This test kit is based on the competitive enzyme immunoassay for the detection of Ampicillin in the sample. The antigens conjugated Ampicillin is pre-coated on the micro-well stripes, Ampicillin in the sample and the conjugated antigens pre-coated on the micro-well stripes compete for the anti-Ampicillin antibodies. 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 Ampicillin concentration in the sample. This value is compared to the standard curve and concentration of Ampicillin in the sample is subsequently obtained.
2. Technical specifications
Sensitivity : 0.1 ppb
tChicken, duck, pork,liver, fish, shrimpt2 ppb
tHoney, milkt2 ppb
tBecause of some interference in honey and milk, the detection limit is 4 ppb.
tChicken, duck, pork, liver, fish, shrimpt75%u00b110%
t1） Micro-well strips: 12 strips with 8 removable wells each
t2） 6u00d7 standard solution (1 mL each): 0 ppb, 0.1 ppb, 0.3 ppb, 0.9 ppb, 2.7ppb and 8.1 ppb
t3） Enzyme conjugate (12 mL)tred cap
t4） Antibody working solution (7 mL)tblue cap
t5） Substrate A solution (7 mL)twhite cap
t6） Substrate B solution (7 mL)tblack cap
t7） Stop solution (7 mL)tyellow cap
t8） 20u00d7 concentrated washing buffer (40 mL)twhite cap
t9） 5u00d7 concentrated redissolving solution (50 mL)ttransparent cap
4. Materials required but not provided
t1) Equipments: microplate reader, printer, homogeniser, nitrogen-drying device, vortex, centrifuge, measuring pipets, balance (a reciprocal sensibility of 0.01 g)
t2) Micropipettors: single-channel 20-200 u00b5L and 100 to 1000 u00b5L, and multi-channel 250 u00b5L
t3) Reagents: NaOH, Acetonitrile(CH3CN), N-Hexane, deionized water, Na2Fe(CN)5u00b7NOu00b72H2O and ZnSO4
5. Sample pre-treatment
The following points must be dealt with before the pre-treatment of any kind of sample:
t1） Only the disposable tips can be used for the experiments and the tips must be changed when used for different reagents.
t2） Before the experiment, each experimental equipment must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.
Solution preparation before sample pre-treatment:
t1 5u00d7concentrated redissolving solution is mixed with deionized water at 1:4 (1 mL 5u00d7 concentrated redissolving solution+ 4 mL deionized water), ued for sample redissolving.
t2 0.1 M NaOH : dissolve 0.4 g NaOH in deionized water to 100 mL.
t3 CH3CN-0.1M NaOH solution: ⅤCH3CN:ⅤNaOH =84:16 (84 mL CH3CN + 16 mL 0.1 M NaOH).
t4 C solution(for milk sample): dissolve 10.7 g Na2Fe(CN)5u00b7NOu00b72H2O in deionized water to 100 mL .
t5 D solution(for milk sample): dissolve 28.8 g ZnSO4u00b77H2O in deionized water to 100 mL.
t6 1 M NaOH solution: dissolve 4g NaOH in deionized water to 100 mL.
5.1 Tissue (Chicken, duck, pork, liver, fish or shrimp)
1 Homogenize the sample.
t2 Take 2u00b1 0.05 g of the homogenized sample into 50 mL centrifugal tube, add 2 mL 1 M NaOH solution, rock it with vortex for 2 min, add 8 mL CH3CN, shake for 10 min, centrifuge at above 4000 r/min at room temperature(25 ℃) for 10 min.
3 Take 1 mL of the supernatant, blow to dry by nitrogen or air at 50-60 ℃.
t4 Dissolve the dry residues in 1 mL of the diluted redissolving solution, then dilute at 1:3 ( 50 u00b5L sample + 150 u00b5L the diluted redissolving solution).
t6 Take 50 u00b5L for further analysis.
tFold of dilution of the sample: 20
t1 Put 1.0 u00b1 0.05 g honey into centrifuge tube, add 3 mL diluted redissolving solution, then static for 20 min after shaking vigorously. centrifuge at above 4000 r/min at room temperature (25 ℃) for 10 min.
t2 Take upper layer, diluted 1:4 in diluted redissolving solution(20 u00b5L sample + 80 u00b5L the diluted redissolving solution).
3 Take 50 u00b5L for further analysis.
tFold of dilution of the sample: 20
a) First method
t1 Take 2 mL fresh milk into 5 mL centrifugal tube, add 50 u00b5L C solution , mix properly.
t2 Add 50 u00b5L D solution ,shake for 1 min , centrifuge at 4000 r/min at 10 ℃ for 10 min.
t3 Take clear liquid(upper layer), dilute with the diluted redissolving solution at 1:19 (20 u00b5L sample+380 u00b5L diluted redissolving solution).
4 Take 50 u00b5L for further analysis.
tFold of dilution of the sample: 20
tNote: Repeat again if centrifuged sample is muddy
b) Second method
1 Put 2 mL milk (removed fat) into centrifugal tube.
t2 Add 8 mL of the CH3CN-0.1 M NaOH solution, shake vigorously for 10min, centrifuge at above 4000 r/min at 15 ℃ for 10 min, take 1 mL supernatant (upper layer), evaporate to dryness by nitrogen at 60 ℃.
t3 Dissolve the dry residues in 1 mL N-Hexane, add 1 mL of the diluted redissolving solution, mix properly for 1 min, centrifuge and remove N-hexane phase.
4 Take the lower to dilute 1:3 ( 50 u00b5L sample + 150 u00b5L the diluted redissolving solution).
5 Take 50 u00b5L for analysis.
tFold of dilution of the sample: 20
6. ELISA procedures
t1 Bring all reagents and micro-well strips to the room temperature (20-25 ℃).
t2 Return all reagents to 2-8 ℃ immediately after use.
t3 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 the procedures of ELISA.
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.
Product Specification / Models
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 Ampicillin in the sample.
7.1 Qualitative determination
The concentration range (ng/mL) can be obtained from comparing the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sampleⅠis 0.310, and that of the sampleⅡ is 0.820, while those of the standard solutions are as the followings: 1.610 for 0 ppb, 1.350 for 0.1 ppb, 1.030 for 0.3 ppb, 0.660 for 0.90 ppb, 0.389 for 2.7 ppb and 0.198 for 8.1 ppb, accordingly the concentration range of the sampleⅠis 2.7 to 8.1 ppb, and that of the sampleⅡ is 0.3 to 0.9 ppb.
7.2 Quantitative determination
The mean values of the absorbance values is 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,
tPercentage of absorbance value =ttBttu00d7100%
tB-the average OD value of the sample or the standard solution
tB0-the average OD value of the 0 ng/mL standard solution
Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithmic values of the Ampicillin standard solutions (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 actual concentration of Ampicillin 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)
t1） The room temperature below 20 ℃ 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;
t2） Dryness of the microplate in the washing process will be accompanied by the situations
tincluding the non-linear standard curves and the undesirable reproducibility;
t3） Mix every reagent and reaction mixture evenly and wash the microplate thoroughly, otherwise there will be the undesirable reproducibility;
t4） The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin;
t5） 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;
t6） 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;
t7） Discard the colouration solution with any color that indicates the degeneration of this solution. The detecting value of standard solution 1（0 ppb） of less than 0.5 indicates its degeneration;
t8） Colouration time is 15 min after the addition of the substrate A and then the B solution, if the color is light, prolong the time, don't exceed 30min;
t9） 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
tStorage: store at 2-8 ℃, not frozen.
tExpiry date: 12 months; please check date of production before use.
Hongkong Wanyuan Technology Limited. was established in early 2013, relying on the core technology of the Group Corporation, Hong Kong and Taiwan as well as Europe and the United States and the industry's most advanced technology combined the successful production of a series of food safety testing reagents for domestic and international inspection standards, the total class mainly covers the following areas: food safety, drug residues (also known as veterinary drugs in foods of animal origin, antibiotic residues) ELISA kit; the mycotoxins class enzyme-linked immunosorbent assay kit; food safety rapid test kit(rapid strips); the mycotoxins class gold standard rapid detection of card. Mainly used for testing samples of food of animal origin: aquatic (fish, shrimp, crab, squid, clam extract), livestock and poultry organizations (chickens, ducks, pigs, etc.), dairy products (raw milk, milk, milk powder) bee products (honey, royal jelly), egg, serum, animal feed, urine, hair, water and the like. "Wanyuan Technology "at the beginning of its establishment, adhering to the" quality first, quality assurance, "the purpose and dedication to provide quality products and services for customers at home and abroad, we hope for all mankind table health escort and contribute our mite to the force!
Our company services customer groups are: honey export enterprises / processing plants, dairy production and processing enterprises, aquaculture enterprises, livestock companies, feed companies; Inspection and Quarantine Bureau, the aquatic testing center, quality inspection center of agricultural products, agricultural products quality supervision and inspection center , the aquatic animal testing center, the quality supervision and Inspection Station of aquatic feed, veterinary drugs and feed monitors, etc.; across the country, the majority of equipment supplies distribution agents.
Hongkong Wanyuan Technology Limited. sincerely look forward to your support and cooperation. Letter to inquire to discuss cooperation!