Microformat Imaging ELISA for Pesticide Determination

Abstract

A flat-well microformat competitive enzyme-linked immunosorbent chemiluminescent assay for the detection of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D) is described. Thick-film technology was used to pattern a hydrophobic layer 100 μm thick onto glass microscope slides to form an array of 2 × 2 mm(2) squares. These flat wells were able to hold 2 μL of reagents, corresponding to a height of ∼500 μm, with minimal contamination risk. The hydrophobic ink used to pattern the surfaces allowed significantly larger volumes of samples to be applied when compared with surfaces patterned with nonhydrophobic inks. This reduced evaporation effects and permitted greater pipetting accuracy, thereby improving assay reproducibility. A competitive immunoassay was developed based on the ability of free 2,4-D hapten to inhibit binding of anti-2,4-D monoclonal antibodies to 2,4-D-bovine serum albumin conjugate adsorbed onto the glass support. The support was subsequently incubated with alkaline phosphatase (AP) labeled anti-mouse IgG. The amount of AP conjugate bound was determined by quantitating the chemiluminescent emission produced from the enzymatic breakdown of CSPD substrate by AP using a cooled CCD camera. The detection limit of the single-sample microformat assay was 2.7 × 10(-)(11) M, or 6 pg of 2,4-D. The linear ranges of the single-sample and multisample assays were 4.5 × 10(-)(8)-4.5 × 10(-)(11) and 4.5 × 10(-)(7)-1.66 × 10(-)(10) M, respectively. In comparison, the detection limits of a tube-based chemiluminescent assay using standard luminometer and of a colorimetric ELISA were 45 × 10(-)(11) and 9.9 × 10(-)(8) M, respectively. The ability to scale the thick-film-based microformat assay makes it an ideal candidate for the development of affinity arrays and high-throughput assay formats. Prospects for further improvements of this imaging ELISA strategy will be discussed.

Use of Charge Coupled Devices for the Simultaneous Detection of Multiple Pesticides by Imaging ELISA Techniques

“…The CCD Camera-Based Cheimluminescence System, luminometer based Chemiluminescence ELISA and microliter plate based Colorimetric ELISA were performed as described earlier (27).…”

Detection of 2,4-Dichlorophenoxyacetic Acid Using a Fluorescence Immunoanalyzer

“…These methods are, for the most part, immunochemically based enzyme-linked immunosorbent assays (ELISA) using absorbance, fluorescence or electrochemical detection. These assays have typically been formatted for microtiter plates, 2 thick film-based planar microwells, 3 microflow cells, 4 test kits, 5 or test tube-type optical spectrometers. 6 Although automation has been incorporated into a number of these method formats, they typically require manual addition of reagents, several incubation steps, and physical manipulations such as changing tubes, trays or capillaries.…”

“…This pesticide is typically monitored in environmental samples using GC methods which require extensive clean-up and derivatization, however, a number of potential field analytical methods have been reported. These methods include: ELISA 3,4,9,11 enzyme immunoassay, 1 immunoagglutination, 12 fluorescence polarization, 13 and enzyme assays 14 as well as several antibody-based biosensor methods. [15][16][17] Although most of these methods are portable and relatively simple to execute, they typically require multiple steps and generate solid wastes (e.g., plates, tubes and vials).…”

Ανάπτυξη Ανοσοδιαγνωστκών Μεθόδων Πόλωσης Φθορισμού Για Τον Προσδιοριμό Τοξικών Ουσιών

“…Το ένζυµο που χρησιµοποιείται για σήµανση είναι συνήθως η υπεροξειδάση (HRP) µε φασµατοφωτοµετρικό προσδιορισµό του χρώµατος από την αντίδραση χρωµογόνουυποστρώµατος. Άλλα ένζυµα είναι η αλκαλική φωσφατάση, ακετυλοχοληνεστεράση, βγαλακτοσιδάση χωρίς όµως να έχουν βρει τόσο ευρεία χρήση όσο η υπεροξειδάση [50,70]. Αποτελούν µια ιδιαίτερη κατηγορία µε εφαρµογές στην περιβαλλοντική ανάλυση.…”

“…Από αντίστοιχες µετρήσεις για τον ανιχνευτή 15α οι συντελεστές µεταβλητότητας ήταν παρόµοιοι µε αυτούς που υπολογίστηκαν για τον 13α ενώ το όριο ανίχνευσης από δεδοµένα πόλωσης ήταν 5 ng/ml, το οποίο ήταν χαµηλότερο ή συµφωνούσε µε ήδη δηµοσιευµένα αποτελέσµατα [60,70,96,116]. ΟΡΙΑ ΑΝΙΧΝΕΥΣΗΣ (Y 0 -3SD) 1ος τρόπος: (α) από mP n : 0,8 ng/ml (β) από *X n : 0,8 ng/ml 2ος τρόπος:…”