ELISA microplate Research Article


activated, as representatives for the assessment. For comparison, Tosylactivated magnetic beads were also employed. A therapeutic protein being developed in Astellas Pharma Inc was used as an evaluation analyte. The results revealed that with careful optimization, microplates could provide results comparable to those of magnetic beads.


Materials & methods Materials & reagents The proprietary therapeutic protein drug, ASP2409, was provided by Astellas Pharma, Inc (Tokyo, Japan), and its capture protein, Cluster of Differentiation 86 (CD86) was provided by Ancell Corporation (MN, USA). Stable isotope-labeled peptide with sequence GIASFVCEYESP(13


standard (IS), was synthesized by Midwest Bio-tech, Inc. (IN, USA). Chemicals, such as dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicar- bonate, O-methylisourea hemisulfate, boric acid and sodium hydroxide, were purchased from Sigma- Aldrich (MO, USA). HPLC grade acetonitrile, metha- nol, acetic acid and formic acid were from Fisher Scien- tific (NH, USA). 10 × phosphate-buffered saline (PBS) was from Teknova (CA, USA). Sequence grade trypsin was from Promega (WI, USA). Tosylactivated mag- netic beads (Dynabeads M-280) were from Life Tech- nologies (CA, USA). Unmodified microplates (named GBO) were from Greiner Bio-One (NC, USA). Maleic anhydride-activated microplates (named MA) were from Pierce (IL, USA). Nunc®


C5,15


(named NIA) were from Thermo Scientific (PA, USA). All plates have 200 μl-capacity.


Sample preparation Calibration standards were prepared fresh daily by diluting 10 mg/ml ASP2409 stock solution with pooled human serum to the desired levels. Quality control samples (QCs) were prepared in pooled human serum from a different ASP2409 stock solution (10 mg/ml) and stored at -20°C before use. QCs at four concentra- tion levels, LLOQ, low (LQC), mid (MQC) and high (HQC), were prepared for sensitivity, accuracy and precision evaluation.


Immunocapture procedures for magnetic beads A 30 μl aliquot of M-280 Tosylactivated beads in a Protein Lobind tube (Eppendorf) was washed two- times with 0.5 ml of 0.1 mol/l borate buffer, pH 9.5 (Buffer A). The beads were separated from solution using a magnetic particle concentrator for microcen- trifuge tubes (Life Technologies). After removal of the washing solution, the beads were added with 10 μl of Buffer A and 5 μl of CD86 at 1 mg/ml and incubated


future science group N)GKA, used as internal Immobilizer™ Amino


for 4 h at 37°C with shaking at 1000 rpm for covalent binding to the beads. The beads were washed three- times with 0.5 ml of PBS buffer containing 0.1% BSA (Buffer B) to remove unbound capture protein. The beads were resuspended in 0.5 ml of Buffer B and incu- bated at 37°C with shaking at 1000 rpm for 2 h to block and prevent nonspecific binding. After remov- ing the washing buffer, the beads were added to 10 μl PBS buffer containing 0.1% BSA (Buffer B) and trans- ferred into a polypropylene plate well. Serum samples (200 μl) were added and incubated at 37°C with shak- ing at 1000 rpm for 1.5 h. After removal of the super- natant, the beads were washed three-times with Buffer B and were ready for digestion after complete removal of the buffer.


Immunocapture procedure for ELISA microplates Aliquots of 150 μl/well (Buffer A for MA and NIA, 0.5 mol/l sodium bicarbonate for GBO), were added into a microplate followed by the addition of 3 μl/well of CD86 at 1 mg/ml and incubated for 1.5 h at 37°C with shaking at 1000 rpm. The plate was emptied and washed three-times with Buffer B, followed by the addition of 150 μl/well of Buffer B and incubation for 1 h at 37°C with shaking at 1000 rpm. After empty- ing the plate, 200 μl/well of each sample was added and incubated at 37°C with shaking at 1000 rpm for 1.5 h. Prior to the digestion step the plate was washed three-times with Buffer B and completely emptied.


Digestion The internal standard (IS) was freshly prepared at 200 ng/ml in 0.5 mol/l ammonium bicarbonate buffer (Buffer C). A 50-μl IS aliquot was added to the beads or the microplate well prepared above followed by spik- ing with 15 μl of 10 mmol/l DTT prepared in Buffer C and incubated at 70°C for 60 min for disulfide bond


www.future-science.com 309 Key terms


Ligand-binding assay: Analytical procedure that relies on the binding of ligand molecules to receptors used for the determination of the target analytes in biological samples.


Protein therapeutics: Proteins that are manufactured in the laboratory for therapeutic use.


Surrogate peptides: Proteotypicpeptides generated from enzymatic digestion of intact proteins; their sequence and quantity can serve as the proxy for the intact proteins.


ELISA: Analytical technique used to quantify proteins, antigens or antibodies in biological samples.


Immunoaffinity-LC–MS/MS: Using a capture protein or antibody to extract target analytes from biological sample matrix prior to LC–MS analysis.


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