Phosphate buffered saline (PBS) (Sigma P4417-199TAB), Tris (50 mM, Sigma T6066) buffered saline (150 mM, Fisher Scientific S/3161/65) with Tween-20 (0.01%, Sigma P1379) (TBST) were prepared and used for the study. Microtiter ELISA plates were procured from Costar 9018, Thermo Fisher Scientific whereas the plate reader and RDS-2500 reader were purchased from FLUOstar Omega Microplate Reader (BMG Labtech) and (DETEKT, USA) respectively. Anti-species alkaline phosphatase (A5187 and SAB3700286) was procured from Sigma, USA. The substrate pNPP was procured from BioPanda, USA. The paired antibodies used in the study are summarized in Table 5.
Immunological testing of the antibody using ELISA:
The microtiter ELISA plates (Costar 9018, Thermo Fisher Scientific) were coated with 100 µL of respective coating antibody at a concentration of 0.5/1 µg/mL in PBS and incubated overnight at room temperature. The plates were washed thrice with 300 µL TBST using a plate washer (BioTek 405). The wells were blocked with 150 µL PBST 0.1% BSA 1% and incubated at room temperature for 1 h on a shaker. The blocking solution was discarded, the plates were washed thrice with 300 µL TBST and incubated at room temperature with respective 100 µL antigen for 1 h on shaker. After discarding the antigen solution, the plates were washed thrice with 300 µL TBST and incubated with the respective 100 µL detection antibody (0.5/1 µg/mL in PBST 0.1% BSA 1%) for 1 h at room temperature on shaker. Post incubation, the antibody solution was discarded and following a final wash, the plates were incubated at room temperature with 100 µL of respective anti-species alkaline phosphatase at a dilution of 1:30 k/1: 10 k for 1 h on shaker at room temperature. The plates were incubated at room temperature in the dark with 100 µL pNPP and the absorbance was read at 405 nm after 30/45 min depending on the assay.
Conjugation buffer pH optimization and conjugation concentration optimization
1.5 µL of the detection antibody (1 mg/mL) was mixed with 5 µL of conjugation buffer (pH spanning from 5.3 to 9.3—20 mM MES pH 5.3, 20 mM BES pH6.4, 20 mM TES pH7.1 and 7.5, 20 mM TAPS pH 7.8 and 8.5, 20 mM Borate pH 9.0 and 9.3) and 0.1 mL colloidal gold (EM.GC40, BBI Solutions) in microtiter ELISA wells (StarLab, E2996-1600) on a plate shaker for 10 min. The absorbance was read at 530 nm, 550 nm and 600 nm on FLUOstar Omega Microplate Reader (BMG Labtech). The aggregation ratio of Abs 550/600 was calculated. To determine the antibody loading concentration, 5 µL of the selected buffer was mixed with varying volumes of the antibody solution (1 mg/mL) to obtain a loading concentration of 0–30 µg/mL. To this mixture, 100 µL of colloidal gold was added and incubated for 10 min. The mixture was subjected to a salt challenge by adding 10 µL of 1 M NaCl. The absorbance was read at 530, 550 and 600 nm on FLUOstar Omega Microplate Reader (BMG Labtech). The aggregation ratio of Abs 550/600 was calculated.
Lateral flow strip development
The lateral flow test strip consisted of a sample pad (Ahlstrom-Munksjo, USA, Product 8964), conjugate pad (Ahlstrom-Munksjo, USA, Product 8964), CN140 nitrocellulose test membrane (Sartorius, 1UN14ER100025NT USA) and a wicking adsorbent pad (Ahlstrom-Munksjo, USA, Product 222) onto a backing card (Kenosha, Netherlands, product KN-2211). For the UMOD assay, a biotin—polystreptavidin (PSA) format was used to design the lateral flow strip. The test and control lines were prepared by spraying (1 mg/mL polystreptavidin-R, BioTez, Berlin). The membranes were incubated at a temperature of 50 °C for 5 min. The membranes were dried at room temperature in a desiccator to remove excess moisture. The LFD was assembled and stored until further use.
For the OPN assay, the test strips with capture antibody at a concentration of 0.5 mg/mL were prepared by mounting the CN140 nitrocellulose membrane (Sartorius, 1UN14ER100025NT USA) onto a backing card (Kenosha, Netherlands, product KN-2211) with a conjugate pad (Ahlstrom-Munksjo, USA, Product 8964) containing the detection antibody (15 µg load level, 5 OD/device) sprayed down as a gold conjugate. The membranes were incubated for 5 min at 50 °C to remove most of the moisture and further dried at room temperature in a desiccator. The LFD was assembled and stored until further use.
Lateral flow device (LFD) testing
Using recombinant protein standards
A recombinant protein standard curve (hereafter referred to as standard) was generated for UMOD (0–2,000,000 pg/mL) and for OPN (0–1,000,000 pg/mL) on a biotin/polystreptavidin based lateral flow device and standard lateral flow device for UMOD and OPN respectively. The UMOD and OPN standards were prepared and filled in a custom-made diluent tube (Fig. 4). Three drops of the respective standards were added to the devices and incubated on a flat surface for 20 min at room temperature. The results were read using a handheld RDS-2500 reader (DETEKT, USA) in the form of control line (CL) and test line (TL) intensity. A ratio of the TL/CL intensity was computed to analyze the results.
Lateral flow device assembly kit for urinary biomarkers Uromodulin (UMOD) and Osteopontin (OPN). (Left to Right) Biotin/Polystreptavidin (PSA) based LFD for UMOD, Standard LFD for OPN, Diluent tube and Disposable pipette.
Human urine samples
Human urine samples (n = 5) were collected from deceased organ donors and tested for UMOD and OPN in duplicates. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by Johns Hopkins Institutional Review Board (IRB no: IRB00248332). Informed consent was obtained from next of kin and family. For the measurement, the urine samples were centrifuged at 2000 rpm for 12 min. 50 µL of the centrifuged sample was pipetted using a custom-made pipette and added to the diluent in the custom-made diluent tube. The diluent tube was inverted 5 times. Three drops of this diluted sample were added to the LFD and incubated for 20 min before being read on the RDS—2500 reader.
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- Source: https://www.nature.com/articles/s41598-024-59104-5