| Bone alkaline phosphatase(BAP)is a biochemical marker of bone metabolism.Detection of neonatal bone alkaline phosphatase(NBAP)levels in serum is often used to diagnose,assess and screen rickets for children.Anti-NBAP monoclonal antibodies(anti-NBAP mAb)with high affinity and specificity are important for establishing an immunological assay for detection of NBAP.In this study,NBAP was used as immunogen to generate anti-NBAP mAb.The titer of serum from BALB/c mice was monitored from the second immunization and reached up to 1,024,000 after the fifth immunization.Then spleen cells were isolated from the immuned BALB/c mice,followed by fusing with mouse myeloma cells(SP2/0).After a series of steps such as HAT medium screening,ELISA screening and cell cloning,73 kinds of monoclonal cell strains were obtained which were able to secrete anti-NBAP mAb stably.35 kinds of ascites containing anti-NBAP mAb were obtained from the cell strains.Then the ascites were purified by octanoic acidammonium sulfate method and the concentration of the purified antibody was determined by ultraviolet spectrophotometry.Most of the anti-NBAP mAbs reached an ideal titer over 10,000 by the indirect ELISA.The cell supernatants,ascites,and purified antibodies were compared,and the results showed that most of the 38 cell strains had good viability and genetic stability.The monoclonal cell strains were able to produce ascites and secrete anti-NBAP mAb.The anti-NBAP antibody with high affinity exhibited a promising application for rapid detection of NBAP.In this study,11 kinds of purified monoclonal antibodies were selected to run a pair matching experiment on the immunochromatographic test strip using the doubleantibody sandwich principle.Three anti-NBAP mAbs(3-19-C8,3-12-B6 and 1-16-B2)with good matching results were selected for fabricating colloidal gold immunochromatographic test strip(CG-ICTS).The mAbs of 3-12-B6 and 1-16-B2 were labeled with colloidal gold,respectively.The mAbs(3-19-C8 or 3-12-B6)and goat anti-mouse IgG were sprayed on nitrocellulose(NC)membrane as test line(T line)and control line(C line),respectively.NBAP was successfully detected by CG-ICTS in phosphate buffer saline(PBS)and artificial serum,respectively.The optimal experimental conditions for CG-ICTS of NBAP were as follows: the labeling pH of the both two mAbs was 6,the concentration of two mAbs was both 5 ?g/mL,the spray volume of two labeled-m Abs conjugates was 7 ?L/cm,the concentration of mAbs(3-19-C8 or 3-12-B6)on the T line was 2.0 mg/m L,and the immunoreaction time of CGICTS with mAbs 3-19-C8 and 3-12-B6,mAbs 3-12-B6 and 1-16-B2 respectively was 15 min and 10 min,respectively.Under optimal conditions,the limit of detection(LOD)of CG-ICTS with mAbs 3-19-C8 and 3-12-B6,mAbs 3-12-B6 and 1-16-B2 was 5 pg/mL and 10 pg/m L in PBS,and 5 pg/m L and 25 pg/mL in artificial serum,respectively.The CG-ICTS with the lowest LOD was selected for the specificity experiment and recovery experiment.The results of specificity experiment showed that CG-ICTS showed a low cross-reactivity rate against with adult bone alkaline phosphatase(ABAP)and intestinal mucosa alkaline phosphatase,and no crossreactivity was shown with placental alkaline phosphatase.The results also revealed that differences may exist in the antigen epitopes between NBAP and ABAP.The average recoveries for intra-and inter-assays ranged from 96.2% to 108.6% and 94.2% to 101.0%,respectively.The coefficient of variations(CVs)for intra-and inter-assay were less than 15%.In conclusion,the established assay was simple,convenient,sensitive,specific,and stable enough.Thus,it possessed a potential application prospect for the detection of NBAP in relevant samples. |
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