Elisa Test Kit for Cephalosporins

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Appearance: Liquid
• Model NO.: KA11001H
• Transport Package: Foam Box with Ice Bag
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000
• Production Capacity: 50000PCS/Year

Product Description

Product Description

 

Competitive Enzyme Immunoassay Kit for 
Quantitative Analysis of Cephalosporins

1. Background
The cephalosporins are a class of β-lactam antibiotics originally derived from Acremonium, which was previously known as "Cephalosporium". Together with cephamycins they constitute a subgroup of β-lactam antibiotics called cephems.
The ELISA kit is a new product based on ELISA technology, which is fast, easy, accurate and sensitive compared with common instrumental analysis and only needs 1.5h in one run, so it can considerably minimize operation error and work intensity.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Cephalosporins residue in the sample competes with the antigen coated on the microtiter plate for the antibody. After the addition of enzyme conjugate, TMB substrate is used to show the color. Absorbance of the sample is negatively related to the cephalosporins in it, after comparing with the Standard Curve, multiplied by the dilution factor, cephalosporins quantity in the sample can be calculated.
3. Applications
This kit can be used in quantitative and qualitative analysis of cephalosporins in animal tissue (pork, chicken, beef, fish and shrimp) and milk.
4. Cross-reactions
Ceftiofur............…....................100%
Ceftriaxone......................….....169%
Cefotaxime........................…...63%
Cefazolin.........................….…<0.1%
5. Materials Required
5.1 Equipments
----Microtiter plate spectrophotometer (450nm/630nm)
----Rotary evaporator or nitrogen gas drying system
----Homogenizer
----Shaker
----Vortex mixer
----Centrifuge
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Polystyrene centrifuge tubes: 2ml, 50ml
----Glass test tube: 10ml
----Volumetric flask: 100ml, 500ml
----Micropipettes: 20ul-200ul,100ul-1000ul,
250ul-multipipette
5.2 Reagents
----Concentrated hydrochloric acid (HCl, AR)
----Acetonitrile (AR)
----N-hexane (AR)
----Deionized water
6. Kit Components

1. Microtiter plate with 96 wells coated with antigen
2. Ceftiofur standard solutions. (1ml×6 bottles)

0 ppb, 0.5ppb, 1.5ppb, 4.5ppb, 13.5ppb, 40.5 ppb

1. Spiking standard solution: 1ml, 1ppm
2. Enzyme conjugate (12ml)….…..…......…...red cap
3. Antibody solution (7ml)..............green cap
4. Solution A (7ml) ................…..white cap
5. Solution B (7ml) ...............…..…red cap
6. Stop solution (7ml) ...............yellow cap
7. 20×Concentrated wash solution (40ml)

........................ transparent cap

1. 2×Concentrated extraction solution (50ml)

 ...........................… blue cap
7. Reagents Preparation
Solution 1: 0.05M HCl
Dilute 2.1ml of concentrated HCl with water to 500ml.
Solution 2: Sample extraction buffer
Mix 80ml of acetonitrile with 20ml of 0.05M HCl solution.
Solution 3: Extraction solution
Dilute the 2×concentrated extraction solution with deionized water in the volume ratio of 1:1(e.g. 10ml of 2×extraction solution + 10ml of deionized water), which will be used for sample extraction, this solution can be stored at 4ºC for 1 month.
Solution 4: Wash solution
Dilute the 20×concentrated wash solution with deionized water in the volume ratio of 1:19(e.g. 5ml of 20×wash solution + 95ml of deionized water), which will be used for washing the plates. This solution can be stored at 4ºC for 1 month.
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 absorbing different reagent.
(b) Make sure that all experimental instruments are clean.
8.2 Tissue (pork, chicken, beef, fish and shrimp):
----Take 2.0±0.05g of homogenized tissue sample into a 50ml tube, then add 8ml of sample extraction buffer (solution 2), shake for 5min, and then centrifuge for separation: 3000g / ambient temperature / 5min..  
----Transfer 1ml of the supernate into a 10ml clean glass tube, dry with 50-60ºC water bath under nitrogen gas stream.
----Add 1ml of n-hexane, vortex for 30s, then add 1ml of extraction solution (solution 3), vortex for 30s to dissolve completely, centrifuge for separation: 3000g / ambient temperature / 5min..
----Remove the upper n-hexane layer, and take 50µl of the lower aqueous layer per well for assay.

Dilution factor:       4

8.2 Milk
----Take 100µl of raw milk into 2ml tube, and add 900µl of extraction solution (solution 3), then mix completely.
----Take 50µl per well for assay.

Dilution factor:       10

9. Assay process
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 The diluted wash solution should be rewarmed to be at room temperature before use.
9.2.4 Number: Numbered every microwell positions and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.5 Add standard solution/sample and antibody solution: Add 50µl of standard solution(Kit provided) or prepared sample to corresponding wells. Add 50µl of antibody solution(Kit provided). Mix gently by rocking the plate manually and incubate for 30min at 4-8ºC with cover.
9.2.6 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl diluted wash solution (solution 4) at interval of 10s for 4-5 times. Absorb the residual water with absorbent paper (the rest air bubble can be eliminated with unused tip).
9.2.7 Add Enzyme conjugate: Add 100μl of enzyme conjugate(Kit provided) to each well, Mix gently by rocking the plate manually and incubate for 30min at 25ºC with cover. Repeat the wash step again.
9.2.8 Coloration: Add 50µl solution A(Kit provided) and 50µl solution B(Kit provided) to each well. Mix gently by rocking the plate manually and incubate for 15min at 25ºC with cover(see 12.8).
9.2.9 Measure: Add 50µl of stop solution(Kit provided) to each well. Mix gently by rocking 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 cefitiofur standards solution (ppb) as x-axis.
----The cephalosporins 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 evaluation of the result, special software has been developed, which can be provided on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.5ppb
Detection limit
Animal tissue...............….......….2ppb
Milk........................….....…5ppb
Accuracy
Tissue(pork, beef, chicken)........….......80±20%
Tissue(fish and shrimp)...….............…80±20%
Milk.......................….......…100±20%
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 reproducibility and operate the next step immediately after tap the microwells holder.
12.3. Shake each reagent gently before use.
12.4. Keep your skin away from the stop solution for it is the 0.5M H₂SO₄ solution.
12.5 Don't use the kits out of date. Don't exchange the reagents of different batches, or else it will drop the sensitivity.
12.6 Keep the ELISA kits at 2-8ºC,do not freeze. Seal rest microwell plates, Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended.
12.7 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(A450nm<0.5).
12.8 The coloration reaction needs 15min after the addition of solution A and solution B. And you can prolong the incubation time from 20min to more if the color is too light to be determined. Never exceed 25min, on the contrary, shorten the incubation time properly.
13. Storage
    Storage condition: 2-8ºC.
Storage period:  12 months

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