Glucuronide and Antibody W on E1G‐OVA Sensor Surface
To investigate enhancement of inhibitive immunoassays, two series of standard estrone glucuronide solutions of different concentration ranges were prepared in HBS‐EP+ buffer. Each standard solution was mixed with an equal volume of antibody W solution (2 g/mL) in HBS‐EP+ buffer, to give a final antibody W concentration of 1 g/mL. The mixture (60 L) was passed over the chip surface for 120 seconds at a flow rate of 30 L/min, immediately followed by A9044 secondary antibody‐peroxidase solution 106 g/mL (60 L, 10 L/min). Regeneration was by two pulses of 10 % v/v CH3CN in 100 mM NaOH (2 x 5 L, 10 L/min).
Five replicates were injected. Concentrations of the standard estrone glucuronide material in the final solutions were 0, 2.5, 10, 50, 100, 250, 1250 and 2500 ng/mL in the higher‐range curve, and 0, 0.1, 0.25, 0.50, 0.75, 1.00, 1.50, 2.5 and 5.0 ng/mL in the lower‐range curve.
A 0.5 g/mL concentration of antibody W with 212 g/mL of A9044 secondary antibody‐peroxidase was also investigated. Five replicates were injected. Concentrations of the standard estrone glucuronide material in the final solutions were 0, 10, 50, 100, 250, 1 250 and 2 500 ng/mL in the higher‐range curve, and 0, 0.1, 0.25, 0.50, 0.75, 1.00, 1.50, 2.5 and 5.0 ng/mL in the lower‐range curve.
A 0.1 g/mL concentration of antibody W with 212 g/mL of A9044 secondary antibody‐peroxidase was also investigated as above. Regeneration was by a single pulse of 10 % CH3CN in 100 mM NaOH (5 L, 10 L/min). The procedure was carried
out in triplicate. Concentrations of the standard estrone glucuronide material in the final solutions were 0, 0.5, 1, 2, 5, 10, 20, 50, 100, 250 and 500 ng/mL in the higher‐ range curve, and 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.15, 0.20, 0.25, 0.50 and 1.0 ng/mL in the lower‐range curve.
A lower concentration of antibody W (0.05 g/mL) with 212 g/mL of A9044 secondary antibody‐peroxidase was also investigated with the same concentrations
of estrone glucuronide as for the 0.1 g/mL antibody concentration assay described above.
3.5.2 Signal Enhancement with Gold nanoparticles: 40 nm Gold‐IgG‐
Peroxidase Conjugate
Signal enhancement using a 40 nm then a 20 nm gold nanoparticle conjugated to the A9044 secondary antibody was investigated.
The 40 nm gold‐IgG‐peroxidase conjugate was prepared according to the method of Kumar [148]. A 1.5 mL aliquot of 40 nm colloidal gold and 150 L of A9044 secondary antibody‐peroxidase were combined in a MaxymumTM recovery Eppendorf tube by manual inversion for 20 minutes. The tube was allowed to stand for one to two hours. The tube was then centrifuged at 2000 x g for 10 minutes at 4oC and the supernatant liquid removed. The supernatant liquid itself was again centrifuged as before, and the supernatant liquid from this second centrifugation was discarded. The fluid pellets from both centrifugations were pooled and reconstituted in 150 L of phosphate buffered saline (PBS) pH 7.4 containing 0.2 % w/v bovine serum albumin (BSA) and 3 % v/v polyethylene glycol 400 (PEG). The conjugate was filtered through a Phenomenex ‘Phenex’ 0.45 m Polyethersulphone (PES) or Advantec 0.2 m cellulose acetate disposable syringe filter immediately before use.
To optimise loading of 40 nm gold‐IgG‐peroxidase conjugate, antibody W in running buffer (1 g/mL) was injected (60 L, 30 L/min) followed immediately by neat 40 nm gold‐IgG‐peroxidase conjugate (60 L, 10 L/min). Regeneration was by injections of 10 % (v/v) acetonitrile in 50 mM NaOH (5 L then 10 L at 10 L/min). The injection was repeated with the conjugate diluted 1:1 with 10 % v/v PEG 400 in Milli‐Q water. To assess non‐specific binding to the surface, the injection was repeated using running buffer in place of the antibody W mAb solution.
3.5.2.1 40 nm Gold Conjugate: Enhanced Binding Curve
Signal enhancement with the 40 nm gold nanoparticle conjugated to the A9044 secondary antibody was investigated as for 3.5.1.1, but with neat 40 nm gold‐IgG‐ peroxidase conjugate (60 L, 10 L/min) used in place of the A9044 secondary antibody‐peroxidase solution. Regeneration was by a single pulse of 10 % v/v CH3CN in 100 mM NaOH (10 L, 10 L/min).
3.5.2.2 40 nm Gold Conjugate: Enhanced Inhibitive Immunoassays
(Standard Curves): Estrone Glucuronide and Antibody W (1 g/mL)
To investigate enhancement of inhibitive immunoassays with 40 nm gold nanoparticles conjugated to the A9044 secondary antibody, standard curves were prepared as for 3.5.1.2 but with neat 40 nm gold‐IgG‐peroxidase conjugate (60 L, 10 L/min) used in place of the A9044 secondary antibody‐peroxidase solution. Regeneration was by two pulses of in 10 % v/v CH3CN in 100 mM NaOH (2 x 5 L, 10
L/min).
The procedure was carried out in triplicate. Concentrations of the standard estrone glucuronide material in the final solutions were 0, 0.5, 2.5, 5, 10, 50, 100, 250, 1250, 2500 ng/mL in the higher‐range curve, and 0, 0.25, 0.50, 1.00, 1.50, 2.0 and 5.0 ng/mL in the lower‐range curve.
3.5.3 Signal Enhancement with Gold nanoparticles: 20 nm Gold‐IgG‐
Peroxidase Conjugate
Signal enhancement using 20 nm gold nanoparticles when preparing a gold nanoparticle: secondary antibody conjugate was investigated. The conjugation method was also altered.
The 20 nm gold‐IgG‐peroxidase conjugate was prepared using the method of Mitchell and Wu [54], i.e., two 25 mL aliquots of 20 nm colloidal gold were dispensed into separate 50 mL polypropylene ‘Oak Ridge Tubes’ and 750 L of polyethylene glycol (3 % v/v) was added to each tube before centrifuging at 14000 x
g for 30 minutes. The supernatant was removed from the liquid pellet and reconstituted in 5 mL of Milli‐Q water with the aid of sonication. The contents of the tube were mixed using a vortex mixer during the addition of 500 L of A9044 secondary antibody‐peroxidase solution (8 mg/mL in Milli‐Q water) then shaken on a mechanical rotator for ten minutes. The solution was stored overnight at 4oC before adding 550 L of a freshly prepared solution of bovine serum albumin (20 % v/v in Milli‐Q water). The resulting conjugate solution was mixed on a mechanical rotator for ten minutes and refrigerated at 4oC for four hours, then filtered as for the 40nm conjugate before use in the surface plasmon resonance instrument.
3.5.3.1 20 nm Gold Nanoparticle‐IgG‐Peroxidase Conjugate Enhanced
Binding Curve
The effect of a secondary binding event using a smaller gold nanoparticle was investigated with the 20 nm gold nanoparticle‐IgG‐peroxidase conjugate. The binding curve was carried out as for 3.5.1.1 with antibody W solutions in the concentration range 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 g /mL. 20 nm gold‐IgG‐peroxidase conjugate diluted 1:1 with 1 % v/v PEG 400 (60 L, 10 L/min) was used in place of A9044 secondary antibody‐peroxidase solution. Regeneration was by one pulse of 20 % v/v acetonitrile in 100 mM NaOH (60 L, 10 L/min).
3.5.3.2 20 nm Gold Nanoparticle‐IgG‐Peroxidase Conjugate: Enhanced
Inhibitive Immunoassays (Standard Curves): Estrone Glucuronide
vs Antibody W (1 g/mL and 0.05 g/mL))
To investigate enhancement of inhibitive immunoassays with 20 nm gold nanoparticles conjugated to the A9044 secondary antibody, standard curves were prepared as for 3.5.2.2, but with 20 nm gold‐IgG‐peroxidase conjugate diluted 1:1 with 1 % v/v PEG (60 L, 10 L/min) used in place of 40 nm gold‐IgG‐peroxidase. Regeneration was as for the binding curve.
Standard curves were also generated as above using a lower antibody W concentration (0.05 g/mL) and with lower concentrations of the standard estrone
glucuronide 0, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 25, 50, 125, 250 ng/mL in the higher‐range curve, and 0, 0.005, 0.01, 0.02, 0.03, 0.04, 0.050, 0.075, 0.10, 0.125, 0.25 and 0.50 ng/mL in the lower‐range curve.