Microsoft word - chloramphenicol.doc

ELISA IMMUNOASSAY FOR THE QUANTITATIVE DETECTION OF CHLORAMPHENICOL IN MILK, LIVER, EGGS AND HONEY I - INTRODUCTION
The Bio-X Diagnostics CHLORAMPHENICOL ELISA KIT is a direct competitive enzyme immunoassay for the quantitative analysis of Chloramphenicol in various matrices. The test is suitable for milk, liver, eggs and honey. Time requirement, analysis: about 2 h and 30 min Time requirement for sample preparation (10 samples): Milk 30 min Eggs, liver and honey 3 h Detection limit in buffer: 0.05 ppb Specificity cross-reactivity (%) Chloramphenicol 100 % Thiamphenicol < 0.1 % Chloramphenicol base < 0.1 % Precision: Intra-assay CV < 10% Inter-assay CV < 20% II – PRINCIPLE OF THE TEST
The assay is performed in plastic microwells, which have been pre-coated with goat anti-rabbit IgG. Chloramphenicol standard or sample, enzyme-marked chloramphenicol and rabbit anti-chloramphenicol antibodies are added to microwells. During the incubation, anti-chloramphenicol antibodies are bound by the immobilized IgG; free and enzyme marked chloramphenicol competes for the chloramphenicol antibodies binding sites. After allowing this reaction to proceed, the unbound material is removed in a washing step. The bound enzyme activity is determined by adding a fixed amount of colorimetric substrate, which develops a blue colour in the presence of peroxidase. The colour development is inversely proportional to the original chloramphenicol concentration in the sample, which is determined by reading off a calibration curve derived from standards of known chloramphenicol concentration. Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] III - COMPOSITION OF THE KIT
96 wells microtiter plate (twelve 8-wells strips) coated with antibodies to rabbit IgG. Chloramphenicol standard solutions, 5 vials containing 1 ml each: Anti-Chloramphenicol antibody, 1 vial containing 11 ml Peroxidase conjugated Chloramphenicol (CAP- HRP) 40X solution, Wash buffer 10x concentrate, 1 bottle containing 50 ml Conjugate diluent buffer, 1 vial containing 10 ml Plastic envelope for plate temporary storage Materials required but not provided: For sample preparation • Liver Samples: Ultrasonic Tissue Homogeneizer (Ultra-Turrax or similar equipment) SpeedVac or other system to dry out solvent from tubes 50, 100 and 200 µl precision micropipettes Microtiter plate spectrophotometer equiped with 450 nm filter and OD range capability 1.0-3.0.
IV – PREPARATION OF WORKING SOLUTIONS
Chloramphenicol standard solutions : Ready-to-use Conjugate CAP-peroxidase (40X): Dilute 1:40 with Conjugate Diluent Buffer (i.e. 50 µl CAP-HRP+ 1950 µl diluent buffer). “PLEASE NOTE” the diluted enzyme has a limited stability, therefore it is advisable to prepare the required amount immediately before the use. After dilution, mix the conjugate solution gently, DO NOT VORTEX. Anti-Chloramphenicol antibody: Ready-to-use. Washing buffer: dilute 1:10 with distilled water (i.e. 1 ml + 9 ml). Substrate Solution: dilute the chromogen 1:10 with citrate buffer (i.e. 1 ml + 9 ml). This solution should be prepared in the required amount immediately before use. Chromogen and Substrate Solution are light sensitive: protect them from direct light. Stop Solution: Ready-to-use. Contains sulfuric acid: handle with care and in case of contact wash thoroughly with tap water. Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] • Add 50 µl of CAP-HRP to each well except the Blank wells. • Add 100 µl of anti-chloramphenicol antibody to each well except the Blank wells. This operation should be completed quickly: the use of multichannel pipette is strongly recommended. When samples have been diluted prior to assay (par.5), the concentrations in ng/ml read from the standard • Gently shake the plate with a rotatory motion. curve must be multiplied by the respective dilution factor to obtain the effective chloramphenicol • Cover the plate with the sealing tape. concentration in samples expressed in ppb (ng/ml or ng/g). The dilution factors are 1 for milk, 2 for liver, • Incubate 2 hours at room temperature (20-25 ºC), protected from direct light • At the end of incubation period, empty all the wells by inverting the plate. VIII - CUT-OFF/RESULT INTERPRETATION
Fill completely all the wells with diluted wash buffer; empty wells by inverting the plate (hold firmly the The biological matrix determines a background absorbance in every immunoassay. The non-specific plate plastic frame to prevent strips falling out of the frame); this operation should be done quickly, inhibition, the inhibition observed in analyte-free sample, is the expression of such a background, also called vigorously and without fear of crosscontaminations: the use of a squeeze bottle is recommended to quickly fill wells with wash buffer. Repeat the washing procedure 4 additional times (for a total of 5 times). Remove As a consequence of the “matrix-effect”, the detection limit (D.L.) has been calculated analyzing blank the residual buffer by vigorously tapping the plate on absorbent laboratory towels. Warning: the reproducibility of this enzyme immunoassay strongly depends on the uniform washing of the The DL (or cut-off value) is calculated as the mean blank values minus 2 standard deviations. wells. Follow carefully the washing procedure as described. The suggested cut-off values in different matrices are the following: • Prepare the Substrate Solution as described (par.4) by diluting 1 volume of chromogen solution with 9 • Using a multichannel micropipette, add 200 µl of Substrate Solution to each well including the Blank The application of these cut-off values allows to avoid false negatives in the screening, and to minimize the • Incubate 30 minutes at room temperature protected from light IX - NOTES
After incubation, add 50 µl stop solution to each well with a multichannel micropipette. Mix thoroughly, measure the absorbance by using a microplate reader fitted with a 450 nm filter and blanked against air. Read • Store the kit at 4-6 ºC and never freeze. absorbance within 1 h after addition of the Stop Solution. • Once open, if properly stored (dry resealed, at 4-8 ºC), the microwell plate will be stable for several VII CALCULATION
• Keep CAP-HRP concentrate (40X) vial in the dark, and don’t leave it at room temperature but store it at 4- • Rabbit liver samples cannot be assayed because of the presence of antibodies to rabbit IgG in the wells. The unknown values for chloramphenicol concentration in samples are determined on the basis of the • Positive results obtained with the immunoassay should be confirmed with analytical methods. - calculate the mean absorbance value for Blank and subtract it from the absorbance value of all the wells. - calculate the mean absorbance value for the Maximum Binding, the standards and the samples. - divide the mean absorbance value of standards and samples (B) by the mean absorbance value of the Binding (Bo) and multiply by 100. Maximum binding is thus made equal to 100% and the absorbance values ------------------------------------------------- x 100 = ----- (%) - enter the B/Bo(%) values calculated for each standard in a semi-logarithmic system of coordinates against the standard chloramphenicol concentration; draw the standard curve. - take the B/Bo(%) value for each sample and interpolate the corresponding concentration from the Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] V – PREPARATION OF SAMPLES
X-Calibration curve of Chloramphenicol ELISA Kit
Whole milk: centrifuge 15' at 13000 g, room temperature (RT) , use the defatted milk directly in the test carefully avoiding to collect the fat. Weigh 2 g of minced tissue in a glass centrifuge tube Homogenize by Ultrasonic Tissue Homogeneizer (few seconds, 20.000 rpm) Transfer 1 ml of supernatant in a clean glass tube, dry out solvent by evaporation (under nitrogen or dry air flow, at 40-50 ºC) Resuspend in 250 µl of distilled water, vortex and use 50 µl/well in the assay 5.C – Eggs (yolk or whole egg) and honey (dilution factor 2) - Weigh 2 g of sample in a glass centrifuge tube Take 1 ml supernatant, transfer into a clean glass tube and dry out solvent by evaporation (under nitrogen or dry air flow, at 40-50 ºC); Resuspend in 500 µl of distilled water, vortex and use 50 µl/well in the assay. VI – IMMUNOASSAY PROCEDURE
Dilute the conjugate CAP-HRP in the amount required for the test and immediately store the concentrated solution at 4- 6ºC. Allow all the other reagents to reach room temperature (23-25ºC) at least for 1 hour. Once started, complete all assay steps without interruption. 6.A- Recommended assay layout Maximum Binding, blank, standards and each sample must be assayed at least in duplicate wells. 6.B- Assay implementation Determine the number of wells required to test the desired number of samples plus appropriate number of wells needed to run standard curve, controls and blanks. Remove sufficient strips from the aluminum pouch immediately before the use. Return any unused wells to the pouch together with desiccant gel and seal tightly. For short time storage, place the remaining strips in the plastic envelope provided. • Use a single disposable tip for each pipetting step to avoid cross contamination. • Avoid tips from scratching the microwells surface or touching other reagents already present in the wells. • Place 200 µl of distilled water into the Blank wells. • Place 50 µl of distilled water into the Maximum Binding wells. • Place 50 µl of each standard into the standard wells. • Place 50 µl of each sample into the sample wells. Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Bio-X Diagnostics – Site du Complexe des Postes – 22, rue J. Wauters – 5580 Jemelle – Belgique Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected] Tél : 0032(0)84.32.23.77 - Fax : 0032(0)84.31.52.63 E-mail : [email protected]

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