(h Collagen IV ELISA, Strip Plate)
The Collagen IV H kit contains all of the components and reagents you need to assay human collagen IV in up to 36 duplicate urine or cell free liquid samples (plasma, serum, cell culture supernatant, etc…).
Kit Contents: Your Collagen IV H kit should contain the following items:
a. 1 Collagen IV H Test Plate
b. 2 NHEBSA (Diluent): 12 mL
c. 1 Human Collagen IV Standard: 0.5 mL
d. 1 Rabbit Anti- Collagen IV Antibody: 6mL
e. 1 Goat Anti-rabbit IgG HRP Conjugate: 12mL
f. 1 Color Developer: 12 mL
g. 1 Color Stopper: 12 mL
h. Instruction manual
Human Collagen IV Standard, NHEBSA, Rabbit anti-Collagen IV Antibody and Anti-rabbit IgG HRP Conjugate preparations contain 0.05% Proclin 300
(active components isothiazolones) as preservative. Color Stopper contains dilute (2.0 N) sulfuric acid.
All kit reagents are supplied in ready to use liquid form. The collagen IV H plates are precoated and ready to use. A wash buffer must be supplied by the user. The kit is subjected to QC analysis using Exocell EIA Wash Buffer with composition: 0.15 M NaCl, 0.01 M triethanolamine (pH 6.8), 0.05% Tween 20 and 0.05 % Proclin 300 (preservative may be omitted if the buffer is freshly prepared). This buffer is recommended.
The wash buffer must not contain azide.
Adjustable pipettors are required that are capable of delivering volumes over the range of 10-1000 ul. Multi-channel pipettors capable of delivering 50 and 100 uL are recommended. Finally, a microplate reader equipped to determine
absorbance at 450 nm is required.
Dilutions for the assay are prepared in tubes, and 1.5 mL microfuge tubes are suitable for this purpose.
Technical Background: Collagen IV is a structural component of basement membranes and other extracellular matrices, and is elaborated in cell culture by various cell types. Collagen IV H is designed to measure human collagen IV in
tissue culture samples or biological specimens.
This is a competitive indirect ELISA. To complete the assay, samples containing collagen IV and rabbit anti-collagen IV are added to wells that are pre-coated with (human) collagen IV. The antibody binds either to the collagen IV in the soluble phase or to that of the solid phase, hence the notion of competition. A wash step eliminates reactants in the soluble phase, and an anti-rabbit IgG HRP antibody conjugate is subsequently added. This conjugate binds to the rabbit antibody that is bound to the solid phase. A wash step removes unreacted conjugate and a chromogenic substrate for HRP (horseradish peroxidase) is then added. After a short incubation period, the color development is stopped with acid, and absorbances in the wells are determined at 450 nm using a microtiter plate reader. Because the assay is a competitive one, color intensity is inversely proportional to the logarithm of collagen IV concentration of the sample (in the soluble phase).
The sensitivity and precision of the assay are dependent upon the duration of the primary incubation period. Using an incubation of at least an hour will lead to a usable assay that will measure down to approximately 0.156 ug collagen
IV/mL. However, increasing the incubation of specimen and rabbit anti-collagen IV Ab to 24 hours will improve the slope of the dose response, and will increase sensitivity down to 0.078 ug/mL or better.
Specimen Collection and Storage: Samples should be collected and frozen/stored at ≤ -50°C (without preservative) until they are analyzed. Assay results are not affected by common extraction reagents or protease inhibitors. They should come to room temperature immediately before analysis. A room temperature waterbath is especially useful for this step. Samples should be vortexed thoroughly after thawing. They should be allowed to settle for an hour, and aliquots for analysis should be drawn from the top of the fluid column to preclude introduction of particulates into the assay plate
Assay Procedure: Allow reagents and samples to come to room temperature before running the assay.