Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	15.63-1000 ng/mL
Sensitivity:	9.38 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human ApoB in samples. No significant cross-reactivity or interference between Human ApoB 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 ApoB. 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 ApoB 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 ApoB, 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 ApoB. The concentration of Human ApoB in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	Function: Apolipoprotein B is a major protein constituent of chylomicrons (apo B-48), LDL (apo B-100) and VLDL (apo B-100). Apo B-100 functions as a recognition signal for the cellular binding and internalization of LDL particles by the apoB/E receptor.
UniProt Protein Details:	Subcellular location: Secreted. Induction: Up-regulated in response to enterovirus 71 (EV71) infection (at protein level). Ref. 34 Post-translational modification: Palmitoylated; structural requirement for proper assembly of the hydrophobic core of the lipoprotein particle. Ref. 31 Involvement in Disease: Defects in APOB are a cause of hypobetalipoproteinemia familial type 1 (FHBL1) [ MIM:107730]. A disorder characterized by highly reduced plasma concentrations of low density lipoproteins, and dietary fat malabsorption. Clinical presentation may vary from no symptoms to severe gastrointestinal and neurological dysfunction similar to abetalipoproteinemia. Ref. 46Defects in APOB are a cause of familial ligand-defective apolipoprotein B-100 (FDB) [ MIM:144010]. FDB is a dominantly inherited disorder of lipoprotein metabolism leading to hypercholesterolemia and increased proneness to coronary artery disease (CAD). The plasma cholesterol levels are dramatically elevated due to impaired clearance of LDL particles by defective APOB/E receptors. Ref. 40 Ref. 42 Ref. 44Note=Defects in APOB associated with defects in other genes (polygenic) can contribute to hypocholesterolemia. Sequence similarities: Contains 1 vitellogenin domain. RNA editing: Edited at position 2180. The stop codon (UAA) at position 2180 is created by RNA editing. Apo B-48, derived from the fully edited RNA, is produced only in the intestine and is found in chylomicrons. Apo B-48 is a shortened form of apo B-100 which lacks the LDL-receptor region. The unedited version (apo B-100) is produced by the liver and is found in the VLDL and LDL. Sequence caution: The sequence AAA51752. 1 differs from that shown. Reason: Frameshift at positions 942, 951, 1139, 1165, 1164, 1371 and 1385.
NCBI Summary:	This gene product is the main apolipoprotein of chylomicrons and low density lipoproteins. It occurs in plasma as two main isoforms, apoB-48 and apoB-100: the former is synthesized exclusively in the gut and the latter in the liver. The intestinal and the hepatic forms of apoB are encoded by a single gene from a single, very long mRNA. The two isoforms share a common N-terminal sequence. The shorter apoB-48 protein is produced after RNA editing of the apoB-100 transcript at residue 2180 (CAA->UAA), resulting in the creation of a stop codon, and early translation termination. Mutations in this gene or its regulatory region cause hypobetalipoproteinemia, normotriglyceridemic hypobetalipoproteinemia, and hypercholesterolemia due to ligand-defective apoB, diseases affecting plasma cholesterol and apoB levels. [provided by RefSeq]
UniProt Code:	P04114
NCBI GenInfo Identifier:	114014
NCBI Gene ID:	338
NCBI Accession:	P04114. 1
UniProt Secondary Accession:	P04114,O00502, P78479, P78480, P78481, Q13779, Q13785 Q13786, Q13787, Q13788, Q4ZG63, Q53QC8,
UniProt Related Accession:	P04114,P78482,Q13789,Q13828,Q59HB3,Q7Z7Q0,Q9UE51,Q9UE52,Q9UE53
Molecular Weight:
NCBI Full Name:	Apolipoprotein B-100
NCBI Synonym Full Names:	apolipoprotein B (including Ag(x) antigen)
NCBI Official Symbol:	APOB
NCBI Official Synonym Symbols:	FLDB; LDLCQ4
NCBI Protein Information:	apolipoprotein B-100; apoB-48; apoB-100; apo B-100; mutant Apo B 100; OTTHUMP00000115994; apolipoprotein B48
UniProt Protein Name:	Apolipoprotein B-100
Protein Family:	Apolipoprotein
UniProt Gene Name:	APOB
UniProt Entry Name:	APOB_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
1000	50118 57232	53675	53650
500	21059 23919	22489	22464
250	11110 9312	10211	10186
125	4643 5159	4901	4876
62.5	2632 2274	2453	2428
31.25	1353 1209	1281	1256
15.63	698 718	708	683
0	25 25	25	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human ApoB 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)	54.88	133.55	375.20	51.94	142.52	342.80
Standard deviation	5.83	12.27	29.34	5.98	14.92	29.76
C V (%)	10.62	9.19	7.82	11.51	10.47	8.68

Recovery

The recovery of Human ApoB 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)	88-104	95
EDTA plasma (n=5)	96-108	103
Cell culture media (n=5)	90-103	96

Linearity

Samples were spiked with high concentrations of Human ApoB 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 (%)	96-110	95-110	90-107
1:2	Average (%)	102	101	98
1:4	Range (%)	92-107	101-112	86-100
1:4	Average (%)	98	106	91
1:8	Range (%)	92-105	100-114	88-101
1:8	Average (%)	97	107	93
1:16	Range (%)	93-104	85-97	94-110
1:16	Average (%)	99	91	102

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.