The UCP-LF antigen assay utilizes 20 μL TCA supernatant (after centrifugation) of a serum/urine sample mixed with an equal volume of 4% TCA (w/v). Note that TCA extraction effectively removes interfering proteins and dissociates potential immune complexes (de Jonge et al. 1987 (link)). In previously described assays, the TCA supernatants were neutralized (in analogy with the CAA- and CCA-ELISAs), but this was not necessary for the UCP-LF assay. Omission of the neutralization step and a two-fold increase of the sample input (20 μL TCA supernatant instead of 10 μL) increased analytical sensitivity of the method by a factor of four as compared with the method used in previous studies (Corstjens et al. 2008 ; van Dam et al. 2013 (link)). Quality control (QC) and standard dilution series were prepared spiking AWA-TCA in PBS or high salt lateral flow assay buffer (HSLF: 100 mm HEPES pH 7·5, 270 mm NaCl, 0·5% v/v Tween-20, 1% w/v BSA), or in negative human serum (NHS) or urine. QC and standards received the same TCA treatment as the clinical samples. In the antigen assay 20 μL TCA-supernatant is mixed with 100 μL HSLF containing 100 ng UCP coated with the appropriate antibody (anti-CAA or antiCCA) and incubated for 1 h, 37 °C at 900 rpm. LF strips with the appropriate capture zones (T lines) are then applied to the tubes or microtitre plate wells with the UCP mixture and immunochromatography is allowed to proceed for at least 20 min. After drying, the LF strips are scanned as described below for the UCP-CF antibody format.
The above described assay format is ‘available’ in a wet and dry format. In the dry format the UCP reporter is provided as a dry material, 100 ng per tube. The dry material is simply hydrated by adding 100 μL HSLF assay buffer. The wet assay format includes a sonication step of the UCP stock solution. The UCP stock is provided as a 1 mg/mL suspension in UCP storage buffer (50 mm glycine, 0·03% v/v Triton X-100, 0·1% w/v NaN3, pH 8·0); after homogenization the desired amount is sonicated (1 min, water bath sonicator, 100 W) in HSLF assay buffer at a concentration of 1 μg per 100 μL (enough for 10 assays). After sonication the UCP mixture is further diluted to 100 ng per 100 μL HSLF.
The above described assay format is ‘available’ in a wet and dry format. In the dry format the UCP reporter is provided as a dry material, 100 ng per tube. The dry material is simply hydrated by adding 100 μL HSLF assay buffer. The wet assay format includes a sonication step of the UCP stock solution. The UCP stock is provided as a 1 mg/mL suspension in UCP storage buffer (50 m
