Test Procedure performed Volume (µL) Age (wk) Data collected 1 Blood collection ~275 9 - 2 Blood is processed for plasma collection ~150 9 - 3 Plasma is frozen for shipment to testing facility ~150 9 4 Measurement of plasma fibronectin using ELISA - 9 plasma fibronectin
- Refrigerated tabletop micro-centrifuge: Eppendorf Centrifuge, Model # 5415C.
- Tecan GENios Pro microplate reader
- Affinity chromatography system using Gelatin-Sepharose
- Microhematocrit tubes: uncoated/plain 75 µL volume size (Fisherbrand®), and equipped with a rubber bulb expunger
- Blood collection tubes: 1.5mL Eppendorf microcentrifuge tubes
- Anticoagulant: 3.2% Sodium citrate in sterile distilled water
- Phosphate-buffered saline (PBS): 10mM NaCl, 155mM KCl, 10 mM glucose, 1 mM MgCl2, 2.5 mM KHPO4, pH 7.4, for anticoagulant
- PBS (7 mM Na/KHPO4, 150 mM NaCl, 2.7 mM KCl, pH 7.4) for coating microtiter plates with antibody
- PBS containing 0.05% Tween detergent (PBS-T)
- 2% Bovine serum albumin (BSA) blocking reagent
- 1 mM EDTA
- EZ-link Sulfo-NHS-Biotin (Pierce, Rockford, IL) is biotinylated with antibodies used in detecting bound mouse fibronectin
- Streptavidin-conjugated alkaline phosphatase (ZYMED, San Francisco, CA)
- p-Nitrophenyl phosphate tablets (Sigma (St. Louis, MO), used as alkaline phosphatase substrate
- Mouse plasma fibronectin standard (Pel-freez, Rogers, AR)
- Microtiter, flat-bottom, high binding 96-well plates (COSTAR, Corning, NY)
Acclimation to test conditions
In general all mice are acclimated in the mouse rooms where blood samples are collected.
I. Plasma sample acquisition and preparation
a. Approximately 275 µL of blood sample is collected from 9-wk old mice via retro-orbital eye bleeding using uncoated hematocrit tubes.
b. The blood is collected into 1.5 mL Eppendorf tubes and is mixed with 30 µL of 3.2% Sodium Citrate in sterile distilled water.
c. The blood samples are then centrifuged for 20 min at maximum speed (15,000 RPM) to separate the plasma containing fibronectin.
d. The top plasma sample layers are carefully transferred into another 1.5 mL microcentrifuge tubes and then frozen twice (first at -20°C then at -70°C) for subsequent shipment to testing facility. The bottom packed red cell layers are discarded.
e. Frozen plasma samples are placed in a warm water bath and completely thawed.
f. Plasma samples are run in duplicates and listed values for each mouse is the average of 2 dilutions that fall on the linear scale of a generated standard curve.
II. Measurement of mouse plasma fibronectin using ELISA
A direct sandwich ELISA was developed for the analysis of mouse fibronectin in plasma that utilized affinity purified rabbit antibodies to human fibronectin. These antibodies recognized intact mouse fibronectin as determined by ELISA and immunoblotting (see figure below).
a. Microtiter plate wells are coated with 0.25 µg per well of affinity-purified anti-fibronectin antibodies in PBS overnight at 4°C.
b. Wells are then washed 3 times with PBS containing 0.05% Tween (PBS-T) and blocked with 2% BSA in PBS-T for 2 h.
c. Blocking and substrate reagents are added at 100 µl per well, whereas all other additions are 50 µl per well.
d. All incubations, except coating plates with capture antibodies are performed at room temperature.
e. Mouse fibronectin used to generate a standard curve and plasma samples from JAX mice are diluted in 2% BSA in PBS-T containing 1 mM EDTA and added to wells coated with capture antibody.
f. Following 2 h of incubation and 4 washes with PBS-T, wells are incubated with 0.1 µg per well of biotinylated anti-human fibronectin IgG for 2 h.
g. Wells are washed 4 times with PBS-T and reacted with 1:2000 dilution of streptavidin conjugated to alkaline phosphatase for 1 h followed by 4 washes and incubation with alkaline phosphatase substrate.
h. Plates are read at 405 nm wavelength in microplate reader.
Schematic representation of the sandwich ELISA method.
Notes from submitting investigator: Dilutions for standard curves were: 500, 250, 125, 63, 32, 16, 8 ng/mL. Samples were extrapolated from the linear portion of the logarithmic standard curve. Typically, the detectable range was from 1.6 to 6.25 ng (3.5 -13.9 pmoles) of mouse fibronectin. Plasma samples were diluted 1:2000 and 1:4000 to be able to assess values in the measurable range of the assay. An internal mouse plasma standard (obtained from Pel-freez, Rogers, AR) was measured on each plate with a mean fibronectin concentration of 174 µg/ml +/- 50 SD, n=23, coefficient of variation= 29%. The value of fibronectin concentration in Pel-freez plasma was validated by both indirect ELISA and immunoblotting.
Mouse plasma fibronectin (µg/mL).