ELISA Kit for Vitamin D Binding Protein (DBP)

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

Specificity

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

Recovery

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

Matrix Recovery range (%) Average(%)
serum(n=5) 98-105 101
EDTA plasma(n=5) 88-101 96
heparin plasma(n=5) 81-103 90

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Vitamin D Binding Protein (DBP) 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) 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) 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) 80-94% 79-104% 80-89% 79-104%
EDTA plasma(n=5) 91-98% 94-101% 95-102% 81-96%
heparin plasma(n=5) 83-90% 83-97% 95-103% 89-98%

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
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 1 hours at 37°C;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37°C;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
8. Add 50µL Stop Solution. Read at 450nm immediately.

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Magazine Citations
Proteome Science Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients PubMed: 23101585
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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