Objective To establish a rapid immunological detection method for MPT64 protein based on red microspheres and select highly-sensitive and highly-specific antibody pairs.Methods A His-tagged prokaryotic expression vector was constructed for expression of MPT64 protein that was used to immunize New Zealand white rabbits after purification and validation.Peripheral blood mononuclear cells(PBMCs)were isolated from the rabbits,and antigen-specific B cells expressing antibodies were sorted using single B-cell flow cytometry.mRNA in B cells was reverse-transcribed into cDNA,and paired antibody heavy-and light-chain sequences were amplified via nested PCR.Expression vectors were constructed,and recombinant antibodies were produced in Expi293F cells.Fluorescent immunochromatography was employed to screen for matched antibody pairs.The selected antibodies were used to establish a rapid detection method based on red microsphere immunochromatography.Results Ten high-affinity monoclonal antibodies against MPT64 were generated.Two antibody pairs were selected for MPT64 immunodetection that reached a sensitivity of 0.0125 ng/mL.Conclusion High-affinity rabbit monoclonal antibodies against MPT64 are obtained via single B-cell technology,and a rapid red microspheres-based immunodetection method is established,enabling highly sensitive detection of Mycobacterium tuberculosis MPT64 protein.

BACKGROUND: Magnetic resonance diffusion tensor imaging (MRDTI) may non-wounded detect damage of fiber in white matter and becomes an effectively way to evaluate upper motor neuron(UMN) impairments.OBJECTIVE: To investigate the clinical significance of MRDTI on amyotrophic lateral selerosis(ALS).DESIGN: Case contrast observation.SETTING: Department of Neurology, Renmin Hospital of Wuhan University.PARTICIPANTS: Twenty ALS patients were selected from Department of Neurology, Renmin Hospital of Wuhan University from April to December 2005. There were 11 males and 9 females, and their ages ranged from 33 to 73 years with the mean age of (51±10) years. All subjects met the diagnostic criteria of ALS set by World Neurology League.Other 15 healthy subjects were collected as control group. There were 8 males and 7 females, and their ages ranged from 31 to 73 years with mean age of (50±11) years. All subjects provided the confirm consent.METHODS: Based on level of upper and lower motor neuron impairments, ALS patients were divided into UMN impairment group (n =16) and lower motor neuron group (n =4). Functional scores of ALS, illness developing velocity and pyramidal sign scores were performed, respectively. All subjects were scanned with DTI at axial view. Regions of interest [subcortical white matter of precentral gyrul and postcentral gyrul (Pre-CG/Post-CG), centrum semiovale and frontal white matter (CS/FWM), peripheral lateral cerebral ventricle, posterior limb of internal capsule (PIC), cerebral peduncle (CP), genu corpus callosum and splenium corpus callosum (GCC/SCC) and dorsal thalamus (DT)] were selected to measure fractional anisotropy (FA) and apparent diffusion coefficient(ADC).MAIN OUTCOME MEASURES: Correlations among FA, ADC, functional score of ALS, illness developing velocity and pyramidal sign scores.RESULTS: Twenty patients and 15 subjects in the control group were involved in the final analysis. ① FA was reduced and ADC increased in the posterior limb of the internal capsule in patients with UMN signs compared to healthy volunteers (t =3.452, 2.670; P ＜ 0.01, 0.05). Nonparametric tests revealed that there was a trend toward reduced FA in the posterior limb of the internal capsule in B group compared to controls (U =11, P =0.057). ② In UMN impairment group, FA in the posterior limb of the internal capsule was positively correlated with the ALS rating scale (r =0.577, P ＜0.05) and negatively correlated with pyramidalsign scores (r = -0.789, P ＜ 0.01 ),CONCLUSION : The impairment of pyramidal tracts can be noninvasively evaluated by diffusion tensor MR in vivo, thus providing useful information in diagnosing and further understanding MND.