anti-human CRP detection Ab (22.5 µg mL^-1)

• Streptavidin-conjugated horseradish peroxidase (SA-HRP) !CAUTION Do not freeze. Store in the dark as streptavidin is light-sensitive.

The Human CRP Duoset kit’s components can also be procured separately, i.e. human CRP antibody (cat. no. MAB17071), human CRP biotinylated antibody (cat. no. BAM17072) and recombinant human CRP (cat. no. 1707-CR).

• Blocker BSA in PBS (10X), pH 7.4, 10% (w/v) (Thermo Scientific, Ireland, cat. no. 37525) CRITICAL Filter with 0.2 µm pore size filter paper prior to use to avoid contamination.

• Dynabeads^® M-280 Tosylactivated (Thermo Fisher Scientific, Ireland)

• 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.

• BupH Phosphate Buffered Saline Packs (0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2) (Thermo Scientific, Ireland, cat. no. 18372) !CAUTION Avoid inhalation. CRITICAL Prepare in autoclaved DIW (18Ω), see REAGENT SETUP.

• TMB substrate kit (Thermo Scientific, Ireland, 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 The TMB to peroxide ratio is critical for color development. Maintain the ratio 1:1.

• Hydrogen peroxide solution (containing 0.02 % v/v H₂O₂ in citric acid buffer) (Thermo Scientific, Ireland). !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 a safety cabinet or fume cupboard.

• Human whole blood (HQ-Chex level 2, cat. no. 232754, Streck) 180 day closed-vial stability and 30 day open-vial stability.

• Human serum (CRP free serum, cat. no. 8CFS, HyTest Ltd., Finland)

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

• 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. Z606340)

• 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 RT for a week and at 4 ºC for up to four weeks.

Binding buffer. 0.1% BSA in PBS, pH 7.2.

Washing buffer. 0.05% Tween^® 20 in PBS, pH 7.2.

Anti-CRP capture Ab-bound Dynabeads^®. The anti-CRP capture Ab was bound to the tosylated Dynabeads^® using the standard immobilization procedure provided by the manufacturer (Invitrogen). The prepared stock solution of anti-CRP capture Ab-bound Dynabeads^® was then stored at 4 °C.

Biotinylated anti-CRP detection Ab conjugated to SA-HRP. Biotinylated anti-CRP detection Ab conjugated to SA-HRP was prepared by adding 1 µL of biotinylated anti-CRP detection antibody (0.5 mg mL^-1) to 1 µL of SA-HRP to 2998 µL of the binding buffer followed by 20 min of incubation at room temperature (RT). As a result, the concentration of biotinylated anti-CRP detection Ab used was 0.17 µg mL^-1, while SA-HRP dilution employed was 1:3000.

• -70 °C freezer (operating range -60 to -86 °C) (New Brunswick)

• 2-8 °C refrigerator (Future, UK)

• Direct-Q^® 3 water purification system (Millipore, USA)

• Tecan Infinite M200 Pro microplate reader (Tecan, Austria GmBH)

• Mini incubator (Labnet Inc., UK)

• Quadermagnet magnetic holder (Supermagnete, Germany)

• PVC fume cupboard Chemflow range (CSC Ltd.)

Preblocking TIMING ~ 30 min

Block the MTP wells by incubating with 300 μL of 5% (w/v) BSA for 30 min a 37 °C and wash with 300 μL of wash buffer five times. Washing can also be performed with an automatic plate washer. The preblocking is essential to prevent non-specific binding of IA reagents to the MTP wells⁴⁹. CRITICAL STEP Use filtered BSA or filter the BSA solution prior to use to remove any microbial or other contaminants. ?TROUBLESHOOTING

One-step kinetics-based CRP sandwich ELISA TIMING 30 min

Dispense in the BSA-blocked MTP wells sequentially 2 µL of the diluted stock solution of anti-CRP capture Ab-bound Dynabeads^® (diluted 1:10 in binding buffer), 38 µL of the binding buffer and 40 µL of biotinylated anti-CRP detection Ab (0.17 µg mL^-1) pre-conjugated to SA-HRP (diluted 1:3000). Finally, dispense 40 µL of CRP (varying concentrations; 0.3-81 ng mL^-1) in the respective MTP wells in triplicate. Place the MTP on the magnetic holder and incubate at 37 °C for 15 min so that the magnets capture the Dynabead^®-bound sandwich immune complex. Take out the excess reagents by sucking back the solution using a 300 µL multi-channel pipette. ?TROUBLESHOOTING
Wash the magnetically-captured sandwich immune complex-bound Dynabeads^® twice by dispensing and sucking back 300 µL of the washing buffer using a 300 µL multi-channel pipette. Thereafter, suspend the washed magnetically-captured sandwich immune complex-bound Dynabeads^® in 50 µL of the binding buffer.
Add 100 µL of the TMB-H₂O₂ mixture to each MTP well and incubate at RT for 4 min to allow the enzymatic reaction to develop color. ?TROUBLESHOOTING
Stop the enzymatic reaction by adding 50 µL of 2N H₂SO₄ to each MTP well.
Measure the absorbance at a primary wavelength of 450 nm and 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 enzymatic reaction.

Steps 1, Preblocking: 30 min

Steps 2-6, One-step kinetics-based CRP sandwich ELISA: 30 min

Troubleshooting advice is provided in Table 1.

The developed CRP sandwich ELISA critically reduced the IA assay duration by 12-fold, from 6 h (commercial CRP sandwich ELISA) to just 30 min, based on the use of Ab-bound Dynabeads^®/MTPs. The developed ELISA is cost-effective and highly-simplified as attested by the minimal process steps and 2.5-fold reduced IA components in comparison to the conventional ELISA procedure (Tables S1, S2). It detects 0.3-81.0 ng mL^-1 of CRP with linearity in the range of 1-81 ng mL^-1 (Figure 2A). LOD, analytical sensitivity and correlation coefficient (R²) are determined to be 0.4 ng mL^-1, 0.7 ng mL^-1, and 0.998, respectively (Table S2). The intraday and interday variability determined from five assay repeats (in triplicate) in a single day and five consecutive days, respectively, are in the ranges of 0.7-10.8 and 1.6-11.2, respectively. The developed ELISA can detect the entire pathophysiological range of hsCRP from 3-80 µg mL^-1 in human whole blood and serum after appropriate dilution, as demonstrated by the detection of CRP spiked in diluted whole blood and plasma (Figure 2A). It has high specificity for CRP, as demonstrated by the use of various experimental process controls (Figure 2B) that shows no detectable interference with the immunological reagents. The optimum duration for the formation of the sandwich immune complex by the one-step kinetics-based procedure is 15 min, while the optimum number of magnet-assisted washings thereafter is only two (Figure S1).

The developed and conventional sandwich ELISAs have the same analytical precision for the detection of CRP in diluted whole blood and serum as the results are in agreement with each other (Table 2). The percentage recovery for the CRP-spiked diluted human whole blood is in the range of 93.3-107.0 and 94.0-103.3 for the developed and conventional sandwich ELISAs, respectively. Similarly, the percentage recovery for CRP-spiked diluted human serum ranges from 103.3 -108.0, compared to 93.3-113.0 for the developed and conventional sandwich ELISAs, respectively. The results obtained for the detection of CRP in the EDTA plasma samples of patients by the developed ELISA are also in good agreement with those obtained by the conventional ELISA (Table 3). The anti-CRP capture Ab-bound Dynabeads^® can be stored for more than 4 months at 4 ºC without compromising the CRP detection response (Figure 2C). The production variability for the preparation of anti-CRP capture Ab-bound Dynabeads^®, using the same lots of Dynabeads^® and anti-CRP capture Ab, is less than 3 percent (Figure 2D). Moreover, the developed ELISA using SA-HRP/biotinylated anti-CRP Ab conjugate is similar to the variant of the developed ELISA procedure that employs the two-step binding of biotinylated anti-CRP Ab and SA-HRP (Figure S2A). The one-step kinetics-based sandwich ELISA solution (comprising anti-CRP capture Ab-bound Dynabeads^® and biotinylated anti-CRP detection Ab preconjugated to SA-HRP, stored at 4 ºC in BSA-preblocked MTPs) exhibited no noticeable decrease in its functional activity for up to 4 weeks (Figure S2B). Therefore, taking into account the attributes of the developed generic sandwich ELISA procedure, it can be reliably employed in clinical and bioanalytical settings. Moreover, it has immense potential for the development of novel and fully automated rapid IVD kits in combination with lab-on-a-chip technologies, microfluidics, nanotechnology and smart system integration.

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We thank Dr. Eberhard Barth for providing and anonymizing the leftover EDTA plasma samples of patients treated by intensive care at University Hospital Ulm, Germany for the validation of the developed sandwich ELISA.

The authors declare no competing financial interests.

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A rapid one-step kinetics-based immunoassay procedure for the highly-sensitive detection of C-reactive protein

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