Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	93.75-6000 pg/mL
Sensitivity:	56.25 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human MASP1 in samples. No significant cross-reactivity or interference between Human MASP1 and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human MASP1. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human MASP1 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human MASP1, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human MASP1. The concentration of Human MASP1 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	MASP1: Functions in the lectin pathway of complement, which performs a key role in innate immunity by recognizing pathogens through patterns of sugar moieties and neutralizing them. The lectin pathway is triggered upon binding of mannan-binding lectin (MBL) and ficolins to sugar moieties which leads to activation of the associated proteases MASP1 and MASP2. Functions as an endopeptidase and may activate MASP2 or C2 or directly activate C3 the key component of complement reaction. Isoform 2 may have an inhibitory effect on the activation of the lectin pathway of complement or may cleave IGFBP5. Defects in MASP1 are the cause of 3MC syndrome type 1 (3MC1). 3MC1 is a disorder characterized by facial dysmorphism that includes hypertelorism, blepharophimosis, blepharoptosis and highly arched eyebrows, cleft lip and/or palate, craniosynostosis, learning disability and genital, limb and vesicorenal anomalies. The term 3MC syndrome includes Carnevale, Mingarelli, Malpuech, and Michels syndromes. Belongs to the peptidase S1 family. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 3. 4. 21. -; Protease; Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 3q27-q28 Cellular Component: extracellular space; extracellular region Molecular Function:peptidase activity; protein binding; protein homodimerization activity; serine-type endopeptidase activity; calcium ion binding; calcium-dependent protein binding Biological Process: receptor-mediated endocytosis; negative regulation of complement activation; innate immune response; proteolysis; complement activation, lectin pathway; complement activation Disease: 3mc Syndrome 1
NCBI Summary:	This gene encodes a serine protease that functions as a component of the lectin pathway of complement activation. The complement pathway plays an essential role in the innate and adaptive immune response. The encoded protein is synthesized as a zymogen and is activated when it complexes with the pathogen recognition molecules of lectin pathway, the mannose-binding lectin and the ficolins. This protein is not directly involved in complement activation but may play a role as an amplifier of complement activation by cleaving complement C2 or by activating another complement serine protease, MASP-2. The encoded protein is also able to cleave fibrinogen and factor XIII and may may be involved in coagulation. A splice variant of this gene which lacks the serine protease domain functions as an inhibitor of the complement pathway. Alternate splicing results in multiple transcript variants. [provided by RefSeq, Apr 2010]
UniProt Code:	P48740
NCBI GenInfo Identifier:	218512135
NCBI Gene ID:	5648
NCBI Accession:	P48740. 3
UniProt Secondary Accession:	P48740,O95570, Q68D21, Q8IUV8, Q96RS4, Q9UF09, A8K542 A8K6M1, B4E2L7,
UniProt Related Accession:	P48740
Molecular Weight:	699
NCBI Full Name:	Mannan-binding lectin serine protease 1
NCBI Synonym Full Names:	mannan-binding lectin serine peptidase 1 (C4/C2 activating component of Ra-reactive factor)
NCBI Official Symbol:	MASP1
NCBI Official Synonym Symbols:	3MC1; MAP1; MASP; RaRF; CRARF; MASP3; MAp44; PRSS5; CRARF1
NCBI Protein Information:	mannan-binding lectin serine protease 1; serine protease 5; complement factor MASP-3; Ra-reactive factor serine protease p100; mannose-binding protein-associated serine protease; mannose-binding lectin-associated serine protease 1; complement-activating component of Ra-reactive factor
UniProt Protein Name:	Mannan-binding lectin serine protease 1
UniProt Synonym Protein Names:	Complement factor MASP-3; Complement-activating component of Ra-reactive factor; Mannose-binding lectin-associated serine protease 1; MASP-1; Mannose-binding protein-associated serine protease; Ra-reactive factor serine protease p100; RaRF; Serine protease 5Cleaved into the following 2 chains:Mannan-binding lectin serine protease 1 heavy chain; Mannan-binding lectin serine protease 1 light chain
Protein Family:	Mannan-binding lectin serine protease
UniProt Gene Name:	MASP1
UniProt Entry Name:	MASP1_HUMAN

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	O.D	Average	Corrected
6000	2.515 2.543	2.529	2.448
3000	1.639 1.651	1.645	1.564
1500	0.936 0.934	0.935	0.854
750	0.494 0.496	0.495	0.414
375	0.26 0.25	0.255	0.174
187.5	0.195 0.171	0.183	0.102
93.75	0.127 0.139	0.133	0.052
0	0.077 0.085	0.081	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human MASP1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human MASP1 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	282.00	505.24	2569.31	299.44	502.26	2755.35
Standard deviation	16.84	20.82	115.88	16.38	27.12	101.40
C V (%)	5.97	4.12	4.51	5.47	5.40	3.68

Recovery

The recovery of Human MASP1 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	87-97	92
EDTA plasma (n=5)	91-104	99
Cell culture media (n=5)	94-106	100

Linearity

Samples were spiked with high concentrations of Human MASP1 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)	Cell culture media (n=5)
1:2	Range (%)	98-111	94-111	93-106
1:2	Average (%)	103	101	101
1:4	Range (%)	90-103	81-92	85-94
1:4	Average (%)	97	86	89
1:8	Range (%)	92-107	83-94	86-100
1:8	Average (%)	99	87	92
1:16	Range (%)	91-105	85-95	86-98
1:16	Average (%)	98	90	91

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 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°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.