Enzyme immunoassay kit

Proteinuria and urine creatinine were determined in urine samples by an autoanalyzer Spin120. Plasma creatinine, urea and glucose were also measured in this instrument. Reagents for proteinuria, urea, and creatinine-Jaffé method were provided by Spinreact (Barcelona, Spain). Plasma and urinary Klotho levels were determined with an enzyme immunoassay kit, Bioassay Technology Laboratory (Shanghai, China). Renal and urinary hydroxyproline was measured with a Hydroxyproline Assay Kit (Sigma-Aldrich, Madrid, Spain). Protein content in renal tissue was analyzed with DC Bio-Rad protein assay (Madrid, Spain).

“Favor Prep Blood Genomic DNA Extraction Kit (Biotech Corp, Taiwan China)” was used for extracting DNA. Polymerase chain reaction (PCR) was employed to amplify DNA using specific primers (Macrogen Inc Korea) designed against the polymorphic cytosine–adenine repeat 1kb upstream of the human IGF-1 gene, i.e., sense: 5’-ACCACTCTGGGAGAAGGGTA-3, antisense: 5-GCTAGCCAGCTGGTGTTATT-3’. PCR amplification reactions were carried out in a 25μL reaction mixture which contained, 1μl (50 ng) of genomic DNA, 0.5 μl of each primer (5pM), 12.5 μl of 2x PCR master mix and 10.5 μl double distilled water. Amplification was performed on BioRad thermal cycler (iCycler, BioRad USA). The cycling conditions for the PCR were a cycle of initial denaturation at 94ºC for five minutes, followed by 35 cycles with denaturation for one minute at 94ºC, annealing at 58ºC for 60 seconds, extension at 72ºC for 60 sec, and with a final extension for 10 minutes. at 72ºC. The amplicons were run for 45 minutes on 3% agarose gel. The PCR product was checked under ultraviolet gel documentation system (Bio-Rad USA) and results were recorded.
IGF-1 levels in serum samples of both RA group and healthy group was measured by Enzyme Immunoassay kit (Bioassay technology Laboratory Shanghai China) following manufacturer’s instructions on automated ELISA CODA (BioRad, USA).