Principle of test

  The Chloramphenicol (CAP) ELISA Test Kit is using indirect competitive enzyme immunoassay method.

The CAP coupling antigen has been coated in the plate wells. During the detection, add Standards or samples to the well, CAP of samples will compete with coated CAP coupling antigen to combine with antibody, after adding antibody working solution, Horseradish Peroxidase (HRP), take coloration with TMB substrate. The samples A value is an inverse relationship with CAP residue content. Lastly, compared with the standard curve can be concluded the CAP residues in sample.

Technique Data

 Kit sensitivity: 0.025 ppb (ng/ml)

 reactive mode: 25℃, 30min～30min～15min

 Detection Limits: 

Sample	Detection Limits
Tissue, Liver, Honey, Milk	0.0125 ppb
Eggs	0.05 ppb
Urine, Serum, Casing, Feed, Milk powder	0.025 ppb

Cross-reaction rate:

Name	Cross-reaction rate
Chloramphenicol	100%
Thiamphenicol	＜0.1%
Florfenicol	＜0.1%

Sample Recovery rate:

Sample	Recovery rate
Tissue, Liver	85% ± 20%
Honey, Casing	85% ± 25%
Milk, Feed	75% ± 25%
Urine, Serum	70% ± 20%

Composition of the Kit

Reagent	Quantity
Microelisa Stripplate	8 well × 12 strips
Standard: 0ppb, 0.025ppb, 0.075ppb, 0.225ppb, 0.675ppb, 2.025ppb	1×1.0ml
High standard 100 ppb (red cap)	1×1.0ml
Antibody working solution (blue cap)	1×5.5ml
Enzyme conjugate (red cap)	1×11ml
Substrate A solution (white cap)	1×6ml
Substrate B solution (black cap)	1×6ml
Stop Solution (yellow cap)	1×6ml
concentrated Wash Solution 20×	1×40ml
concentrated Redissolving solution 2× (yellow cap)	1×50ml
Instructions	1
Adhesive Membrane	1
Sealed bag	1

Materials required but not supplied

Equipments: microplate reader, printer, mixer or stomacher, nitrogen-drying device, oscillator, centrifuge, measuring pipets, and balance with a reciprocal sensibility of 0.01g;

Micropipettors: single-channel 20 to 200µl and 100 to1000µl, and multi-channel 300µl;

Reagents: ethyl acetate, N-hexane, acetonitrile, NaAc, acetic acid, K₂Fe(CN)₅(NO)▪2H₂O, glucuronidase , ZnSO₄•7H2O .

Sample pre-treatment

    1.Instructions  

    Labware must be clean and the use of disposable pipette tips to avoid contamination of interference results 

  2.Solution preparation before sample pre-treatment: 

Liquor 1:  0.36 M solution of K₂Fe(CN)₅(NO)▪2H₂O (Milk, Milk powder samples)

Take 11.9g K₂Fe(CN)₅(NO)▪2H₂O into deionized water to 100ml.

Liquor 2:  1.04 M solution of ZnSO₄•7H2O (Milk, Milk powder samples)

Take 29.8g ZnSO₄•7H2O into deionized water to 100ml.

Liquor 3:  0.1 M PH4.8 buffer solution of NaAc (Urine samples)

Take 2.4g NaAc into deionized water to 500ml, then add 1.2ml acetic acid, mix.

Liquor 4:  acetonitrile-water solution

V acetonitrile: V water solution=84:16

Liquor 5:  redissolving solution: (If the test sample is water, please don’t dilute, use the concentrated redissolving solution directly)

2 times dilute the2× concentrated redissolving solution with deionized water to be used for sample redissolving, it can be stored at 4 ℃ environment up to a month. 

 3.Sample pretreatment step:

3.1 tissue, fish, shrimp, liver samples:

Weigh 3.0±0.05g Homogeneous sample into 50ml centrifuge tube

Add 3ml deionized water, mix with vortex, then add 6ml ethyl acetate, mix with vortex for 2min, centrifuge at 4000 r/min at room temperature (20 - 25 ℃) for 10 min

Wipe out 2.5ml supernatant to another centrifuge tube and blow dry at 50 to 60 ℃ with nitrogen or air.

Add 1ml N-hexane to dissolve the dried residue, and then add 1 ml redissolving solution, oscillate 30s, centrifuge at 4000 r/min at room temperature for 5 min.

Wipe out the upper N-hexane; take 50µl Lower water phase to be analyses.

dilution times of the sample: 0.5  detection Limits: 0.0125ppb

3.2 Serum, Plasma samples:

Take 1 ml serum or plasma into centrifuge tube, add 2ml ethyl acetate, oscillate 1min, then centrifuge at 4000 r/min at room temperature for 5 min or place naturally to separate the Water and organic phase.

Wipe out all supernatant to another centrifuge tube and blow dry at 50 to 60℃ with nitrogen or air.

Add 1ml N-hexane to dissolve the dried residue, and then add 1 ml redissolving solution, oscillate 30s, centrifuge at 4000 r/min at room temperature for 5 min.

Wipe out the upper N-hexane; take 50µl Lower water phase to be analyses.

dilution times of the sample: 1  detection Limits: 0.025 ppb

3.3 Urine sample:

Take 2 ml urine into centrifuge tube , add 0.1 M PH4.8 buffer solution of NaAc 0.5ml , mix, then add 40µl glucuronidase, mix, hydrolyze at 37℃ at above 2h (or stay overnight )

Return the solution to room temperature, add 8 ml ethyl acetate, oscillate 1min, centrifuge at 4000 r/min at room temperature for 10 min

Wipe out 4 ml supernatant to another centrifuge tube and blow dry at 50 to 60℃ with nitrogen or air.

Add 1 ml redissolving solution to dissolve the dried residue, mix.

Use 50µl for the assay.

dilution times of the sample:0.1  detection Limits: 0.025ppb

3.4 Honey sample:

Weigh 2.0 ± 0.05g Homogeneous samples into centrifuge tube, add 4 ml deionized water to dissolve, and then add 4ml ethyl acetate, oscillate 1min, centrifuge at 4000 r/min at room temperature for 10min

Wipe out 2ml supernatant to another centrifuge tube and blow dry at 50 to 60 ℃ with nitrogen or air

Add 0.5 ml redissolving solution to dissolve the dried residue, mix.

Use 50µl for the assay.

dilution times of the sample: 0.5  detection Limits: 0.0125 ppb

(Detection Limits is 0.0125 ppb, quantitate Limits is 0.05ppb, because of the disturbance in some samples, please take 0.05ppb as the positive decision value) 

3.5 Casing sample: 

Cleanout the casing

Weigh 1.0 ± 0.02g Homogeneous sample into 50ml centrifuge tube, add 10 ml ethyl acetate, oscillate 2 min, then centrifuge at 4000 r/min at room temperature for 10 min

Wipe out 5 ml supernatant to another centrifuge tube and blow dry at 50 to 60 ℃ with nitrogen or air

Add 1ml N-hexane to dissolve the dried residue, and then add 0.5 ml redissolving solution, oscillate 30s, centrifuge at 4000 r/min at room temperature for 5 min

Wipe out the upper N-hexane; take 50µl Lower water phase to be analyses.

dilution times of the sample: 1   detection Limits: 0.0025 ppb

3.6 Milk sample:

Centrifuge the milk sample at 4000 r/min at 15℃ for 10 min, wipe out the upper fat

Take 5 ml sample into 50ml centrifuge tube, add 250µl K₂Fe(CN)₅(NO)▪2H₂O solution (Liquor 1), oscillate 30s; then add 250µl ZnSO₄•7H2O solution (Liquor 2), oscillate 30s, centrifuge at 4000 r/min at 15℃ for 10 min

Take upper liquid 2.2ml (amount to 2ml milk) into another centrifuge tube, add 4ml ethyl acetate, mix and oscillate 2min, centrifuge at 4000 r/min at room temperature for 10 min

Take 2ml supernatant to blow dry at 50 to 60 ℃ with nitrogen or air

Add 0.5 ml redissolving solution to dissolve the dried residue, mix.

Use 50µl for the assay.

dilution times of the sample: 0.5  detection Limits: 0.0125 ppb

(Detection Limits is 0.0125 ppb, quantitate Limits is 0.025ppb, because of the disturbance in some samples, please take 0.075ppb as the positive decision value)

3.7

 Milk powder sample:

Weigh 2.0 ± 0.05g milk powder into 50 ml centrifuge tube, dissolve with 10 ml deionized water

Add 1 ml K₂Fe(CN)₅(NO)▪2H₂O solution (Liquor 1),and add 1 ml ZnSO₄•7H2O solution (Liquor 2), oscillate and mix,then centrifuge at 4000 r/min at 15℃ for 10 min

Take upper liquid 3.6 ml (amount to 0.6g milk powder) into another centrifuge tube, add 4ml ethyl acetate, mix and oscillate 5min, centrifuge at 4000 r/min at room temperature for 10 min

Take 4 ml supernatant to blow dry at 50 to 60 ℃ with nitrogen or air

Add 0.4 ml redissolving solution to dissolve the dried residue, mix.

Use 50µl for the assay.

dilution times of the sample: 1   detection Limits: 0.025 ppb

(Detection Limits is 0.05 ppb, quantitate Limits is 0.15ppb, because of the disturbance in some samples, please take 0.15ppb as the positive decision value)

3.8 Eggs sample:

Weigh 3.0 ± 0.05g Homogeneous eggs sample into 50ml centrifuge tube, add 9 ml acetonitrile-water solution (Liquor 4), oscillate 2 min, then centrifuge at 4000 r/min at 15℃ for 10 min

Take upper liquid 3 ml into another centrifuge tube, add 3ml deionized water

Then add 4.5 ml ethyl acetate, mix and oscillate 1 min, centrifuge at 4000 r/min at 15℃ for 10 min

Take all the supernatant to blow dry at 50 to 60 ℃ with nitrogen or air

Add 1ml N-hexane to dissolve the dried residues, then add 1 ml redissolving solution, oscillate 30s, centrifuge at 4000 r/min at room temperature for 5 min

Wipe out the upper N-hexane; take 50µl Lower water phase to be analyses.

dilution times of the sample: 2  detection Limits: 0.0.05 ppb

(Detection Limits is 0.05 ppb, quantitative Limits is 0.15 ppb)

3.9 Feed sample:

Weigh 2.0 ± 0.05g Homogeneous feed sample into 50ml centrifuge tube, add 2ml deionized water, then add 6 ml ethyl acetate, oscillate 2 min, centrifuge at 4000 r/min at 15℃ for 10 min

Take upper liquid 3 ml into another centrifuge tube, blow dry at 50 to 60 ℃ with nitrogen or air

Add 1ml N-hexane to dissolve the dried residues, and then add 1 ml redissolving solution, oscillate 30s, centrifuge at 4000 r/min at room temperature for 5 min

Wipe out the upper N-hexane; take 50µl Lower water phase to be analyses. 

dilution times of the sample: 1  detection Limits: 0.025 ppb

  3. 10 Water sample:

Take 0.5 ml clear water into a 1.5 ml centrifuge tube, add 0.5 ml 2× concentrated Redissolving solution, and oscillate 1min

Use 50µl for the assay.

dilution times of the sample: 2  detection Limits: 0.05 ppb

Enzyme-linked immune test steps

   Take out the microtiter plate and the reagents required from 4 ℃ cold storage environment , put it in a place with room temperature for over 30 min , The wash buffer would crystallize in refrigeration,in order to make the Wash Buffer dissolved thoroughly, it needs indoor temperature. Shake the reagent bottle before usage. Take out required quantity of microwell plates and frames. The remaining strips are stored in the foil bag and zip-locked closed. Store the remaining kit in the refrigerator (2-8°C).

   Before experiment :  dilute 40 ml of the concentrated washing buffer (20×concentrated) with the distilled or deionized water to 800ml (or just to the required volume) for using;
microwell plates and frames. The remaining strips are stored in the foil bag and zip-locked closed. Store the remaining kit in the refrigerator (2-8°C).

Step 1: Number: determine the number of well (samples and standards) to be used and store unused wells in 4 ℃. Every sample and standard must be parallel well (2well), and record their location

Step 2: Addition reaction: Add 50µl standard or sample into marked well, then add 50µl antibody working solution into each well

Step 3: Incubate: Cover with the adhesive Membrane, oscillate gently for 5s, avoid the light to incubate for 30 min at 25℃.

Step 4: Washing: Uncover the adhesive Membrane, discard liquid, pipette 250µl washing buffer to every well, still for 30s then drain, repeat 5 times, Pat dry with a blotting paper

Step 5: Addition Enzyme reaction: Add 100µl antibody working solution into each well; avoid the light to incubate for 30 min at 25℃.

Step 6: Washing: The same with Step 4

Step 7: Color: Pipette 50ul Substrate A solution , then pipette 50ul Substrate B solution to each well, oscillate gently for 5s, avoid the light preservation for 15 min at 25℃

Step 8: Stop the reaction: Pipette Stop Solution 50μl to each well, oscillate gently, stop the reaction (the blue change to yellow).

Step 9: Calculate: Read absorbance at 450nm with microplate reader (Recommend reading the OD value at the dual-wavelength 450/630nm).finish this step within 10min.

Interpretation of result

1. Calculate the percentage of absorbance value:

百分吸光度值（%）＝	A	×100%
	A0

A—the average (double wells) OD value of the sample or the standard solution;

A0—the average OD value of the 0 ppb standard solution.

2. Draw the standard curve and calculate

Take absorbance percentage of standards as Y-axis, the corresponding log of standards concentration (ppb) as X-axis.

Draw the standard semilog curves with X-axis and Y-axis.

Take absorbance percentage of samples substitute into standard curve, then can get the corresponding concentration from standard curve; last, Multiplied by the corresponding dilution times is the actual concentration of CAP of samples

It is more convenient for a large amount of samples to use professional analyzing software to calculate, this will be accurate and rapid. (Welcome to contact us for this software)

Attention

Before test, the reagents and samples should be balanced to room temperature (25℃).if below 25℃, it will lead to all the standard OD value is low

In washing process, the dry micro plate will lead to the non-linear standard curves and undesirable reproducibility, so continue to next step immediately after washing.

The reproducibility is largely determined by consistency of washing step, so please mix uniformly and wash thoroughly.

On Incubate step, cover micro plates with adhesive Membrane to avoid light.

Do not mix reagents with those from other lots

Substrate A/B solution is colorless, if not, please discard.

If absorbance value of 0ppb is below 0.5, it means that the reagent may be metamorphic.

Stop solution is corrosives, please avoid contact with skin.

Storage conditions

The kit shall be stored at [2-8 ℃]. Expiry date: 12 months

The opened Microelisa Stripplate can be stored at [2-8 ℃] and avoid damp. Use for at least 2 months.

 

Manufacturing Enterprise

Company name: Shenzhen Finder Biotech Co.,Ltd.

Address: F6, Block B, 1st Building, Senyang High Tech Park, 7th Rd, Gaoxinyuan West Area, Guangming New District, Shenzhen, Guangdong, China.

Tel: +86-0755-23499189

Fax: +86-0755-23499025

ZIP: 518132   

    Http://www.finderbio.com