Preparation Of Monoclonal Antibody Against Recombinant Human Augmenter Of Liver Regeneration And Preliminary Exploration Of Clinical Detection

Objective In the model of rat with acute kidney injury, ALR (augmenter of liver regeneration, ALR) could reduce renal injury and renal pathological changes, and promote regeneration of renal tubular epithelial cells. In order to establish a specific and effective method to detect hALR, we tried to prepare monoclonal antibody (McAb) against recombinant human augmenter of liver regeneration (rhALR), detect the hALR levels in Serum and urine of patients with AKI preliminarily.Methods Under the induction of methanol the rhALR was expressed in GS115 and purified with ultrafiltration. We used SDS-PAGE and Western blot to identify the purified product. The proliferation of HepG2 hALR effect was evaluated with vitro 3H-TdR methods. Balb/c mice were immunized with rhALR. We used the cells of mouse myeloma cell line SP2/0 to fuse the of the immunized mice Splenocytes. We used limited dilution method to clone the hybridoma cells, and the cells that secreted anti-rhALR protein McAb antibodies. The affinity of the obtained McAbs and the ascites titers were determined with indirect ELISA. The McAbs specificity was tested by which the western blot analysis and indirect ELISA. The immunoglobulin (Ig) subtype was test by IsoQuick strips , then analysis the Chromosome Assay of the hybridomas. Serum and urine hALR levels in patients with AKI was detected by indirect ELISAResults The rhALR was efficiently expressed as a secretive protein in GS115.the molecular weight of it was 1.5×10~4 dalton. after ultrafiltration,single band of rhALR was found in Western blot and SDS-PAGE. the proliferation of HepG2 was promoted by the rhALR with a dose-dependent way within vitro. 1 of hybridomas was screened out, designated 2A6. The McAb to rhALR was stably secreted by the hybridoma for more than three months. Analysis of Chromosome discovered that the obtlained hybridomias were with the unioversal characlteristics of the monclonal hybridoma cells,the cell which secreted McAb, and the 2A6 McAb subtype of Ig was IgG2b, and the lightchains was kappa. The ascity titers of 2A6 McAb was 1:106. The 2A6 McAb relative affinity was determined as 105.Indirect ELISA and Western blot suggested that the McAbs specifically reacted with rhALR protein. The hALR levels of Serum and urine by indirect ELISA of patients with AKI was negative.Conclusion One in high titer, specific McAb which against rhALR protein had been successful acquired. The preliminary exploration in Serum and urine hALR levels of patients with AKI was negative. With only one hybridoma, the colloidal gold test strip could not eatablise. The indirect ELISA could not serve as an sensitive detection measure for clinic. We need to eatablish a specific and effective method to detect hALR, which could apply to clinical patients.