Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.16-10 ng/mL
Sensitivity:	0.09 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human C1INH in samples. No significant cross-reactivity or interference between Human C1INH and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human C1INH. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human C1INH 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 C1INH, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human C1INH. The concentration of Human C1INH in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	SERPING1: a protein protease inhibitor (C1-inhibitor) that forms a proteolytically inactive stoichiometric complex with the C1r or C1s proteases. May play an important role in regulating complement activation, blood coagulation, fibrinolysis and the generation of kinins. Very efficient inhibitor of FXIIa. Mutations of the SERPING1 gene is associated with adult macular degeneration can also cause hereditary angioedema. Binds to E. coli stcE which allows localization of SERPING1 to cell membranes thus protecting the bacteria against complement-mediated lysis. Belongs to the serpin family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 11q12. 1 Cellular Component: extracellular space; extracellular region Molecular Function:serine-type endopeptidase inhibitor activity; protein binding Biological Process: negative regulation of complement activation, lectin pathway; platelet activation; fibrinolysis; platelet degranulation; blood circulation; innate immune response; blood coagulation; complement activation, classical pathway; blood coagulation, intrinsic pathway; aging Disease: Complement Component 4, Partial Deficiency Of; Angioedema, Hereditary, Type I
NCBI Summary:	This gene encodes a highly glycosylated plasma protein involved in the regulation of the complement cascade. Its protein inhibits activated C1r and C1s of the first complement component and thus regulates complement activation. Deficiency of this protein is associated with hereditary angioneurotic oedema (HANE). Alternative splicing results in multiple transcript variants encoding the same isoform. [provided by RefSeq, Jul 2008]
UniProt Code:	P05155
NCBI GenInfo Identifier:	124096
NCBI Gene ID:	710
NCBI Accession:	P05155. 2
UniProt Secondary Accession:	P05155,Q16304, Q547W3, Q59EI5, Q7Z455, Q96FE0, Q9UC49 A6NMU0, A8KAI9, B2R6L5, B4E1F0, B4E1H2,
UniProt Related Accession:	P05155
Molecular Weight:
NCBI Full Name:	Plasma protease C1 inhibitor
NCBI Synonym Full Names:	serpin peptidase inhibitor, clade G (C1 inhibitor), member 1
NCBI Official Symbol:	SERPING1
NCBI Official Synonym Symbols:	C1IN; C1NH; HAE1; HAE2; C1INH
NCBI Protein Information:	plasma protease C1 inhibitor; serpin G1; C1-inhibiting factor; C1 esterase inhibitor; complement component 1 inhibitor; serine/cysteine proteinase inhibitor clade G member 1
UniProt Protein Name:	Plasma protease C1 inhibitor
UniProt Synonym Protein Names:	C1 esterase inhibitor; C1-inhibiting factor; Serpin G1
Protein Family:	Para-Rep
UniProt Gene Name:	SERPING1
UniProt Entry Name:	IC1_HUMAN

As the RLU 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 (ng/mL)	RLU	Average	Corrected
10	31500 33442	32471	32447
5	14380 16560	15470	15446
2.5	7647 7359	7503	7479
1.25	3581 3725	3653	3629
0.63	1857 1665	1761	1737
0.31	864 784	824	800
0.16	335 379	357	333
0	24 24	24	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human C1INH were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human C1INH 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 (ng/mL)	0.55	1.17	4.78	0.57	1.21	4.47
Standard deviation	0.05	0.12	0.51	0.06	0.10	0.27
C V (%)	9.09	10.26	10.67	10.53	8.26	6.04

Recovery

The recovery of Human C1INH 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)	86-97	92
EDTA plasma (n=5)	90-102	96
Cell culture media (n=5)	86-97	91

Linearity

Samples were spiked with high concentrations of Human C1INH 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 (%)	91-105	90-101	100-112
1:2	Average (%)	97	95	107
1:4	Range (%)	94-106	91-104	90-104
1:4	Average (%)	100	97	97
1:8	Range (%)	100-116	97-110	98-114
1:8	Average (%)	109	102	105
1:16	Range (%)	97-111	99-110	97-110
1:16	Average (%)	103	104	103

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 CLIA 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 A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.