Development of a competitive lateral flow immunoassay for progesterone: influence of coating conjugates and buffer components

• Published in: Analytical and bioanalytical chemistry 2008-11, Vol.392 (6), p.1215-1223
• Main Authors: Posthuma-Trumpie, Geertruida A, Korf, Jakob, van Amerongen, Aart
• Format: Article
• Language: English
• Subjects: Adsorption; Analytical Chemistry; Antibodies; Antibodies - chemistry; Antibodies - immunology; Biochemistry; Biotin - chemistry; Blocking agents; Buffers; Carbon; Carbon - chemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Coating; Collodion - chemistry; Colloids - chemistry; Computer programs; Conjugates; elisa; Enzyme-Linked Immunosorbent Assay - methods; Food Science; Format; Haptens - chemistry; Immunoassay; Immunochromatography; Laboratory Medicine; Lateral flow immunoassay; linked-immunosorbent-assay; mass-spectrometry; Membranes, Artificial; milk; Monitoring/Environmental Analysis; optimization; Original Paper; Ovalbumin - chemistry; plasma; Polyethylene Glycols - chemistry; progesterone; Progesterone - analogs & derivatives; Progesterone - analysis; Reproducibility of Results; samples; Sensitivity and Specificity; Serum Albumin, Bovine - chemistry; Signal generation; water
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-008-2362-8

Several aspects of the development of competitive lateral flow immunoassays (LFIAs) are described. The quantitation of progesterone is taken as an example. The LFIA format consisted of a nitrocellulose membrane spotted with various progesterone conjugates as the test line. A mixture of primary antibody and secondary antibody adsorbed to colloidal carbon was used for signal generation. A digital scanner and dedicated software were used to quantitate the response. A reappraisal of the checkerboard titration, often used in the optimisation of immunoassays, is discussed. Surprisingly, the highest sensitivity of the LFIA format (IC₅₀ of 0.6 μg L⁻¹ progesterone in buffer) was achieved by using a high coating concentration of the analyte-protein conjugate and a high dilution of the antibody solution. Immediate addition of all reagents in LFIA was superior to premixing the components and allowing prereaction. Of several blocking agents tested bovine serum albumin was superior in performance, whereas the combination of ovalbumin and progesterone substantially influenced test results.