Human Thyrotropin Releasing Hormone (TRH) ELISA Kit

two product lines: Traditional ELISA Kit and Ready-to-Use ELISA Kit.

	Traditional ELISA Kit	Ready-to-Use ELISA KIT
Product name:	Human Thyrotropin Releasing Hormone (TRH) ELISA Kit
Method:	Competition
Synonyms:	TRF; Thyrotropin-Releasing Factor; Thyroliberin; Protirelin; Pro-tirelin; Prothyroliberin; TSH-releasing factor
Detection range:	7.812-500pg/mL
Target Protein:	TRH
Size:	96T/48T
Quality guarantee period:	for 12 months, 16 months
Catalog number:	DL-TRH-Hu (traditional)	(ready-to-use)
Assay length	1-2.5Hours	1-2.5Hours
Advantages:	Competitive price. High sensitivity. High stability. 12 months shelf life.	Pre-diluted Detection Reagent A and B Reduction in the number of steps when conducting the test All the reagents can be stored at -20℃ Faster reaction compare to other brands 16 months shelf life
Instruction Manual

1.Overview：

Other names：TRF; Thyrotropin-Releasing Factor; Thyroliberin; Protirelin; Pro-tirelin; Prothyroliberin; TSH-releasing factor

Function: As a component of the hypothalamic-pituitary-thyroid axis, it controls the secretion of thyroid-stimulating hormone (TSH) and is involved in thyroid hormone synthesis regulation. It also operates as modulator of hair growth. It promotes hair-shaft elongation, prolongs the hair cycle growth phase (anagen) and antagonizes its termination (catagen) by TGFB2. It stimulates proliferation and inhibits apoptosis of hair matrix keratinocytes.

Sequence：

10         20         30         40         50
MPGPWLLLAL ALTLNLTGVP GGRAQPEAAQ QEAVTAAEHP GLDDFLRQVE
        60         70         80         90        100
RLLFLRENIQ RLQGDQGEHS ASQIFQSDWL SKRQHPGKRE EEEEEGVEEE
       110        120        130        140        150
EEEEGGAVGP HKRQHPGRRE DEASWSVDVT QHKRQHPGRR SPWLAYAVPK
       160        170        180        190        200
RQHPGRRLAD PKAQRSWEEE EEEEEREEDL MPEKRQHPGK RALGGPCGPQ
       210        220        230        240
GAYGQAGLLL GLLDDLSRSQ GAEEKRQHPG RRAAWVREPL EE

2．Features
INTENDED USE
The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of TRH in human serum, plasma or other biological fluids.

DETECTION RANGE
7.81-500pg/mL. The standard curve concentrations used for the ELISA’s were 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.2pg/mL, 15.6pg/mL, 7.81pg/mL.

SENSITIVITY
The minimum detectable dose of TRH is typically less than 3.25pg/mL.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

SPECIFICITY
This assay has high sensitivity and excellent specificity for detection of TRH.
No significant cross-reactivity or interference between TRH and analogues was observed.

You can reference link of the kit as following
https://www.dldevelop.com/uploadfile/data/DL-TRH-Hu.pdf
https://www.dldevelop.com/uploadfile/data/DL-TRH-Mu.pdf
https://www.dldevelop.com/uploadfile/data/DL-TRH-Ra.pdf

Introduction

Item	Standard		Test
Description	The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of TRH in human serum, plasma and other biological fluids.		Conform
Identification	Colorimetric		Positive
Composition	Traditional ELISA Kit	Ready-to-Use ELISA KIT	Conform
	Pre-coated, ready to use 96-well strip plate 1	Pre-coated, ready to use 96-well strip plate 1
	Plate sealer for 96 wells 2	Plate sealer for 96 wells 2
	Standard 2	Standard 2
	Diluents buffer 1×45mL	Standard Diluent 1×20mL
	Detection Reagent A 1×120μL	Detection Solution A 1×12mL
	Detection Reagent B 1×120μL	Detection Solution B 1×12mL
	TMB Substrate 1×9mL	TMB Substrate 1×9mL
	Stop Solution 1×6mL	Stop Solution 1×6mL
	Wash Buffer (30 × concentrate) 1×20mL	Wash Buffer (30 × concentrate) 1×20mL
	Instruction manual 1	Instruction manual 1

Test principle

This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to the index has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled the index and unlabeled the index (Standards or samples) with the pre-coated antibody specific to the index. After incubation the unbound conjugate is washed off. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of the index in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of the index in the sample.

Recovery

Matrices listed below were spiked with certain level of recombinant TRH and the recovery rates were calculated by comparing the measured value to the expected amount of the index in samples.

Matrix	Recovery range (%)	Average(%)
serum(n=5)	81-93	86
EDTA plasma(n=5)	80-97	88
heparin plasma(n=5)	90-101	95

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of the index and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1：2	1：4	1：8	1：16
serum(n=5)	82-96%	83-98%	81-99%	93-101%
EDTA plasma(n=5)	88-101%	86-95%	90-102%	80-93%
heparin plasma(n=5)	80-91%	82-90%	95-104%	79-95%

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Stability

The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
Note:
To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity, and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 50µL standard or sample to each well.
And then add 50µL prepared Detection Reagent A immediately.
Shake and mix. Incubate 1 hour at 37℃;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 1 hour at 37℃;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
7. Add 50µL Stop Solution. Read at 450 nm immediately.