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Multiplex Assay Kit for Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay)

GC; VDBG; VDBP; Group-Specific Component; Gc-globulin

(Note: Up to 8-plex in one testing reaction)

Specificity

This assay has high sensitivity and excellent specificity for detection of Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 92-99 96
EDTA plasma(n=5) 78-95 84
heparin plasma(n=5) 97-104 101

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Vitamin D Binding Protein (DBP) ,etc. by FLIA (Flow Luminescence Immunoassay) 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) 87-94% 78-102% 80-102% 81-90%
EDTA plasma(n=5) 95-102% 99-105% 94-101% 93-101%
heparin plasma(n=5) 85-99% 79-96% 79-89% 78-95%

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.

Reagents and materials provided

Reagents Quantity Reagents Quantity
96-well plate 1 Plate sealer for 96 wells 4
Pre-Mixed Standard 2 Standard Diluent 1×20mL
Pre-Mixed Magnetic beads (22#:DBP) 1 Analysis buffer 1×20mL
Pre-Mixed Detection Reagent A 1×120μL Assay Diluent A 1×12mL
Detection Reagent B (PE-SA) 1×120μL Assay Diluent B 1×12mL
Sheath Fluid 1×10mL Wash Buffer (30 × concentrate) 1×20mL
Instruction manual 1

Assay procedure summary

1. Preparation of standards, reagents and samples before the experiment;
2. Add 100μL standard or sample to each well,
    add 10μL magnetic beads, and incubate 90min at 37°C on shaker;
3. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60min at 37°C on shaker;
4. Wash plate on magnetic frame for three times;
5. Add 100μL prepared Detection Reagent B, and incubate 30 min at 37°C on shaker;
6. Wash plate on magnetic frame for three times;
7. Add 100μL sheath solution, swirl for 2 minutes, read on the machine.

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Toxins (Basel). Increased Circulating Levels of Vitamin D Binding Protein in MS Patients Pubmed:25590278
Endocrine Vitamin D-binding protein and free vitamin D concentrations in acromegaly PubMed: 26547217
Proteomics Clinical Applications Comparative mass spectrometric and immunoassay‐based proteome analysis in serum of Duchenne muscular dystrophy patients Pubmed:26680509
PLoS One. 25-Hydroxivitamin D Serum Concentration, Not Free and Bioavailable Vitamin D, Is Associated with Disease Activity in Systemic Lupus Erythematosus Patients. pubmed:28085957
PROTEOMICS - Clinical Applications Comparative mass spectrometric and immunoassay-based proteome analysis in serum ofDuchenne muscular dystrophy patients. pubmed:26680509
Plos One 25-Hydroxivitamin D serum concentration, not free and bioavailable vitamin D, is associated with disease activity in systemic lupus erythematosus patients 10.1371/journal.pone.0170323
Nutrients Gene Expression of Sirtuin-1 and Endogenous Secretory Receptor for Advanced Glycation End Products in Healthy and Slightly Overweight Subjects after … Pubmed:30037068
BBA- Molecular Basis of Disease Enhanced remedial effects for vitamin D3 and calcium co-supplementation against pre-existing lead nephrotoxicity in mice: The roles of renal calcium homeostatic … Pubmed: 30553018
J Clin Res Pediatr Endocrinol Is Bioavailable Vitamin D Better Than Total Vitamin D to Evaluate Vitamin D Status in Obese Children? 34013709
PLoS One Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism Pubmed:35202418
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