Preparation Of Monoclonal Antibodies Against Human ARMET And Establishment Of Detection Method By ELISA

In public database, ARMET (Arginine rich, mutated in early stage of tumors) was also designated as ARP (arginine-rich protein) or MANF (mesencephalic astrocyte-derived neurotrophic factor). So far, only few reports associated with ARMET are availabe and its detailed function is still unclear. Among the 30 000 genes we screened by microarray, ARMET was the most sensitive one upregulated by endoplasmic reticulum stress (ER-stress). Our recent data indicates that ARMET, induced by cerebral ischemia, is widely expressed in neurons and glia cells. However, not like its denomination, astrocytes are not the main source of ARMET. Therefore, we think MANF is not a proper name for ARMET. Since ER stress is involved in the on-set of many diseases, ARMET, as a secreted protein, may be an serological marker for the early diagnosis. Therefore, it is very important to establish a good rapid clinic serological detection method for ARMET.OBJECTIVE:To prepare monoclonal antibodies against human ARMET (mAb) and establish standard procedure for ARMET detection by ELISA.METHODS:pET28a-ARMET was transformed into E.coli BL21. The His-tagged fusion protein was expressed under IPTG induction and further processed for affinity purification by nickle beads. After running SDS-PAGE gel and staining Commassie Blue, the images were taken and the purity of ARMET was analyzed by BandScan. The concentration of ARMET was measured by bicinchoninic acid protein assays (BCA).Female BALB/c mice were immunized with purified ARMET protein. The mAb against ARMET was prepared by hybridoma technology. The hybridoma cells producing mAbs against ARMET were injected into mouse peritoneal cavity and mAbs were harvested from ascitic fluid. Indirect ELISA was used to confirm the titrations and subtypes of the mAbs. The mAbs were purified by caprylic acid-ammonium sulfate precipitation (CA-AS) and affinity chromatography. Antibody specificity was analyzed by immunofluorescence and western blot.Regular sandwich ELISA test procedure was carried out by using serial diluted mAb from clone 2G4 for coating, serial diluted purified ARMET, serial diluted rabbit anti-ARMET polyclonal antibodies, and HRP labeled anti-rabbit antibody. Equal volumne of PBS was used as negative control for each step. The optimal dilution for ARMET mAb, purified ARMET protein and ARMET polyclonal antibodies were used to establish the sandwich ELISA test kit.The efficacy and sensitivity of the established kit were tested by detecting peripheral blood samples from normal male and female rats, the model rats with cerebral ischemia and arthritis, and human beings. RESULTS:1. Expression and purification of ARMET We successfully expressed soluble ARMET from E.coli and obtained a higher purity and concentration of fusion protein by affinity purification.2. Preparation and characterization of monoclonal antibodies against human ARMET We obtained 7 hybridoma cell lines which can secret specific anti-ARMET mAbs. The titers of 7 mAbs in ascitic fluid were 1×10–5~1×10–7.3. Preparation of ELISA test kit The ARMET minimum detection limit of sandwich-antibody ELISA is 5ng/ml, the standard curve has a good linearity in the range of 5ng/ml ~ 100ng/ml.4. The levels of ARMET in normal male and female SD rats The serum ARMET levels of normal specific pathogen free (SPF) male SD rats were 1.25μg/ml~2.3μg/ml, mean level was (1.67±0.46)μg/ml (n = 5). The serum ARMET levels of normal female SD rats of SPF class were 3.18μg/ml ~ 5.08μg/ml, mean level was (4.22±0.77)μg/ml (n = 5).5. ARMET levels in the peripheral blood of cerebral ischemia rats The SD rats used in cerebral ischemia experiments were conventional. In the sham group, serum ARMET values were 0.4μg/ml~2.8μg/ml, average value was (1.66±1.01)μg/ml (n = 4). In the ischemic group, serum ARMET values were 0.23μg/ml~3.91μg/ml, average value was (1.74±0.93)μg/ml (n = 15).6. ARMET levels in the peripheral blood of arthritic rats The SD rats used in experimental arthritis were conventional. ARMET levels were measured on 2, 7, 14, 21 and 28 days after inflammation, respectively. The corresponding ARMET values were 0.5μg/ml~1.2μg/ml, 0.7μg/ml~1.8μg/ml, 1.49μg/ml~2.42μg/ml, 0.7μg/ml~2.05μg/ml, 0.6μg/ml~1.70μg/ml. The corresponding mean values of the blood ARMET concentration were (1.02±0.39)μg/ml, (1.2±0.42)μg/ml, (1.92±0.41)μg/ml, (1.25±0.53)μg/ml, (1.16±0.41)μg/ml ( n = 4~6).7. ARMET levels in the peripheral blood of the arthritic rabbits ARMET levels of normal female rabbits were 1.67μg/ml~5.78μg/ml. Mean level was (4.21±2.21)μg/ml (n = 3). The range of ARMET levels in model rabbits with arthritis were 178μg/ml~621μg/ml. Mean was (319.46±197)μg/ml (n = 5). The range of ARMET in arthritis models treated with dexamethasone was 134μg/ml~554μg/ml. Mean was (272.3±190)μg/ml (n = 5). The range of ARMET in arthritis models treated with gadolinium chloride was 87μg/ml~550μg/ml. Mean was (206.45±179.39 )μg/ml (n = 5).8. The levels of ARMET in human peripheral blood ARMET in the peripheral serums of normal adult men and women was not detectable (20 males, 20 females), ARMET in the peripheral serums of 100 cases of cancer patients was not detectable too.CONCLUSIONS:The mAbs against ARMET have been prepared successfully. We also set up standard procedure for the detection of ARMET in blood samples using sandwich ELISA kit we've made.