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Product Description
Competitive enzyme immunoassay Kit for
Quantitative analysis of Chloramphenicol
1. Background
Chloramphenicol (CAP) is a broad spectrum antibiotic which is frequently employed in animal production for its excellent antibacterial and pharmacokinetic properties. However, in humans it leads to hematotoxic side effects. It can cause aplastic anaemia and agranulocytopenia, thus it has been banned or restrictively used in EU and US.
This kit is a new product based on ELISA, which is fast (only 50min in one operation), easy, accurate and sensitive compared with common instrumental analysis, and so it can considerably minimize operation error and work intensity.
2. Test Principle
This ELISA kit is designed to detect chloramphenicol based on the principle of "direct-competitive" enzyme immunoassay. The microtiter wells are coated with coupling antigen. Chloramphenicol in the sample competes with the coating antigen for binding to the limited number of antibody added. After the addition of a ready-to-use TMB substrate the signal is measured in an ELISA reader. The absorption is inversely proportional to the chloramphenicol concentration in the sample.
3. Applications
This kit can be used in quantitative and qualitative analysis of CAP residue in animal tissue (muscle, liver fish & shrimp), cooked meat,honey, royal jelly and egg.
4. Cross-reactions
Chloramphenicol..................….....100%
Chloramphenicol palmitate.........…......…<0.1%
Thiamphenicol..................….......<0.1%
Florfenicol...........................<0.1%
Cetofenicol.....................….....<0.1%
5. Materials Required
5.1 Equipments
----Microtiter plate spectrophotometer (450nm/630nm)
----Rotary evaporator / Nitrogen drying instrument
----Homogenizer / stomacher
----Shaker
----Vortex mixer
----Centrifuge
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Volumetric flask: 100ml, 500ml
----Glass test tube: 10ml
----Polystyrene Centrifuge tube: 2ml, 50ml
----Micropipettes: 20μl-200μl, 100μl-1000μl
250μl- multipipette
5.2 Reagents
----Ethyl acetate (AR)
----n-hexane (AR)
----Acetonitrile (AR)
----Alumina B (AR, for royal jelly)
----Deionized water
6. Kit Components
- Microtiter plate coated with antigen, 96wells
- Standard solutions (6×1ml/bottle)
0ppb,0.025ppb,0.075ppb,0.3ppb,1.2ppb,4.8ppb
- Spiking standard solution: (1ml/bottle) ...…100ppb
- Concentrated enzyme conjugate 1ml…..….red cap
- Enzyme conjugate diluent 10ml..................green cap
- Solution A 7ml...........….......white cap
- Solution B 7ml.........…......…...…red cap
- Stop solution 7ml.........…....…yellow cap
- 20×Concentrated wash solution 40ml
.....................….…transparent cap
- 2×Concentrated extraction solution 50ml......blue cap
7. Reagents Preparation
Solution 1: Ethyl acetate- Acetonitrile solution
Mix 70ml of ethyl acetate and 30ml of acetonitrile completely;
Solution 2: Extraction solution:
Dilute the 2×concentrated extraction solution with deionized water in the volume ratio of 1:1(e.g. 10ml of 2×concentrated extraction solution + 10ml of deionized water), which will be used for dissolving the extracted sample. The diluted extraction solution can be conserved for 1 month at 4ºC.
Solution 3: Wash solution:
Dilute the 20×concentrated wash solution with deionized water in the volume ratio of 1:19(e.g. 5ml of 2×concentrated wash solution + 95ml of deionized water), which will be used for dissolving the extracted sample. The diluted extraction solution can be conserved for 1 month at 4ºC.
8. Sample Preparations
8.1 Notice and precautions before operation
(a) Please use one-off tips in the process of experiment, and change the tips when absorb different reagent.
(b) Make sure that all experimental tools are clean.
(c) If blank controls is taken, and then subtract the test result of the blank control from the test result of sample, the real quantity of CAP residue in sample is obtained.
Preparations of blank tissue sample:
----Dry 4ml of ethyl acetate completely with nitrogen gas flow;
----Then dissolve the leftover with 1ml of n-hexane;
----Add 1ml of extraction solution (solution 2), vortex for 1ml fiercely, keep steady till the liquid layer separates, and then remove the supernatant n-hexane phase;
----Take 50μl of the prepared solution for assay.
(d) Keep untreated sample in freeze.
(e) Treated samples can be stored at 2-8ºC for 24h, it's better to keep the dry samples (before adding n-hexane).
(f) Remove part of the supernatant organic phase and keep in 60ºC water bath for 5-10min to eliminate the emulsion if the treated sample become emulsified, and then repeat the centrifuge step.
8.2 Tissue (chicken/chicken liver, pork/pig liver, shrimp and fish etc.)
----Homogenize the tissue sample with homogenizer;
----Weigh 3.0±0.05g homogenate into a 50ml polystyrene centrifuge tube, then add 6ml of ethyl acetate, shake for 5min with shaker;
----Centrifuge for separation: 3000g / 5min / at room temperature (20-25ºC).
----Transfer 4ml of the supernatant organic phase (corresponding to 2g sample) into a 10ml clean glass test tube, dry with 50-60ºC water bath under nitrogen gas flow;
----Add 1ml of n-hexane, vortex for 1min with vortex mixer, then add 1ml of extraction solution (solution 2), vortex for 30s;
----Centrifuge for separation: 3000g / 5min / at room temperature (20-25ºC);
----Remove the supernatant organic phase, and take 50ml of the substrate phase for assay;
Notice: Add 1ml of n-hexane to dissolve the dry leftover when animal liver sample is detected, then add 1ml of extraction solution, shake manually for 10s, centrifuge and take the substrate phase for assay; Increase the volume of n-hexane to 2ml when sample high in fat is tested.
Dilution Factor: 0.5
8.3 Cooked meat
----Homogenize the sample with homogenizer;
----Weigh 3.0±0.05g homogenate in to a 50ml polystyrene centrifuge tube, then add 3ml of deionized water, 6ml of ethyl acetate, shake for 10min with shaker;
----Centrifuge for separation: 3000g / 10min / at room temperature (20-25ºC);
----Transfer 4ml of the supernatant organic phase (corresponding to 2g sample) into a 10ml clean glass test tube, dry with 50-60ºC water bath under nitrogen gas flow;
----Add 1ml of n-hexane, vortex for 1min with vortex mixer, then add 1ml of extraction solution (solution 2), vortex for 15s;
----Centrifuge for separation: 3000g / 5min / at room temperature (20-25ºC);
----Remove the supernatant organic phase, take 50μl of the substrate phase for assay;
Dilution Factor: 0.5
8.2 Honey:
----Weigh 2.0±0.05g of honey sample into a 50ml polystyrene centrifuge tube;
----Add 4ml of deionized water, shake with shaker to dissolve the honey completely;
----Add 4ml of ethyl acetate, shake manually up and down for 10min, then centrifuge: 3000g, 20-25ºC, 10min.
----Transfer 2ml of organic supernate(corresponding to 1g of sample ) to 10ml dry clean glass tube, dry under 50-60ºC nitrogen water bath flow.;
----Add 0.5ml of extraction solution (solution 2), vortex for 2min to dissolve the dry leftover;
----Take 50µl of the prepared solution for assay.
Dilution factor: 0.5
8.5 Royal jelly
----Weigh 1.0±0.01g royal jelly sample into a 50ml polystyrene centrifuge tube;
----Add 3.0±0.01g ml of Alumina B to the centrifuge tube;
----Add 1ml of deionized water, then add 8ml of e Ethyl acetate- Acetonitrile solution(solution 1), shake for 10min;
----Centrifuge for separation: 3000g / 5min / at room temperature (20-25ºC);
----Take 2ml(corresponding to 2g samples) of the supernatant organic phase into a 10ml glass tube, dry with 50-60ºC water bath under nitrogen gas flow;
----Add 0.5ml extraction solution (solution 2), vortex for 2min to dissolve the dry leftover;
----Take 50μl of the prepared solution for assay.
Dilution Factor: 2
8.7 Egg
----Homogenize the egg sample with homogenizer;
----Weigh 1.0±0.05g homogenate into a 50ml polystyrene centrifuge tube;
----Add 8ml of ethyl acetate;
----Centrifuge for separation: 3000g / 5min / room temperature (20-25ºC);
----Take 4ml of the supernatant organic phase (corresponding 0.5g sample) into a 10ml dry clean glass tube, dry with 50-60ºC water bath under nitrogen gas flow;
----Add 1ml of n-hexane, vortex for 1min, then add 1ml of extraction solution (solution 2), vortex for 15s to dissolve the dry leftover;
----Centrifuge for separation: 3000g / 5min / room temperature (20-25ºC);
----Remove the supernate organic phase, take 50μl of the lower layer solution for assay.
Dilution Factor: 2
9 Assay Steps
9.1 Notice before assay
9.1.1 Make sure all reagents and microwells are all at room temperature (20-25ºC).
9.1.2 Return all the rest reagents to 2-8ºC immediately after used.
9.1.3 Washing the microwells correctly is an important step in the process of assay; it is the vital factor to the reproducibility of the ELISA analysis.
9.1.4 Avoid the light and cover the microwells during incubation.
9.2 Assay Steps
9.2.1 Take all reagents out at room temperature (20-25ºC) for more than 30min, homogenize before use.
9.2.2 Get the microwells needed out and return the rest into the zip-lock bag at 2-8ºC immediately.
9.2.3 All reagents should be rewarmed before use.
9.2.4 Number: Number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.5 Add standard solution / sample: Add 50µl of standard solution(kit insert) or prepared sample to corresponding wells.
9.2.6 Dilute the concentrated enzyme conjugate :Dilute Concentrated enzyme conjugate(kit insert) with enzyme conjugate diluent(kit insert) in the volume ratio of 1: 10(e.g. 0.5ml of the concentrated enzyme conjugate + 5ml of enzyme conjugate diluent ), which should be used immediately.
9.2.7 Add the mixture of enzyme conjugate: Add 50ul of the mixture of the enzyme conjugate per well, Mix gently by shaking the plate manually and incubate for 30min 25ºC with cover.
9.2.8 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl of wash solution(solution 3) at interval of 10s for 5-6 times. Absorb the residual water with absorbent paper (eliminate the air bubble with unused tips).
9.2.9 Coloration: Add 50µl of solution A(kit insert) and 50µl of solution B(kit insert) to each well. Mix gently by shaking the plate manually and incubate for 20 min at 25ºC with cover (see 12.8).
9.2.10 Measure: Add 50µl of the stop solution(kit insert) to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm (It's suggested measure with the dual-wavelength of 450/630nm. Read the result within 5min after addition of stop solution.).
10. Results
10.1 Percentage absorbance
The mean values of the absorbance values obtained for the standards and the samples are divided by the absorbance value of the first standard (zero standard ) and multiplied by 100%. The zero standard is thus made equal to 100% and the absorbance values are quoted in percentages.
B --absorbance standard (or sample)
B0 --absorbance zero standard
10.2 Standard Curve
To draw a standard curve: take the absorbance value of standards as y-axis, semi logarithmic of the concentration of the CAP standards solution (ppb) as x-axis.
The CAP concentration of each sample (ppb), which can be read from the calibration curve, is multiplied by the corresponding Dilution factor of each sample followed, and the actual concentration of sample is obtained.
Please notice:
For data analysis of the ELISA kits, special software has been developed, which can be ordered on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.025ppb
Detection limit
Tissue, cooked meat............….…..…0.025ppb
Cooked meat.....................…0.0125ppb
Honey...............….....…...…0.05ppb
Royal jelly........................0.2ppb
Egg.....................…....0.05ppb
Accuracy:
Tissue...........................100±30%
Cooked meat.....................…. 100±30%
Honey.........…...............…100±30%
Royal jelly...............…...... 100±30%
Egg........................…....95±30%
Precision
Variation coefficient of the ELISA kit is less than 10%.
12. Notice
12.1 The mean values of the absorbance values obtained for the standards and the samples will be reduced if the reagents and samples have not been regulated to room temperature (20-25ºC).
12.2 Do not allow microwells to dry between steps to avoid unsuccessful repetitiveness and operate the next step immediately after tap the microwells holder.
12.3 Shake each reagent gently before use.
12.4 Don't use the kits out of date. Don't exchange the reagents of different batches, or else it will drop the sensitivity.
12.5 Keep the ELISA kits at 2-8ºC, do not freeze. Seal rest microwell plates Avoid straight sunlight for the standard sample and the colorless chromogenic reagent are sensitive to light.
12.6 Substrate solution should be abandoned if it turns colors. The reagents may be turn bad if the absorbance value (450/630nm) of the zero standard is less than 0.5(OD450nm<0.5).
12.7 The coloration reaction needs 20min after adding Solution A and Solution B. And you can prolong the incubation time to 25min if the color is too light to be determined. Never exceed 30min, On the contrary, shorten the incubation time properly.
12.8 The optimal reaction temperature is 25ºC. Higher or lower temperature will lead to the changes of sensitivity and absorbance values.
13. Storage condition and storage period
Storage condition: 2-8ºC.
Storage period: 12 month
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