Isotope GFR was performed at the Department of Nuclear Medicine at IALCH by a qualified radiographer. Fasting plasma glucose (FPG) was assayed by a hexokinase method. Serum creatinine was assayed by a kinetic Jaffé method standardised against isotope dilution-mass spectrometry (ID-MS). Albumin was assayed by a colorimetric method. Microalbumin was assayed by an immunoturbidometric method. All assays were performed on a Hitachi 917 analyser (Roche Diagnostics, Indianapolis, USA). Haemoglobin A1c (HbA1c) was assayed using ion-exchange high-performance liquid chromatography on a VARIANT II analyser
(BIO-RAD, CA). Urinary protein was assayed using SIEMENS Multistix ® 10SG strips (Siemens Healthcare Diagnostics Inc, Tarrytown, USA). Clinical proteinuria was indicated if protein excretion was greater than 0.5 g (500 mg) per day (test strip result of 0.3 g/L). Patients were screened for proteinuria by dipstick; if this was negative, then MA was tested. In control subjects it was assumed that proteinuria was absent so all controls were tested for MA.
2.1.6 Sample collection and storage
At each visit, blood and urine specimens were collected from each patient as follows: two whole blood samples were collected in ethylenediaminetetraaceticacid (EDTA). In addition, two serum samples, one sodium fluoride (glucose) sample and a spot urine sample were collected. Bloods were collected aseptically using standard phlebotomy techniques and collected in the order:
serum, EDTA, glucose. The EDTA and sodium fluoride tubes were gently mixed to prevent clot formation while the serum specimens were allowed to stand at room temperature (20 to 25°) for 15 minutes to clot completely. Spot urine samples were collected aseptically and frozen within two hours of collection at –20 C. Serum, EDTA and glucose specimens were centrifuged (Biofuge primo, Heraues) at 1000 g for five minutes. Plasma from the EDTA and sodium fluoride tubes were separated into Nunc cryovials (Denmark), appropriately labelled and frozen at – 20C.
2.3.1 Assay principle
The Collagen IV H assay is a competitive indirect ELISA. Briefly, equal volumes of samples containing collagen type IV and rabbit anti-human collagen type IV were added to vials
(Figure 5). Following incubation overnight (18 to 21 hours) at room temperature (20 to 25°C), the pre-incubation step allowed binding of the antibody and antigen in solution, thereby limiting binding to coated antigen when competing antigen was present in low concentrations. Following overnight incubation an aliquote of the solution was added to the well of the ELISA plate. The antibody binds either to the collagen IV in solution or to that coating the plate (Figure 6).
This allowed measurements of collagen type IV in unconcentrated urine samples
(Cohen et al. 2001). The addition of a chromogenic substrate, 3,3',5,5’ tetramethyl-benzadine (TMB, Exocell) allowed for colour development. The reaction was stopped by the addition of 2 N sulphuric acid and the absorbance in the wells was read at 450 nm. Colour intensity of the wells was inversely proportional to the logarithm of human collagen type IV concentration in the sample. The assay was sensitive to 0.0024 µg/mL. Intra-assay and inter-assay coefficients of variation were < 10 %.
Figure 5:Schematic diagram showing the antigen-antibody complex formation following overnight incubation. Adapted from www.genwaybio.com.
Figure 6: Schematic diagram showing the binding of uncomplexed collagen IV antibody to collagen IV antigen coated on ELISA microtiter plates. Adapted from
www.genwaybio.com.
2.3.2 Assay of standards and samples
All assays were performed in 96-well microtitre plates pre-coated with collagen type IV antibody (Exocell, Philadelphia, USA) at room temperature (20 to 25°C). Plates were incubated in a moistened plastic chamber with tightly fitting lid unless otherwise specified. The stock human collagen type IV standard (Exocell, 20 µg/mL) was diluted 1 in 8 using EIA diluent (360 µL: 2 520 µL). The dilution yielded a final collagen IV concentration of 2.5 µg/mL.
The standard was serially diluted one in two using EIA diluent to yield the following final collagen IV concentrations as follows: 1.25, 0.625, 0.313, 0.156, 0.078 and 0.039 µg/mL.
To each vial was added a volume of 625 µL of goat anti-human collagen IV antibody and the tubes were vortexed briefly. The final volume (1 250 µL) for each standard concentration was sufficient for six wells (200 µL/well). The standards were incubated overnight (21 hours) at room temperature and assayed the following day.
Samples (patient and control) were removed from the freezer and thawed for 10 minutes in a water bath containing tap water at room temperature (20 to 25°C). Samples were centrifuged (Biofuge primo, Heraues) for five minutes at 1000 g. Equal volumes of sample supernatant and primary antibody (Exocell) were incubated in an Eppendorf tube (Denmark) and allowed to react overnight (18 to 21 hours).
To start the assay, pre-coated plates were washed 10 times using EIA buffer (as specified previously) and blotted by inverting onto absorbent paper towel. A total volume of 200 µL of the standard or sample mixtures were added to the wells. Each standard was pipetted into six wells, samples were tested in duplicate. The plates were incubated for one hour. Following incubation, the plates were washed as described above, 100 µL of rabbit anti-goat IgG horseradish peroxidase (HRP) conjugate was added to each well and the plates incubated for one hour.
The plates were washed as described above and 100 µL of TMB substrate was added to each well. The plates were incubated for a further five to ten minutes for colour development. The colour development was stopped by the addition of 100 µL of acid stopper (2 N sulphuric acid).
The assay plate was read on a PowerWaveXS microplate reader (BIOTEK, Lionheart Technologies Inc, USA) at 450 nm with blanking against wells A1 and A2. The blank wells containing EIA diluent were used as a negative control to ensure that there was no non-specific binding of reagents to the coated plate.
A standard curve was plotted on a log scale, using the KC4 software provided with the microplate reader to generate a dose-response curve. The average optical density (OD) of six wells for the standards were used to generate a standard curve, which was used for subsequent assays to determine the concentration of collagen type IV in each sample. In subsequent assays, one of the dilutions of the stock standard used to generate the standard curve was used as internal quality control to check assay validity. Blanks were included in each assay to ensure that there were no non-specific reactions occurring between reagents. Patient samples tested previously were run with the next assay to confirm assay reproducibility.