IVDT_In Vitro Diagnostics Technology

IVD Technology, Fall 2013

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Methods Manufacture of the test components, assembly of tests, performance studies, including all data and conclusions, were provided by DCN Diagnostics (Carlsbad, CA). A colloidal gold myoglobin assay was previously developed at DCN Diagnostics for internal use and demonstration purposes. Te assay, utilizing the same conjugate pads, antibodies, colloidal gold conjugate, and processes, was transferred to the FF120HP membrane. All tests were performed in DCN Diagnostics' laboratories using standards with various concentrations of myoglobin prepared in myoglobin-free serum. Tests were done by pipetting 100 μl of the myoglobin standard into a test tube, then adding a test strip and waiting for 15 minutes. After 15 minutes, the strip was removed and peak heights were determined using an ESEQuant Lateral Flow Immunoassay Reader (Qiagen). Results and Discussion Te best line appearance on the FF120HP membrane was obtained at a dispensing rate of 0.6 μl/cm. Te antibody mass was 0.099 μg per test strip. Reproducibility of the FF120HP membrane was tested using the conditions described above. Concentrations of myoglobin in myoglobin-free serum were 0, 0.25, 2.5 and 10 ng/ml. Twenty strips were prepared for each concentration of myoglobin. Test strips were read using a refectance reader (Table I), and peak heights were obtained. Te % CV was determined by dividing the standard deviation (SD) by the mean times 100%. Te results of this limited testing of a single roll of membrane showed that the peak heights (Figure 3) had low variability. Intra-lot Variability of the Whatman FF120HP Membrane Te intra-lot variability of the FF120HP membrane was evaluated by striping the membranes from three different rolls (7A/Q, 7A/R, 7A/S) from a single casting lot (G1471608). A test line of 0.2 mg/ml of anti-human Average peak height 800 700 600 Avg peak Ht water, pouring this casting mix onto a solid belt-like support, and evaporating the solvents under controlled conditions of temperature, humidity, and belt speed within the membrane manufacturing machine. As the organic solvents evaporate, the water concentration in the casting mix increases to the point that the nitrocellulose fbers precipitate and form a random meshor sponge-like structure. Te consistency and reproducibility of this structure is key to the reproducibility of the LFA devices manufactured using this type of membrane. Using new membrane formulations and improved manufacturing conditions, GE Healthcare Life Sciences has developed a new family of highly consistent membranes. One of these membranes is Whatman FF120HP. 500 400 (1A/T) 300 (15A/T) 200 (7A/Q) 100 0 0 2 4 6 8 10 12 ng/ml Myoglobin Figure 5. Inter-lot variability of FF120HP based on the testing of three rolls from three different casting lots. myoglobin antibody was dispensed at 0.6 μl/cm and 50 mm/sec. Twenty tests were performed for each roll using 0, 0.25, 2.5, and 10 ng/ml of myoglobin in myoglobinfree serum. Te results show that the three rolls within the same casting lot produced similar peak heights when striped with the same antibody solutions (Figure 4). Inter-lot Variability of Whatman FF120HP Membrane Te inter-lot variability of the FF120HP membrane was evaluated by striping membranes from three diferent casting lots (D013024, roll 15A/T; G1471608, roll 7A/Q; G1471610, roll1A/T) with a test line of 0.2 mg/ ml of anti-human myoglobin antibody dispensed at 0.6 μl/cm and 50 mm/s. Twenty tests were performed for each roll using 0, 0.25, 2.5 and 10 ng/ml of myoglobin in myoglobin-free serum. Te results showed that the three rolls of FF120HP membrane produced very similar peak heights when striped with the same antibody solution (Figure 5). Conclusion Te FF120HP membrane provides a very high level of consistency. Additionally, the FF120HP membrane demonstrated remarkable intra- and inter-lot reproducibility with very low CV values that provide assay developers better control of manufacturing processes, reduced QC burden, and lower scrap rates in assay manufacturing. IVD Dr Klaus Hochleitner is global lead technical product specialist, diagnostics, at GE Healthcare Life Sciences. He can be reached via e-mail at klaus.hochleitner@ge.com. Brendan O'Farrell is president and founding partner at DCN Diagnostics. He can be reached via e-mail at bofarrell@dcndx.com. Also contributed to this article: Vincent Sy is global lead applications specialist, diagnostics, at GE Healthcare Life Sciences. Wei Sun is research scientist, polymer and analytical chemistry, at GE Healthcare Life Sciences. I VD T EC H N O LO G Y | FALL 2 0 1 3 1 7 magenta cyan yellow black ES323924_IV1309_017.pgs 09.24.2013 06:19 UBM

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