General Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) ELISA Kit

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

	Traditional ELISA Kit	Ready-to-Use ELISA KIT
Product name:	General Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) ELISA Kit
Method:	Competition
Synonyms:
Detection range:	0.156-10ng/mL
Target Protein:	NAADP
Size:	96T/48T
Quality guarantee period:	for 12 months, 16 months
Catalog number:	DL-NAADP-Ge (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

Introduction

Item	Standard	Test Result
Description	This immunoassay kit allows for the specific measurement of this index in serum, Plasma , Urine ,tissue homogenates and Cell culture supernates and Other biological fluids..	Conform
Identification	Colorimetric	Positive
Composition	Pre-coated, ready to use 96-well strip plate Standard (freeze dried) Standard Diluent Detection Reagent A Detection Reagent B Assay Diluent A Assay Diluent B TMB Substhumane Stop Solution Wash Buffer(30 x concenthumane) Plate sealer for 96 wells Instruction manual	1 2 1 × 20ml 1× 120μl 1× 120μl 1 × 12ml 1 × 12ml 1 × 9ml 1 ×6ml 1 ×20ml 2 1	Conform

Test principle
This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to this index has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled the index and unlabeled this 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 the index and the recovery humanes 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 concenthumanion of the index and their serial dilutions. The results were demonsthumaned by the percentage of calculated concenthumanion 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 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.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator 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 37oC;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37oC;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37oC;
7. Add 50µL Stop Solution. Read at 450 nm immediately

Introduction

Item	Standard		Test
Description	The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of NAADP in 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 NAADP 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.