1. Sample Handling

Upon receipt, samples are visually inspected for problems of shipping, specifically the absence of dry ice in the package and evidence of sample thawing. The number of tubes is checked against the enclosed order form along with the labels on the tubes. The order is then assigned a unique log-in number, placed in a box clearly marked with the log-in number, customer's name and date received. This information is also entered into our sample log book.


2. Assay Procedures

• A protocol is written for each assay and is present in our procedure manual. This includes the sources of the antibodies, standards, and the streptavidine-biotin conjugate, the working dilution of each reagent, the standard curve range, the concentration of the internal control and the assay detection limit.

• A template is made for each assay containing the order log-in number, customer name, date, assay type and the ELISA plate well-address of each sample. This is placed with a hard copy of the assay results in the customer's file. Samples are identified by the log-in number, customer name and date. The tube ID numbers are double checked, thawed at room temperature, are immediately vortexed and placed on ice. The contents of the tubes are checked for sufficient volume for robotic dispensing and the presence of a solid phase that may interfere with dispensing. The tubes are kept on ice until they are dispensed in a Biomek robot workstation. The samples are kept in the workstation are packed with ice and the samples kept in the workstation for no more than 45 minutes in order to avoid sample degradation.

• When stored as frozen aliquots , antibodies and standards are freshly thawed and diluted.

• All assays are performed in Nunc Maxisorb ELISA strips which are freshly coated with capture antibody for 16 hours the night before the assay is performed.

• All assays are performed by specially trained and 100% dedicated personnel under the direction of Dr. Jeffrey Hasday.

• The standard is diluted and all samples and controls are plated by the Biomek robot workstation. The plates are visually inspected by the technician to detect any plating errors. If any errors are suspected, the volumes will be checked manually with a pipetter, quantified and noted.

• All subsequent additions to the ELISA plate, specifically the detecting antibody and the streptavidin-peroxidase conjugate are freshly prepared and stored at 4° until added manually with a multichannel pipetter.

• Plate washing is performed using an automatic strop washer (Biotek) under visual inspection.

• Premixed substrate solution (Neogen) is then added.

• The plate is read on a Molecular Divices ELISA plate reader. Curve fitting is selected among linear, quadratic and 4-point based on the best regression coefficient(s) using the SoftPro software package.