Chloramphenicol ELISA test Kit

Catalog No. LSY-10007

1. Principle

This test kit is based on the indirect competitive enzyme immunoassay for the detection of Chloramphenicol in the sample. The coupling antigen is pre-coated on the micro-well stripes. The Chloramphenicol in the sample and the coupling antigen pre-coated on the micro-well stripes compete for the anti-Chloramphenicol antibody. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with the Chloramphenicol in it. This value is compared to the standard curve and the Chloramphenicol concentration is subsequently obtained.

 

2. Technical specifications

Sensitivity:  15 ppt

Incubation Temperature:  25℃

Incubation Time:  30min～15min

Detection limit:

Tissue/Aquatic(method 1)    7.5ppt

Tissue/Aquatic(method 2), egg, honey   15ppt

Milk, milk powder    50ppt

Feed    200ppt

Recovery rate

Tissue,Aquatic, egg   95±25%

Feed   90±30%

Milk, milk powder, honey   100±30%

Cross-reaction rate:

Chloramphenicol   100%

Thiamphenicol  < 0.1%

Florfeniol   < 0.1%

 

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	7× standard solution (1mL each)	0ppt	15ppt
		45ppt	135ppt
		405ppt	1215ppt  10ppb
3	Enzyme conjugate	7ml	red cap
4	Antibody working solution	7ml	blue cap
5	Substrate A	7ml	white cap
6	Substrate B	7ml	black cap
7	Stop solution	7ml	yellow cap
8	20× concentrated washing buffer	15ml	white cap
9	2×concentrated redissolving solution	50ml	transparent cap
10	Sample extracting buffer	50ml	yellow cap

 

4. Materials required but not provided

1)    Equipments: microplate reader, homogenizer, nitrogen-drying device, vortex, centrifuge, measuring pipets, and balance( a sensibility reciprocal of 0.01 g), incubator.

2)      Micropipettors: single-channel 20-200 µL, 100-1000 µL, and multi-channel 30～300 μl.

3)    Reagents: Ethyl acetate, N-hexane, NaCl, H₂O.

 

5. Sample pre-treatment

Instructions

The following points must be dealt with before the pre-treatment of any kind of sample:

1)  Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;

2)  Before the experiment, each experimental equipment must be checked to be clean and should be re-cleaned if necessary, in order to avoid the contamination which interferes with the experimental results.

Solution preparation before sample pre-treatment

1)    Sample redissolving solution: the 2×concentrated redissolving solution is diluted with deionized water at 1:1.

5.1 Tissue (Chicken, duck, pork, fish, shrimp, beef, lamb) Method 1

1.    Take 3± 0.05 g of the homogenized sample into a 50ml centrifuge tube. Firstly add 3 mL deionized water, then add 6mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

2.    Take 4mL of the supernatant, blow to dry by nitrogen in 50-60℃.

3.    Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature (20-25℃) for 10 min, remove the up-layer organic phase.

4.    Take 50 µL of the down-layer for analysis.

Fold of dilution of the sample: 0.5

(If cannot take 4ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 9ml ethyl acetate then take 6ml supernatant blow to dry, the dilution factor is same)

5.2 Tissue (Chicken, duck, pork, fish, shrimp, beef, lamb) Method 2

1.    Take 2± 0.05 g of the homogenized sample into a 50ml centrifuge tube. Firstly add 3 mL deionized water, then add 6mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

2.    Take 3mL of the supernatant, blow to dry by nitrogen in 50-60℃.

3.    Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min, remove the up-layer organic phase.

4.    Take 50 µL of the down-layer for analysis.

Fold of dilution of the sample: 1

(If cannot take 3ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 10ml ethyl acetate then take 5ml supernatant blow to dry, the dilution factor is same)

5.3 Other tissue samples with high fat content (kidney, liver, intestine, skin, heart, pork belly, etc.)

1.    Take 2± 0.05 g of the homogenized sample into a 50ml centrifuge tube. Add 10mL N-hexane, shake for 3 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min, discard N-hexane.

2.    Add 6mL ethyl acetate, shake for 1min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

3.    Take 3mL of the supernatant, blow to dry by nitrogen in 50-60℃.

4.    Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min, remove the up-layer organic phase.

5.    Take 50 µL of the down-layer for analysis.

Fold of dilution of the sample: 1

5.4 Egg

1.    Take 2± 0.05 g of the fresh homogenized sample into a 50ml centrifuge tube. Add 6mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

2.    Take 3mL of the supernatant, blow to dry by nitrogen in 50-60℃.

3.    Dissolve the dry residues in 1 mL N-hexane, add 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min, remove the up-layer organic phase.

4.    Take 50 µL of the down-layer for analysis.

Fold of dilution of the sample: 1

(If cannot take 3ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 10ml ethyl acetate then take 5ml supernatant blow to dry, the dilution factor is same)

5.5 Feed

1. Take 1±0.05g of the homogenized sample into a 50ml centrifuge tube, firstly add 9 mL deionized water, then add 5mL ethyl acetate, shake properly for 3 min.

2. Centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

3. Take 5mL of the middle-layer water phase, transfer into another clean 50ml centrifuge tube, add 10mL ethyl acetate,shake properly for 10S.

4. Centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

5. Take 5mL of the up-layer liquid into another clean centrifuge tube, blow to dry by nitrogen or air at 50℃.

6. Dissolve the dry residues in 2 mL N-hexane, add 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature (20-25℃) for 10 min, remove the up-layer organic phase.

4. Take 50 µL down-layer liquid for analysis.

Fold of dilution of the sample: 4     

5.6 Honey

1.    Take 2± 0.05g fresh homogenized honey sample into a 50mL centrifuge tube, add 2mL deionized water to dissolve, then add 6mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

2.    Take 3mL of the supernatant into a new centrifuge tube, blow to dry by nitrogen in 50-60℃.

3.    Add 0.5mL of the sample redissolving solution to dissolve the dry residues, mix for 30 seconds;

4.    Take 50 µL for analysis.

Fold of dilution of the sample: 0.5

5.7 Raw milk, pasteurized milk, sterilized milk, modified milk, reconstituted milk, fermented milk, milk beverages

1. Take 2±0.05ml fresh sample into a 50ml centrifuge tube, add 0.1mL Sample extracting buffer, then add 6mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.

2. Take 3mL of the up-layer liquid into a new centrifuge tube, blow to dry by nitrogen in 50-60℃.

3. Dissolve the dry residues in 1mL N-hexane, then add 1mL of the sample redissolving solution, mix for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25℃) for 10 min, discard up-layer organic phase;

4. Take 50 µL down-layer liquid for analysis.

Fold of dilution of the sample: 1

5.8 Milk powder

1. Take 2±0.05g fresh milk powder sample into a 50ml centrifuge tube, add 5mL deionized water, then add 0.5mL Sample extracting buffer, shake properly for 1 min;

2. Add 3g NaCl solid, then add 10mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min;

3. Take 5mL of the up-layer liquid into a new centrifuge tube, blow to dry by nitrogen in 50-60℃.

4. Dissolve the dry residues in 1 mL N-hexane, then add 1mL of the sample redissolving solution, mix for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min, discard up-layer organic phase;

5.    Take 50 µL down-layer liquid for analysis.

Fold of dilution of the sample: 1

 

6. ELISA procedures

6.1 Instructions

1       Bring all reagents and micro-well strips to the room temperature (20-25 ℃) before use;

2       Return all reagents to 2-8 ℃ immediately after use;

3       The reproducibility of the ELISA analysis, to a large degree, depends on the consistency of plate washing. The correct operation of plate washing is the key point in ELISA the procedures;

4       For the incubation at constant temperatures, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane.

6.2 Operation procedures

1.    Take out all the necessary reagents from the kit and place at the room temperature (20-25 ℃) for at least 30 min. Note that each reagent must be shaken to mix evenly before use.

2.    Take the required micro-well strips and plate frames. Re-sealed the unused microplate, store at 2-8℃, not frozen.

3.    Solution preparation: dilute 15 mL of the concentrated washing buffer (20 × concentrated) with the deionized water at 1:19 (1 part of 20X concentrated washing buffer + 19 parts of deionized water), or prepare as quantity needed.

4.    Numbering: number the micro-wells according to samples and standard solution; each sample and standard solution should be performed in duplicate, record their positions.

5.    Add 50 µL of the sample or standard solution to separate duplicate wells; then add 50 µL enzyme conjugate into each well, at last add 50 µL of antibody working solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, and incubate at 25 ℃ for 30 min.

6.    Pour the liquid, wash the microplate with the diluted washing buffer at 250 µL/well for 4-5 times. Each time soak the well with the washing buffer for 15-30 sec, flap to dry with absorbent paper (if there are the bubbles after flapping, cut them with the clean tips).

7.    Coloration: add 50 µL of the substrate A solution and then 50 µL of the B solution into each well. Mix gently by shaking the plate manually, and incubate at 25 ℃ for 15 min at dark for coloration.

8.    Determination: add 50 µL of the stop solution into each well. Mix gently by shaking the plate manually. Set the wavelength of microplate reader at 450 nm to determine the OD value. (recommend to read the OD value at the dual-wavelength 450/630 nm within 5 min).

 

7. Result judgment

There are two methods to judge the results; the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with the content of Chloramphenicol.

7.1 Qualitative determination

The concentration range (ng/mL) can be obtained from the comparison the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sampleⅠ is 0.3, and that of the sampleⅡ is 1.0, while those of the standard solutions are as the followings: 2.243 for 0ppt, 1.816 for 15ppt, 1.415for 45ppt, 0.74 for 135ppt, 0.313 for 405ppt and 0.155 for 1215ppt, accordingly the concentration range of the sampleⅠis 405 to 1215ppt, and that of the sampleⅡ is 45 to 135ppt.

7.2 Quantitative determination

The mean values of the absorbance values obtained for the average OD value (B) of the sample and the standard solution divided by the OD value (B₀) of the first standard solution (0 standard) and subsequently multiplied by 100%, that is,

 

       B—the average OD value of the sample or the standard solution

       B₀—the average OD value of the 0ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solution and the semilogarithm values of the Chloramphenicol standard solution (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, thus finally obtaining  the Chloramphenicol concentration in the sample.

Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software) .

 

8. Precautions

1.    The room temperature below 25 ℃ or the temperature of the reagents and the samples being not returned to the room temperature (20-25 ℃) will lead to a lower standard OD value.

2.    Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility.

3.    Mix every reagent and reaction mixture evenly and wash the microplate thoroughly, otherwise there will be the undesirable reproducibility.

4.    The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin;

5.    Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colourless color former is light sensitive, and thus they cannot be directly exposed to the light.

6.    Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use.

7.    Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value of the standard solution 1 (0 ppb) of less than 0.5 indicates its degeneration.

8.    The optimum reaction temperature is 25 ℃, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.

 

9. Storage and expiry date

Storage: store at 2-8 ℃, not frozen.

Expiry date: 12 months; date of production is on the box.

Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.

Shenzhen Lvshiyuan Biotechnology Co.,Ltd

D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China

Tel. 86-755-28438788

Fax 86-755-28938800

Email:   info@lsybt.com 

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