Product Description
Competitive enzyme immunoassay Kit for
Quantitative analysis of ß-fructofuranosidase
1. Introduction
ß-fructofuranosidase is a natural enzyme existing in honey. The normal content of ß-fructofuranosidase is very rare. However, ß-fructofuranosidase (invertase) from industrial applications is used illicitly to produce adulterated honey.
This kit is based on sandwich immunoassay to detect ß-fructofuranosidase in honey quantitatively.
2. Materials Required
2.1 Equipments
----Microtiter plate spectrophotometer (450nm/630nm)
----Vortex mixer
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Volumetric flask: 100ml, 500ml,
----Polystyrene centrifuge tubes: 2ml,10ml,50ml
----Micropipettes:
20μl-200μl, 100μl-1000μl, 250μl-multichanel
2.2 Reagents:
----Sodium Chloride( AR)
----Deionized water
3. Kit components
- Microtiter plate coated with antibody (96wells)
- Standard Solution×5bottle (2ml/bottle)
0U/kg, 1U/kg, 3U/kg, 9U/kg, 27U/kg
- Enzyme conjugate 12ml..............red cap
- Antibody solution 12ml ............green cap
- Substrate solution A 7ml.............white cap
- Substrate solution B 7ml............…red cap
- Stop solution 7ml...............…yellow cap
- 20×concentrated wash solution 40ml
........................transparent cap
- Sample diluent 50ml…....….......…blue cap
4. Reagents Preparation
Solution 1: NaCl solution
Take 1.0g Nacl, and add 100ml deionized water to make it
mix completely.
Solution 2: wash solution
Dilute 20×Concentrated wash solution with deionized water in the volume ratio of 1: 19, which will be used to wash the plates. This diluted solution can be stored for 1 month at 4ºC.
5. Sample Preparation
5.1 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.
5.2 Honey:
----Weigh 1.0±0.05g sample into a 10ml polystyrene tube;
----Add 4ml NaCl solution (solution 1); Vortex for 1min till dissolved completely, incubate at room temperature for 30min;
----Take 500μl into a 2ml polystyrene tube and mix with 500μl Sample diluent, mix completely;
----Take 100μl per well for assay.
6. Assay process
6.1 Notice before assay:
6.1.1 Make sure all reagents and microwells are all at room temperature (20-25ºC).
6.1.2 Return all the rest reagents to 2-8ºC immediately after use.
6.1.3 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.
6.1.4 Avoid the light and cover the microwells during incubation.
6.2 Assay Steps:
6.2.1 Take all reagents out at room temperature (20-25ºC) for more than 30min, shake gently before use.
6.2.2 Get the microwells needed out and return the rest into the zip-lock bag at 2-8ºC immediately.
6.2.3 The wash solution should be rewarmed to room temperature before use.
6.2.4 Number: number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions.
6.2.5 Add standard solution/sample: add 100µl of standard solution or prepared sample to corresponding wells. Shake gently and then incubate at 37oC for 30min with cover.
6.2.6 Wash: remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl wash solution (solution 2) 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).
6.2.7 Add antibody: add 100µl of antibody solution, and shake gently and then incubate at 37oC for 30min with cover.
6.2.8 Repeat Wash Step 6.2.6.
6.2.9 Add enzyme conjugate: Add 100µl enzyme conjugate, mix gently and incubate for 30min at 37ºC with cover.
6.2.10 Repeat Wash Step 6.2.6.
6.2.11 Coloration: add 50µl solution A and 50µl solution B to each well. Mix gently by shaking the plate manually and incubate for 10min at 37oC with cover (see 9.8).
6.2.12 Measure: add 50µl the stop solution 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 adding stop solution)
7. Results
7.1 Percentage absorbance
The mean values of the absorbance values obtained from the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100%.
= [i][ii]
B --absorbance of standards or samples
B5 --absorbance of standard 6 solution (81U/kg)
7.2 Standard Curve
----To draw a standard curve: The absorbance value of standards as y-axis, semi-logarithmic of the concentration of the standards (U/kg) as x-axis.
----The beta fructofuranosidase concentration of each sample (U/kg), 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:
Special software has been developed for all data interpretation, which can be provided on request.
Dilution factor:
Honey...............…...........10
8. Sensitivity, accuracy and precision
Test Sensitivity: 1 U/kg
Detection limit.....................10 U/kg
Accuracy......................…....100±20%
Precision
Variation coefficient of the ELISA kit is less than 10%.
9. Notice
9.1 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).
9.2 Do not allow microwells to dry between steps to avoid unsuccessful reproducibility and operate the next step immediately after tap the microwells holder.
9.3 Shake each reagent gently before use.
9.4 Keep your skin away from the stop solution for it is the 0.5M H2SO4 solution.
9.5 Don't use the kits out of date. Don't exchange the reagents of different batches, or else it will drop the sensitivity.
9.6 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.
9.7 Substrate solution should be abandoned if it turns colors. The reagents may turn bad if the absorbance value (450/630nm) of the zero standard is less than 0.5 (A450nm<0.5).
9.8 The coloration reaction needs 10min after the addition of solution A and solution B. And you can prolong the incubation time ranges, never exceed 15min, on the contrary, shorten the incubation time properly.
9.9 The optimal reaction temperature is 37 oC. Higher or lower temperature will lead to the changes of sensitivity and absorbance values.
10. Storage
Storage condition: 2-8ºC.
Storage period: 12 months
Certifications
Certificate
ISO9001: 2008 & ISO13485: 2003;
CNAS (China National Accreditation Service for Conformity Assessment);
CQC (China Quauty Certification Center);
CE (Communate Europpene).
Awards
High-Tech Enterprise;
Key High-Tech Enterprise of China Torch Program;
Beijing Engineering Research Center;
Industrialization Demonstration Project of China Torch Program;
Beijing Science and Technology Award;
Tianjin Science and Technology Award;
Shandong Science and Technology Award;
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