AGROS Enzyme Immunoassay Kit
The AGROS Enzyme Immunoassay Kit is available for the detection and quantification of sex hormone, progesterone. The immunoassay based on AGROS Kit is fast, cost effective, precise, specific, sensitive and easy to perform. In little over 1 hour total time, progesterone can be measured at 18 pmol – 0,6 nmol/10 mL (0.6 – 20 ng/ml) using only 10 mL of milk sample. The Kit is cheaper than other immunoassays and suitable for a large number of samples.
The AGROS ELISA Kit utilises the principle of competitive binding to measure the concentration of hormone in milk. The progesterone-specific antibodies are pre-coated onto the surface of the reaction wells. The milk sample, containing an unknown amount of hormone, is mixed in the reaction well with a known amount of progesterone-enzyme conjugate (tracer) to react with a limited number of antibodies in the reaction wells. During incubation, the hormone in the sample competes with the tracer for the antibody binding sites. After incubation, the unbound reagents are washed out of the reaction wells. Substrate is then added to each reaction well and incubated for 10-15 min. The substrate reacts with the tracer that is bound to the antibody in the reaction wells producing a yellow-coloured product measurable in 450nm. The absorbance of the colour, inversely proportional to the amount of hormone present in the sample, is measured with a vertical spectrophotometer. The absorbance of the sample is converted to concentration of hormone by means of a standard curve, which is produced by simultaneously treating standards along with the samples.
1) Reaction Wells 5 pcs (480 assays)
2) Tracer 30 mL
3) Tracer Diluent 5 x CONC, 0.02% Thiomerosal
4) Reaction Solution 5 x CONC, 0.02% Thiomerosal
5) Wash solution 10 x CONC
6) Stopping Reagent 4 x CONC
7) Substrate Diluent 5 x CONC
8) Substrate 1000 mL
9) Standards 300 mL of each, 0.0 ng/ml, 0.625 ng/ml, 1.25 ng/ml, 2.5 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml.
Directions for Use
1. Preparation of tracer Solution
Dilute Progesterone Tracer Diluent (2 ml CONC + 8 ml red. water). Add 5 mL of Tracer to this solution. Diluted tracer can be stored at 2-4oC for up to 2 days. For one Elisa plate approximately 10 ml of solution is needed.
2. No further preparation of standards is needed.
3. Dilute reaction solution in the same proportion as tracer diluent (2 ml CONC + 8 ml redestil. water). 10 ml of solution per plate is needed.
4. Dilute wash solution before use (6ml CONC + 54 ml red. Water). 60 ml per plate is needed.
5. Remove plastic cover from desired number of wells from the refrigerator, decant and dry the plate against absorbent cellulose. Wash with 300 μL of Wash Solution. Shake the plate for a few seconds, decant wash mixture from the wells and tap the plate firmly against absorbent cellulose. Add 100 mL of diluted Reaction Solution to each well.
6. Add 10 mL of standard and 10 mL of milk samples, following the scheme and then add 100 mL of diluted Progesterone tracer solution to each well. Only Blank well is left pure of Tracer Solution (100 mL Reaction Solution only).
7. Mix in hand, better by tapping plate.
8. Cover wells with plate sealer.
9. Incubate sealed wells in refrigerator for 50 min.
10. After 50 min incubation decant solution and dry the plate against absorbent cellulose.
11. Wash wells by adding 300 mL of the Wash Solution to each well. Shake the plate for a few seconds, decant wash mixture from the wells and tap the plate firmly against absorbent cellulose.
12. To prepare substrate solution (during the incubation), mix 20 ml of diluted substrate solution (4 ml CONC + 16 ml red. water) with 200 mL of substrate per plate. Recommended temperature of the Substrate Solution is 25 oC.
13. Add 200 mL of the Substrate Solution to each well using a multichannel pipette. Warning! Don’t leave strips without solution long. This period has to be as short as possible, because the activity of tracer could be destroyed.
14. Cover wells with plate sealer.
15. Incubate at 25 oC for 10-15 minutes.
16. Stop the reaction by adding 100 mL diluted Stopping Reagent (2,5 ml CONC + 7,5 ml redest. H2O, mix) to each well. Wait for 5 min.
17. Read colour absorbance at 450 nm.
18. Calculate progesterone concentrations calculated by AGROS Elisa Software.
Reagent Storage and Precautions
PGR Reaction Wells These are very sensitive to temperature and must be stored at + 4 oC (stable for more than 6 months).
Tracer Reconstituted tracer is stable for 2 days at 2 - 5 oC. Stock solution must be stored at - 20 oC (stable for more than 6 months).
Substrate Store at 2 - 5 oC in dark. The working solution is stable for 8 hours at 2 - 5 oC.
Standards Store at +4 oC (stable for more than 6 months).
Do not use reagents after their marked respiration date.
All reagents should be stored as indicated on label.
Precise pipetting of the sample and tracer is critical to the accuracy and reproducibility of the assay.
It is important that a standard curve must be included in each run when all strips are not processed at one time.
AGROS Enzyme Immunoassay Kits are for research use only. Some reagents in the kit contain 0.02% Thiomerosal as a preservative. If reagents containing thimerosal are to be disposed via the sink, flush with generous amounts of water to prevent accumulation of toxic compounds.
Avoid skin contact with stop solution! Stop solution is caustic agent.
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Fig.1. Scheme of application of progesterone standards and milk samples on ELISA plate.