This kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. The purified anti-Estrogen receptor antibody was pre-coated onto 96-well plates. And the HRP conjugated anti-Estrogen receptor antibody was used as detection antibodies. The standards, test samples and HRP conjugated detection antibody were added to the wells subsequently, mixed and incubated, then, unbound conjugates were washed away with wash buffer. TMB substrates (A & B) were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the Estrogen receptor amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of Estrogen receptor can be calculated.
1.Sample Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20℃ for long term. Avoid multiple freeze-thaw cycles.
Serum: Coagulate at room temperature for 10-20 min, then, centrifuge at the speed of 2000-3000 r.p.m. for 20 min to collect supernatant. If precipitation appeared, centrifuge again.
Plasma: Collect plasma using EDTA or citrate plasma as an anticoagulant, and mix for 10-20 min, centrifuge at the speed of 2000-3000 r.p.m. for 20 min of collection. If precipitation appeared, centrifuge again.
Urine: Collect urine using a sterile container, centrifuge at the speed of 2000-3000 r.p.m. for 20 min to collect supernatant. If precipitation appeared, centrifuge again. For collection of hydrothorax and cerebrospinal fluid, take reference to this operation.
Cell culture supernatant: For secretory components: use a sterile container to collect. Centrifuge at the speed of 2000-3000 r.p.m. for 20 min to collect supernatant. For intracellular components: Dilute cell suspension with PBS(pH7.2-7.4) to make the cell concentration reached 1 million / mL. Damage cells and release of intracellular components through repeated freeze-thaw cycles. Centrifuge at the speed of 2000-3000 r.p.m. For 20 min to collect supernatant. If precipitation appeared, centrifuge again.
Tissue samples: Cut samples and weight, add certain volume of PBS (pH7.4), rapidly frozen with liquid nitrogen. After melting, store samples at 2-8 ℃ . Add certain volume of PBS (pH7.4), homogenize thoroughly, centrifuge at the speed of 2000-3000 r.p.m. for 20 min to collect supernatant.
1.Coagulate blood samples completely, then, centrifuge, and avoid hemolysis and particle.
2.NaN 3 can not be used as test sample preservative, since it is the inhibitor for HRP.
3.After collecting samples, analyze immediately or aliquot and store frozen at -20°C. Avoid repeated freeze-thaw cycles.
2.Wash buffer Dilute concentrated Wash buffer (Kit Component 4) 30-fold (1:30) with distilled water (i.e. add 20 mL of concentrated wash buffer into 580 mL of distilled water).
3.Standard Dilution of the Human Estrogen receptor standard (Kit Component 2): standard solution should be prepared no more than 2 hours prior to the experiment. (
Note: Do not dilute the standard directly in the plate) a. 10 ng/mL of standard solution: Add 0.5 mL of the 13.5 ng/mL standard (Kit Component 2) into 0.175 mL Standard diluent buffer (Kit Component 3) and mix thoroughly. b. 5 ng/mL -> 0.3125 ng/mL of standard solutions: Label 5 Eppendorf tubes with 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, respectively. Aliquot 0.2 mL of the Standard diluent buffer (Kit Component 3) into each tube. Add 0.2 mL of the above 10 ng/mL standard solution into 1st tube and mix thoroughly. Transfer 0.2 mL from 1st tube to 2nd tube and mix thoroughly. Transfer 0.2 mL from 2nd tube to 3rd tube and mix thoroughly, and so on. 0.2mL 0.2 mL 0.2 mL 0.2 mL 0.2 mL
1.Equilibrate kit components for 15-30 min at room temperature.
2.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. Add 50µl of diluted standards (10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL) into the standard wells. Add 100µl of Standard diluent buffer (Kit Component 3) into the control (zero) well. Do not add sample and HRP conjugated antibody into the control (zero) well.
3.For test sample wells, add 40µl of Sample diluent buffer (Kit component 5) first, then, add 10µl of sample. Add the solution at the bottom of each well without touching the side wall. Shake the plate mildly to mix thoroughly.
4.Cover the plate with Plate sealer (Kit Component 10) and incubate at 37℃ for 30 min.
5.Remove the sealer, and wash plate using one of the following methods: Manual Washing: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers. Fill each well completely with Wash Buffer (1×) and vortex mildly on ELISA shaker for 2 min, then aspirate contents from the plate, and clap the plate on absorbent filter papers. Repeat this procedure four more times for a total of FIVE washes. Automated Washing: Aspirate all wells, then wash plates FIVE times using Wash Buffer (1×). After the final wash, invert plate, and clap the plate on absorbent filter papers until no moisture remained. It is recommended that the washer be set for a soaking time of 10 seconds or shaking.
6.Add 50µl of HRP conjugated anti-human Estrogen receptor antibody (Kit Component 6) into each well (except control well).
7.Cover the plate with Plate sealer (Kit Component 10) and incubate at 37℃ for 30 min.
8.Remove the sealer, and wash the plate. (See Step 5)
9.Add 50µL of TMB chromogenic reagent A (Kit Component 8) into each well, and then, add 50µL of TMB chromogenic reagent B (Kit Component 9), vortex gently the plate on ELISA shaker for 30 seconds (Or shake gently by hand for 30 seconds), and incubate in dark at 37°C for 15 min. The shades of blue can be seen in the wells.
10.Add 50µL of Stop solution (Kit Component 7) into each well and mix thoroughly. The color changes into yellow immediately.
11.Read the O.D. absorbance at 450nm in a microplate reader within 15 min after adding the stop solution. For calculation, (the relative O.D. 450 ) = (the O.D. 450 of each well) – (the O.D. 450 of Zero well). The standard curve can be plotted as the relative O.D. 450 of each standard solution (Y) vs. the respective concentration of the standard solution (X)