Rat MT(Melatonin) ELISA Kit

Product name: Rat MT(Melatonin) ELISA Kit

Catalog E-EL-R0031

Size: 96T

Reactivity: Rat

Detection Range: 0.16~10 ng/mL

Sensitivity: 0.10 ng/mL

Lead time: 7~10days

Intended use

This ELISA kit can be applied to the in vitro quantitative determination of Rat MT concentrations in serum, plasma and other biological fluids.

Test principle

This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with Rat MT. During the reaction, Rat MT in the sample or standard competes with a fixed amount of Rat MT on the solid se supporter for sites on the Biotinylated Detection Ab specific to Rat MT. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by adding Stop Solution and the color change is measured spectrotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Rat MT in the samples is then determined by comparing the OD of the samples to the standard curve.

Assay type	Competitive
Format	96T
Assay time	2.5h
Reactivity	Rat
Detection Method	Colormetric
Detection Range	15.63—1000 pg/mL
Sensitivity	9.38 pg/mL
Sample Volume	50μL
Sample Type	Serum, plasma and other biological fluids

Specificity

This kit recognizes some recombinant and natural Rat MT. No significant cross-reactivity or interference was observed.

Typical data

For convenience in result calculation, absorbance can be used as ordinate and standard concentrations as abscissa. The standard curve provided in the manual is only for reference, and experimenters should draw the standard curve based on data of themselves.

(pg/mL)O.DAverage

1000	0.394 0.408	0.401
500	0.483 0.527	0.505
250	0.71 0.676	01
125	0.98 1.01	99
62.5	1.404 1.39	1.397
31.25	1.826 1.812	1.819
15.63	2.158 2.168	2.163
0	2.655 2.661	2.658

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Rat MT were tested 20 times on one plate, respectively.Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Rat MT were tested on 3 different plates, 20 replicates in each plate.

Intra-assay PrecisionInter-assay Precision

Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean(pg/mL)	51.50	95.00	358.50	52.50	88.90	364.10
Standard deviation	2.70	4.30	18.60	2.70	4.40	19.30
C V (%)	5.24	4.53	5.19	5.14	4.95	5.30

Recovery

The recovery of Rat MT spiked to three different levels in samples throughout the range of the assay in various matrices was evaluated.

Sample TypeRange (%)Average Recovery (%)

Serum (n=5)	97-110	102
EDTA plasma (n=5)	92-106	100
culture media (n=5)	91-105	96

Samples were spiked with high concentrations of Human ACTH and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5)EDTA plasma (n=5)culture media (n=5)

1:2	Range (%)	95-112	88-99	97-109
	Average (%)	103	94	102
1:4	Range (%)	89-100	89-104	103
	Average (%)	94	97	103
1:8	Range (%)	87-98	88-100	98-110
	Average (%)	92	94	104
1:16	Range (%)	88-100	91-102	96-109
	Average (%)	93	96	102

Kit components & Storage

The unopened kit can be stored at 4℃ for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions since the kit is received.

ItemSpecificationsStorage

Micro ELISA Plate(Dismountable)	8 wells ×12 strips	-20℃, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 μL
Concentrated HRP Conjugate (100×)	1 vial, 120 μL	-20℃(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4℃, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4℃(shading light)
Stop Solution	1 vial, 10 mL	4℃
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution.The volume of reagents in partial shipments is a little more than the volume marked on the label, please use in measuring instead of directly pouring.

Other supplies required

• Microplate reader with 450nm wavelength filter
• High-precision transferpettor, EP tubes and disposable pipette tips
• 37℃ Incubator
• Deionized or distilled water
• Absorbent paper
• Loading slot for Wash Buffer

Assay Procedure

	1.Add 50 μL of standard or sample to each well.
	2.Immediately add 50 μL of Biotinylated Detection Ab to each well.
	3.Incubate for 45 min at 37℃. Aspirate and wash 3 times.
	4.Add 100 μL of HRP Conjugate to each well. Incubate for 30 min at 37℃. Aspirate and wash 5 times.
	5.Add 90 μL of Substrate Reagent. Incubate for 15 min at 37℃.
	6.Add 50 μL of Stop Solution.
	7.Read the OD at 450 nm immediately. Calculation of the results.

Citations

Publication: Liu Y, Ni C, Li Z, et al. Prolactic Melatonin Attenuates Isoflurane-Induced Cognitive Impairment In Aged Rats Through Hippocampal Melatonin Receptor 2-CREB asic & Clinical rmacology & 016.Species: RatSample Type: plasma and tissue homogenates