[Experimental study on ultra-filtration in reducing matrix effects of recombination human acellular dermal matrix immersion on residual bovine serum albumin measurement by ELISA].
OBJECTIVE
To investigate the effect of ultra-filtration on reducing the matrix effects of the immersion of recombination human acellular dermal matrix (rhADM) on detecting residual bovine serum albumin (BSA) by ELISA.


METHODS
Preparation of rhADM immersion: rhADM were rinsed, and then rhADM immersion were prepared. Physiological saline was used as immersion medium. Presaturation and ultra-filtration: marked the ultra-filtration tubes as PR1 (presaturation protocol 1), PR2 (presaturation protocol 2) and rhADM, respectively, added 2 mL of 1 mg/mL and 10 microg/mL BSA solution into PR1 and PR2 respectively, and added 2 mL of rhADM immersion into rhADM tubes (rhADM1 and rhADM2). The tubes were then centrifuged at 1500 x g for 20 minutes. The above steps were repeated for 3 times. Take the inner-tube of ultra-filtration into unused centrifuge tube. Added 4 mL of 10 microg/mL BSA solution in PR1 and PR2 tubes, 4 mL of rhADM immersion in rhADM tubes, centrifuged at 1500 x g for 20 minutes, and then the filtration was collected. Detecting BSA concentration: the BSA concentrations of all samples were detected by using the quantitative measure of residual BSA ELISA kit. The recoveries of 10 microg/mL BSA solution treated by presaturation protocol 1 and 2 were calculated (untreated 10 microg/mL BSA solution was as the basic sample, marked R10 and R20 respectively). The correlation coefficient between the logarithm of the filtrate dilution and the absorbance (A) value was calculated and compared with that of water exact without ultra-filtration.


RESULTS
The BSA concentration of PR1 and R10 was (23.80 +/- 1.58) microg/mL and (9.04 +/- 0.24) microg/mL, respectively. The BSA concentration of PR2 and R20 was (8.64 +/- 0.24) microg/mL and (8.12 +/- 1.01) microg/mL, respectively. The average recovery of 10 microg/mL BSA was 263.4% +/- 16.9% and 106.5% +/- 3.0% when the ultra-filtration tubes were presaturaed by PR1 and PR2 (P < 0.01), respectively. The BSA recovery of PR2 met the detecting demand. The correlations between A value and sample dilution were increased, the correlation coefficient was raised from -0.727 to -0.960 after rhADM immersion were treated by ultra-filtration.


CONCLUSIONS
The results show that the matrix effects can be reduced effectively by ultra-filtration, indicating that an acceptable recovery of BSA can be acquired when ultra-filtration tube is presaturated by sample water extract.
Fang
Yu
Y
Center of Medical Devices, National Institutes Control for Pharmaceutical & Biological Products, 100050, P.R. China. fangyu@nicpbp.org.cn
Du
Xiaodan
X
Xi
Tingfei
T
Feng
Xiaoming
X
D000818:Animals; D002417:Cattle; D020405:Dermis / Q000737:chemistry*; D004350:Drug Residues; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005109:Extracellular Matrix / Q000737:chemistry; D006801:Humans; D011995:Recombination, Genetic; D012710:Serum Albumin, Bovine / Q000032:analysis*; D014461:Ultracentrifugation
D012710:Serum Albumin, Bovine