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Melamine ELISA test kit instructions
1. Overview
The principle of this kit is the enzyme-linked immunoassay, which can be used for qualitative or quantitative detection of melamine in all samples. HPLC, GC/MS techniques can be utilized if further measurements are required.
2. Safety guidance
The kit standard contains a small amount of melamine. In addition, the substrate solution contains tetramethylbenzidine, and the stop solution contains diluted hydrochloric acid. Try not to contact these reagents. If these reagents come into contact with the skin, rinse them off immediately.
3. Storage and stability
The kit should be stored at 4–8 ° C. All reagents in the kit should be warmed to room temperature (20-25 ° C) before use. The reagent can be used continuously during the validity period.
4 . principle
Enzyme-linked immunosorbent assay for the quantitative determination of melamine residues. The standard, sample extract and melamine label were added to the wells that had been coated with the melamine antibody to initiate the reaction. During the 30 minute incubation period, the melamine in the sample extract competed with the melamine label for binding to the melamine antibody in the microwell, and after incubation for 30 minutes, all unbound melamine and melaminease markers in the wells were washed away. After washing with the diluted wash solution, a clear substrate solution is added to each well and the bound enzyme label converts the colorless substrate to a blue material. After 20 minutes of incubation, the reaction was stopped and data was read according to the color depth of each well. The concentration of melamine in the sample is derived from the standard color.
5. Limitations
We have tested many organic and inorganic substances that may be present in the test sample and found that they do not cross-react with this kit. However, there are also some perishable compounds in the sample that interfere with the assay by matrix effects, such as fat-containing foods, which are diluted to eliminate some matrix effects prior to measurement. Mistakes in the course of use can also affect the results, such as the conditions under which the kit is stored, the procedure for drawing the liquid, the inaccuracy of the ingested liquid, and the inaccurate incubation time during the immunization and substrate reactions.
6 . Working data
Sensitivity: The detection limit of melamine is 10 μg/L, and the value of B/B 0 is 50% when it is 50 μg/L. The measurement result is the most accurate in the middle of the standard curve.
Repeatability:
Standard coefficient of variation (CVs) <10%; sample coefficient of variation (CVs) <15%.
Specificity:
A kit composition
1. Vacuum packed microplate (8×12 strips) coated with melamine polyclonal antibody
2, 6 bottles of melamine standard solution, the concentration is 0, 20, 50, 100, 200, 500ng / mL (ppb)
3, 1 bottle of 7mL melamine enzyme label
4, 5 × concentrated washing liquid 100ml; need to use distilled water to dilute 1:5 before use
5, 1 bottle of 14mL substrate (TMB)
6, 1 bottle of 14mL stop solution. (Note: hydrochloric acid, do not touch the skin!).
B test preparation
Micropipettes and tips are mandatory. It is recommended to use a row of guns. Use the same box of reagents and standards for the same test.
1. Bring all reagents to room temperature (19 ° C - 25 ° C) before use.
2. Take out the required number of microporous strips from the aluminum foil bag, place the desiccant and reseal the bag to prevent the microporous strip from getting wet. Store at 4-8 ° C.
3. Standard solution, enzyme conjugate, substrate and stop solution are used without dilution.
4. The lotion should be diluted 1:5 before use (eg 10ml lotion + 40ml distilled water).
5. Be careful when using hydrochloric acid in the stop solution.
C. Steps
1. Add 100 μL of standard or sample to the corresponding microwell. It is best to repeat.
2. Add 50 μL of the enzyme label to each well with a lance, then cover the well with a piece of paper, and gently shake the plate for 30 seconds to mix the liquid in the well.
3. Incubate for 30 minutes at room temperature.
4. Pour the liquid in the well into a suitable container. Add at least 250 μL of 1× wash solution to each well, then discard the wash solution into a suitable container and repeat the wash 3 times. Clapper on a thick layer of absorbent paper to remove any residual lotion.
5. Add 100 μL of substrate color solution to each well using a lance and incubate for 20 minutes at room temperature. Try to keep it out of sight.
6. Add 100 μL of Stop Solution to each well and the color will change from blue to brownish yellow.
7. Read the absorbance value (OD) of each well at 450 nm.
D. Evaluation of results
The ELISA results can be evaluated using commercial ELISA software programs such as Logit/Log or 4-Parameter. It is also possible to calculate the %B/B0 value of each standard by using %B/B0 as the y-axis and melamine concentration as the x-axis as a standard curve. The sample concentration can be calculated from the standard curve. When the melamine concentration in the sample is higher than the standard 5 (500 μg / L), it is necessary to re-measure after dilution to obtain more accurate results.
The determination of the semi-quantitative result can be determined according to the absorbance value of the sample and the standard absorbance value. The absorbance value of the sample is lower than the absorbance value of a certain standard, indicating that the melamine content of the sample is greater than the concentration of the standard, and the absorbance value of the sample is high. The absorbance value of a standard indicates that the melamine content of the sample is less than the concentration of this standard.
E. Also need equipment
1, 50μL ~ 200μL single or multichannel pipettes and tips
2, microplate washing machine
3, the oscillator
4. Distilled or deionized water
5, absorbent paper
6, centrifuge
7, 450nm filter microplate reader
If necessary, this product, please contact Kim Yung technology companies in Shenzhen Â
 Contact: Qi Qicheng
 Phone: 15323787737
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