Ⅳ型膠原(COL4)檢測試劑盒
適用生物 Rattus norvegicus (Rat，大鼠)
Ⅳ型膠原(COL4)檢測試劑盒檢測范圍 0.625-40ng/mL 靈敏度 0.274ng/mL
樣本類型 Serum, plasma, tissue homogenates and other biological fluids.
實驗時長 4.5h 實驗方法 雙抗夾心法 Ⅳ型膠原(COL4)檢測試劑盒規格 96T
Ⅳ型膠原(COL4)檢測試劑盒 ELISA Kit for Collagen Type IV (COL4)
FOR IN VITRO AND RESEARCH USE ONLY, NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES!
| Organism species | Rattus norvegicus (Rat) |
| Product No. | SEA180Ra |
| Sample type | Serum, plasma, tissue homogenates and other biological fluids. |
| Format | 96-well strip plate |
| Assay length | 4.5 hours |
| Detection range | 0.625-40ng/mL The standard curve concentrations used for the ELISA’s were 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL |
| Sensitivity | The minimum detectable dose of this kit is typically less than 0.274ng/mL. |
Specificity
This assay has high sensitivity and excellent specificity for detection of Collagen Type IV (COL4).
No significant cross-reactivity or interference between Collagen Type IV (COL4) and analogues was observed.
Recovery
Matrices listed below were spiked with certain level of recombinant Collagen Type IV (COL4) and the recovery rates were calculated by comparing the measured value to the expected amount of Collagen Type IV (COL4) in samples.
| Matrix | Recovery range (%) | Average(%) |
| serum(n=5) | 93-101 | 97 |
| EDTA plasma(n=5) | 80-90 | 84 |
| heparin plasma(n=5) | 92-99 | 95 |
Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Collagen Type IV (COL4) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Collagen Type IV (COL4) 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 Collagen Type IV (COL4) 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) | 79-101% | 80-95% | 89-105% | 97-105% |
| EDTA plasma(n=5) | 82-101% | 78-96% | 84-101% | 91-101% |
| heparin plasma(n=5) | 89-103% | 92-104% | 99-105% | 79-103% |
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 2 hours at 37oC;
3. Aspirate and add 100μL prepared Detection Reagent A. Incubate 1 hour at 37oC;
4. Aspirate and wash 3 times;
5. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37oC;
6. Aspirate and wash 5 times;
7. Add 90μL Substrate Solution. Incubate 15-25 minutes at 37oC;
8. Add 50μL Stop Solution. Read at 450nm immediay.
Test principle
The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Collagen Type IV (COL4). Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Collagen Type IV (COL4). Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Collagen Type IV (COL4), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of Collagen Type IV (COL4) in the samples is then determined by comparing the O.D. of the samples to the standard curve.
