Publications by authors named "Mei-Ping Zhao"

16 Publications

  • Page 1 of 1

[The role of endoplasmic reticulum stress in pulmonary hypertension in rat induced by chronic hypoxia and hypercapnia].

Zhongguo Ying Yong Sheng Li Xue Za Zhi 2018 Apr;34(4):327-333

Department of Pathophysiology, Wenzhou Medical University, Wenzhou 325035.

Objective: To observe the pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia, and to explore the role of endoplasmic reticulum stress in pulmonary hypertension.

Methods: Forty SD rats were random-ly divided into four groups:normoxic control group (N), hypoxia hypercapnia group (HH), ERS inhibitor 4-phenylbutyric acid group (4-PBA), endoplasmic reticulum stress (ERS) pathway agonist tunicamycin group (TM), ten rats in each group.The mean pulmona-ry artery pressure (mPAP), mean carotid artery pressure (mCAP) and right ventricular hypertrophy index of rats in each group were measured.Pulmonary artery smooth muscle cells were identified by immunofluorescence α-smooth muscle actin (α-SMA).Morphologi-cal changes of lung tissue and pulmonary artery were observed by electron microscope.The apoptotic index of pulmonary artery smooth muscle cells in each group was detected by TUNEL.Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), c-Jun N-terminal kinase (JNK) and cysteinyl aspartate specific proteinase-12 (caspase-12) mRNA and protein in each group.

Results: ①Compared with the N group, the mPAP, the ratio of right ventricle weight to left ventricle plus ventricular septum weight[RV/(LV+S)]and the ratio of pulmonary artery wall area to total tube area (WA/TA) were increased (<0.01), and the ratio of pulmonary artery luminal area to total tube area (LA/TA) were decreased (<0.01), pulmonary artery smooth muscle cell apoptosis index were decreased (<0.05 or <0.01) in HH group, 4-PBA group and TM group.ERS related protein and mRNA expressions were increased, the differences were statistically significant.②Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of 4-PBA group were decreased ( <0.01), LA/TA and pulmonary artery smooth muscle cell apoptosis index were increased (<0.01, <0.05).The expressions of ERS related protein and mRNA were all decreased (<0.05 or <0.01).③Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of TM group were increased (<0.05 or <0.01), pulmonary artery middle layer thickened, LA/TA and pulmonary artery smooth muscle cell apoptotic index were decreased (<0.01).ERS related protein and mRNA expressions were increased with statistical significance except GRP78 protein.

Conclusions: Pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia may be related to the excessive proliferation of pulmonary artery smooth muscle cells and too little apopto-sis;ERS related factors (JNK, caspase-12 and CHOP) are involved in the regulation of pulmonary hypertension induced by hypoxia hypercapnia.
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http://dx.doi.org/10.12047/j.cjap.5644.2018.075DOI Listing
April 2018

[The regulation of MAPK signaling pathway on cell proliferation and apoptosis in hypoxic PASMCs of rats].

Zhongguo Ying Yong Sheng Li Xue Za Zhi 2017 Mar;33(3):226-230

Department of Pathophysiology, Wenzhou Medical University, Wenzhou 325035, China.

Objective: To explore the relationship between hypoxic pulmonary arterial smooth muscle cells(PASMCs)proliferation, apop-tosis and mitogen-activated protein kinases(MAPK) signal pathway in rats.

Methods: PASMCs were obtained from male SD rats by the enzyme digestion method and primarily cultured; PASMCs were identified through two methods:immunofluorescence staining and light microscopy; the 4~6th generation PASMCs of logarithmic growth state of good growth period were selected, and randomly divided into 7 groups:normoxic con-trol group (N), hypoxia group (H), DMSO group (D), extracellular signal-regulated kinase1/2(ERK1/2) inhibitor-U0126 group (U) and p38MAPK inhibitor-SB203580 group (S), the p38MAPK activator-Anisomycin group (A), the ERK1/2 activator-Staurosporine Aglycone group (SA). When all the models were completed, the all groups joined the CCK-8 to measure cell proliferation; cell apoptosis of each group was detected by TUNEL kit after the modeling.

Results: Compared with N group, the expression of OD value in H group was up-regulated (0.990 ±0.041 1.143 ±0.033, < 0.01). There was no statistical significance on PASMCs apoptosis index(AI) in H group (4.913 ±0.451 5.452 ±0.557, > 0.05); Compared With H group, there were no statistical significance on the expression of PASMCs OD value and apoptosis index(AI)in D group (1.143 ±0.033 1.142 ±0.049,5.452 ±0.557 5.402 ±0.651, > 0.05); the expression of OD value in U group was down-regulated, and the expression of AI was up-regulated (1.143 ±0.033 0.985 ±0.078, 5.452 ±0.557 10.145 ±2.545, < 0.01); the expression of OD value in S group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 1.295 ±0.039, 5.452 ±0.557 3.093 ±0.409, < 0.01); the expression of OD value in A group was down-regulated, and the expres-sion of AI was up-regulated (1.143 ±0.033 0.347 ±0.067, 5.452 ±0.557 25.753 ±1.262, < 0.01); the expression of OD value in SA group was up-regulated, and the expression of AI was down-regulated (1.143 ±0.033 1.685 ±0.100, 5.452 ±0.557 1.700 ±0.095, < 0.01).

Conclusions: The regulation of PASMCs' proliferation and apoptosis under hypoxia condition have a relationship with the participation of MAPK signal pathway.
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http://dx.doi.org/10.12047/j.cjap.5422.2017.056DOI Listing
March 2017

[Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia].

Sheng Li Xue Bao 2017 Feb;69(1):47-54

Department of Pathophysiology, Wenzhou Medical University, Wenzhou 325035, China.

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO, 21% O, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO, 5% O, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca] in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca] were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca].
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February 2017

Identification of Larvicidal Constituents of the  Essential Oil of Echinops grijsii Roots against the  Three Species of Mosquitoes.

Molecules 2017 Jan 27;22(2). Epub 2017 Jan 27.

Department of Entomology, China Agricultural University, Haidian District, Beijing 100193, China.

The screening of Chinese medicinal herbs for insecticidal principles showed that the essential oil of Echinops grijsii Hance roots possessed significant larvicidal activity against mosquitoes. The essential oil was extracted via hydrodistillation and its constituents were determined by gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS analyses revealed the presence of 31 components, with 5-(3-buten-1-yn-1-yl)-2,2'-bithiophene (5-BBT, 27.63%), αterthienyl (α-T, 14.95%),1,8-cineole (5.56%) and cis-β-ocimene (5.01%) being the four major constituents. Based bioactivity-directed chromatographic separation of the essential oil led to the isolation of 5-BBT, 5-(4-isovaleroyloxybut-1-ynyl)-2,2'-bithiophene (5-IBT) and αT as active compounds. The essential oil of E. grijsii exhibited larvicidal activity against the fourth instar larvae of Aedes albopictus, Anopheles sinensis and Culex pipiens pallens with LC50 values of 2.65 μg/mL, 3.43 μg/mL and 1.47 μg/mL, respectively. The isolated thiophenes, 5-BBT and 5-IBT, possessed strong larvicidal activity against the fourth instar larvae of Ae. albopictus(LC50 = 0.34 μg/mL and 0.45 μg/mL, respectively) and An. sinensis(LC50 = 1.36 μg/mL and 5.36 μg/mL, respectively). The two isolated thiophenes also had LC50 values against the fourth instar larvae of C. pipiens pallens of 0.12 μg/mL and 0.33 μg/mL, respectively. The findings indicated that the essential oil of E. grijsii roots and the isolated thiophenes have an excellent potential for use in the control of Ae.albopictus, An. sinensis and C. pipiens pallens larvae and could be used in the search for new, safer and more effective natural compounds as larvicides.
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http://dx.doi.org/10.3390/molecules22020205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155871PMC
January 2017

[The effects of ERK1/2 pathway on the expression of calcium activated chloride channel in hypoxia in PASMCs rat model].

Zhongguo Ying Yong Sheng Li Xue Za Zhi 2017 Jan;33(1):47-50

Department of Pathophysiology, Wenzhou Medical University, Wenzhou 325035.

Objective: To investigate the expression of mRNA and protein of Calcium activated chloride channel (CLCA2) in hypoxic pulmonary artery smooth muscle cell (PASMCs) of rat and it's relationship with ERK1/2 signal pathway.

Methods: PASMCs were randomly divided into 5 groups including normal group(N group), hypoxia group(H group), DMSO group(D group), U0126 group (U group) and Staurosporine aglycone group(SA group). The protein expression of CLCA2 in PASMCs was detected by Western blot.The mRNA expression of CLCA2 was detected by half quantitative reverse transcription polymerase chain reaction (RT-PCR).

Results: The mRNA and protein expressions of CLCA2 in H group were significantly higher than N group (<0.01). Comparing with D group,the mRNA and protein expressions of CLCA2 were significantly increased in U group (<0.01),the mRNA expression of CLCA2 in SA group was obviously decreased (<0.01) with slightly decreasing of its protein expression.

Conclusions: Hypoxia promotes the expressions of mRNA and protein of CLCA2 in rat PASMCs. The ERK1/2 pathway activator Staurosporine aglycone reduces the mRNA and protein expression of CLCA2 in rats PASMCs and the ERK1/2 pathway inhibitor U0126 induces the upregulation of the mRNA and protein expressiosn of CLCA2 in rats PASMCs.
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http://dx.doi.org/10.12047/j.cjap.5448.2017.011DOI Listing
January 2017

[The effects and mechanisms of ligustrazine injection on pulmonary arterial hypertension in COPD patients].

Zhongguo Ying Yong Sheng Li Xue Za Zhi 2016 May;32(5):408-412

Department of Pathophysiology, Wenzhou Medical University, Wenzhou 325035.

Objective: To observe the effects of ligustrazine hydrochloride injection(LHI) on pulmonary arterial hypertension in chronic obstructive pulmonary disease(COPD) patients and to investigate its possible mechanisms.

Methods: Twenty-two cases of patients with COPD were randomly divided into conventional treatmentgroup (group C) and ligustrazine treatment group(group L), 11 persons were randomly selected from healthy subjects without lung disease served as normal control group(group N). Group C was given bed rest, low flow oxygen inhalation, bronchial diastolic agent, glucocorticoid and antibiotics and other conventional treatment, and group L was added with ligustrazine hydrochloride injection on the above mentioned basis treatment, group N was given no treatment. After 2 weeks, lung function, blood gas analysis and pulmonary arterial pressure were compared among the three groups, and the content of HS in plasma was tested with sensitive sulfur electrode method.

Results: ①After two weeks treatment, in group L and group C pulmonary function, blood gas analysis, pulmonary artery pressure were obviously improved, and group L was better than group C (<0.05); ② In group L the content of HS was increased (<0.01), group C had no significant difference (>0.05), and there was a significant difference between the two groups (<0.01).

Conclusions: Combination with LHI can effectively improve lung function. LHI mayrelieve hypoxic hypercapnia pulmonary hypertension induced by COPD through raising the content of HS.
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http://dx.doi.org/10.13459/j.cnki.cjap.2016.05.006DOI Listing
May 2016

[Effect of ERK1/2 on rat pulmonary artery smooth muscle cells Kv1.5 channel in the process of hypoxia].

Zhongguo Ying Yong Sheng Li Xue Za Zhi 2015 Sep;31(5):418-21, 426

Objective: To explore the effect of ERK1/2 MAPK pathway on the expression of Kv1.5 channel, a voltage-gated potassium ion channel, in rat pulmonary artery smooth muscle cells (PASMCs) and its mechanisms during the process of hypoxia.

Methods: The PASMCs derived from SD rats were cultivated primarily. The third to sixth generation of PASMCs were divided into 5 groups randomly: (1) Normal group (N); (2) Hypoxic group (H); (3) Demethy sulfoxide(DMSO) group (HD); (4) U0126 group (HU): 10 micromol/L U0126; (5) Anisomycin group (HA): 10 micromol/L anisomycin. There were three dishes of cells in each group. The cells in normal group were cultured in normoxic incubator (5% CO2, 37 degrees C), the cells in other groups were added to 0.05% DMSO in the hypoxic incubator (5% CO2, 2% O2, 37 degrees C), all cells were cultured for 60 h. RT-PCR and Western blot were used to detected the espressions of Kv1.5 mRNA and protein in PASMCs.

Results: Compared with N group, the expressions of Kv1.5 mRNA and protein in H, HD and HA groups were reduced significantly (P < 0.05); Compared with H group and HD groups, Kv1.5 mRNA and protein expressions in HU group were increased sharply (P < 0.05). Compared with the HU group, Kv1.5 mRNA and protein expressions in HA groups were significantly lower (P < 0.05).

Conclusion: Low oxygen reduced Kv1.5 mRNA and protein expressions, U0126 could resistant the Kv1.5 channel lower expression caused by hypoxia. Anisomycin had no significant effect on Kv1.5 channel expression under hypoxia, but the expression of Kv1.5 was still significantly lower than the normal oxygen group. These data suggest that hypoxia may cause hypoxic pulmonary hypertension by interfering ERK1/2 signaling pathway to inhibit Kv1.5
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September 2015

A simple and rapid competitive enzyme-linked immunosorbent assay (cELISA) for high-throughput measurement of secretory immunoglobulin A (sIgA) in saliva.

Talanta 2010 Jun 22;82(1):432-6. Epub 2010 Apr 22.

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry & Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

A simple competitive enzyme-linked immunosorbent assay (cELISA) was established for rapid measurement of secretory immunoglobulin A (sIgA) in saliva. The method was based on competitive reaction between the immobilized IgA and free IgA in the solution for the limited amount of horseradish peroxidase-conjugated rabbit anti-human IgA. In comparison with the conventionally used Sandwich ELISA, the cELISA is simpler, low-cost, and shows better reproducibility since it is not affected by the variation of capture antibodies from different batches. The assay time was also significantly reduced from more than 5h to less than 3h. Different curve-fitting models were compared, among which the fully specified logit-log model gave the best results. The linear working range and limit of detection were found to be 0.1-100 microg mL(-1) and 0.05 microg mL(-1), respectively. Matrix effects of saliva samples were investigated and a reasonable range of dilution factors were proposed. The developed method offers a very practical approach for high-throughput measurement of sIgA in saliva samples.
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http://dx.doi.org/10.1016/j.talanta.2010.04.040DOI Listing
June 2010

Development of a high-throughput screening platform for DNA 3'-phosphatases and their inhibitors based on a universal molecular beacon and quantitative real-time PCR.

Chem Asian J 2010 May;5(5):1146-51

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871 China.

DNA 3'-phosphatases play a unique role in the repair of strand breaks induced by DNA damaging agents, such as ionizing radiation or oxidative stress. In this paper, we present an efficient detection system for rapid screening of DNA 3'-phosphatases and their inhibitors. A unique template substrate has been designed to hybridize with the universal molecular beacon (U-MB), and the detection process is carried out in a quantitative real-time PCR. The method is successfully applied to monitor the activity and kinetics of two typical 3'-phosphatases, that is, T4 polynucleotide kinase phosphatase (PNKP) and calf intestinal alkaline phosphatase (CIP). The inhibition effect of heparin on T4 PNKP and theophylline on CIP is also quantitatively characterized. The proposed method is demonstrated to be very useful for sensitive, high-throughput, and precise measurement of various 3'-phosphatases and their inhibitors.
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http://dx.doi.org/10.1002/asia.200900661DOI Listing
May 2010

Cupric ion enhanced molecular imprinting of bovine serum albumin in hydrogel.

J Sep Sci 2009 Oct;32(19):3359-63

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.

A novel molecularly imprinted hydrogel for bovine serum albumin (BSA) was prepared using cupric ion as the bridge between the template BSA and the functional monomer 4-vinylpyridine. N-Isopropylacrylamide (NIPA) was used as an assistant monomer to provide the stimuli-responsibility of the polymer. The adsorption conditions of BSA on the BSA-Cu(II)-imprinted hydrogel were optimized considering the influences of pH, temperature, and salt concentration. The proteins bound on the imprinted hydrogel can be easily recovered under mild conditions by using 10 mmol/L ethylene diamine tetraacetic acid (EDTA) (pH 7.0) containing 150 mmol/L NaCl as the eluting solution. The imprinting effect and adsorption capacity of the polymer were found to be significantly improved compared to the hydrogel prepared in the absence of cupric ion. The results demonstrated the advantages of using a template-metal ion-monomer coordination system to strengthen the interaction between the protein and monomer. The effects of different metals ions including Zn(II), Ni(II), Co(II), Cd(II), and Al(III) on the recognition ability of the BSA-Cu(II)-imprinted hydrogel were also investigated. The polymer showed high selectivity toward both the template protein and the cupric ion.
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http://dx.doi.org/10.1002/jssc.200900250DOI Listing
October 2009

Development of an indirect competitive ELISA for the determination of papaverine.

Talanta 2005 May;66(4):1005-11

College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China.

Papaverine (1-(3,4-dimethoxybenzyl)-6,7-dimethoxyisoquinoline, PAP) is a member of the benzylisoquinoline sub-group of the opium alkaloids. It has been widely used for treating diseases like pulmonary arterial embolism and renal or biliary colic. In this paper, a specific conjugate of mono-demethylated papaverine-O-carboxylmethyl ether (MDMPAP-O-CME) and bovine serum albumin (BSA) was synthesized and used as the complete antigen (PAP-BSA), with which we successfully obtained a high-titer anti-PAP polyclonal antibody (pAb) by immunization of rabbits. The anti-PAP pAb showed high affinity to papaverine with an affinity constant (K(aff)) of 7.3x10(7)L/mol. With this antibody, we established a sensitive immunochemical method for the determination of papaverine based on indirect competitive enzyme-linked immunosorbent assay (ELISA). The optimal concentrations of the coated antigen (PAP-OVA) and purified pAb used in the ELISA were 5 and 1.2mug/mL, respectively. The cross reactivity of other benzylisoquinoline derived substances, including 1-(3,4-dihydroxybenzyl)-7-hydroxy-6-methoxy-isoquinoline (6-methoxy-papaveroline, MPAPO), morphine (MP) and codeine (CD) were all lower than 1%. The linear range of the calibration curve was 0.1-1000ng/mL. Normal human serum samples were spiked with known amount of papaverine and measured by the ELISA. Recoveries were between 102% and 105%. Papaverine content in a commercial papaverine hydrochloride injection sample was also determined using the established ELISA. Compared with the results given by the control experiment of HPLC, the recoveries of ELISA to detect injection samples were 102-110%. The limits of detection for synthetic serum samples and injection samples of papaverine hydrochloride were 0.25 and 0.06ng/mL, respectively.
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http://dx.doi.org/10.1016/j.talanta.2005.01.001DOI Listing
May 2005

Biotin-avidin amplified enzyme-linked immunosorbent assay for determination of isoflavone daidzein.

Talanta 2005 Feb;65(3):621-6

The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, PR China.

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed and optimized for the determination of a weakly estrogenic isoflavone daidzein in serum, urine and Puerariae radix. Specific polyclonal antibody was produced against daidzein by immunization of rabbits with a conjugate of 7-O-(carboxymethyl)-daidzein and bovine serum albumin (BSA). The polyclonal antibody showed specific recognition of daidzein, while cross-reactivities to coumarin, 4-hydroxycoumarin, phenol, and other isoflavones such as puerarin and rutin were all lower than 1%. The linear range of daidzein calibration curve was 0.1-1000ngmL(-1). The detection limit was found to be 0.04ngmL(-1), and the intra-assay and inter-assay coefficients of variation were 7 and 16%, respectively. Human serum and urine samples were spiked with known amounts of daidzein and measured by the established BA-ELISA. Recoveries were between 91 and 107%. Daidzein in P. radix was determined by the BA-ELISA method and HPLC method, and the content of daidzein was determined to be 0.0219 and 0.0194%, respectively. The results indicated that there was a good agreement between the two methods. The established method is very useful for monitoring daidzein in biological samples and traditional Chinese medicine.
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http://dx.doi.org/10.1016/j.talanta.2004.07.027DOI Listing
February 2005

A new competitive enzyme-linked immunosorbent assay (ELISA) for determination of estrogenic bisphenols.

Talanta 2002 Jul;57(6):1205-10

The Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, People's Republic of China.

Bisphenol A and other hydroxylated diphenylalkanes (generally known as bisphenols) have been identified as potential estrogenic substances. In this paper, a specific polyclonal antibody was produced against these compounds by immunization of rabbits with a conjugate of 4,4-bis (4-hydroxyphenyl) valeric acid and bovine serum albumin (BHPVA-BSA). The polyclonal antibody showed specific recognition of the bisphenol structure, while the cross reactions of other common phenolic compounds such as phenol, hydroquinol and p-hydroxybenzoic acid were all lower than 1%. The linear range of bisphenol A calibration curve was 1-10 000 ng ml(-1). Real water samples and mouse serum samples were spiked with known amount of bisphenol A and measured by the competitive ELISA. Recoveries were between 92 and 105%. The detection limits were found to be 0.1 ng ml(-1) for real water samples and 2 ng ml(-1) for serum samples. The method is very useful for monitoring bisphenol compounds in environmental and biological samples.
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July 2002

Production and characterization of monoclonal antibody against recombinant human erythropoietin.

Biomed Environ Sci 2007 Jun;20(3):184-8

Beijing National Laboratory for Molecular Sciences (BNLMS), College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

Objective: To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids.

Methods: rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western blot.

Results: The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1 x 10(8) L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.

Conclusions: The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.
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June 2007

Improvement of the performance of an immunoaffinity extraction method via region-specific immobilization of IgG.

J Chromatogr A 2006 Jan 4;1103(2):350-5. Epub 2006 Jan 4.

Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

Using papaverine as a model target, an immunoaffinity column of high selectivity and binding capacity was prepared by utilizing covalent linkage between the Fc portion of IgG and the surface of Sepharose 4B support. Compared with the commonly used random coupling method, the binding capacity of the region-specific immobilized antibodies was increased from 0.04 to 0.2 mol of antigens/mol of antibodies and a much larger concentration factor was thus achieved. The obtained immunoaffinity column has been successfully used in pretreatment of pericarpium papaveris samples. The method offers an improved approach to immunoaffinity extraction that should be useful for purification and concentration of other targeted compounds in highly complex mixture.
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http://dx.doi.org/10.1016/j.chroma.2005.12.026DOI Listing
January 2006

An efficient immunoaffinity chromatographic method for extraction and purification of papaverine from samples of pericarpium papaveris and food products.

J Sep Sci 2005 Jul;28(11):1163-70

Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, PR China.

A highly selective immunoaffinity column was obtained by coupling anti-papaverine polyclonal antibodies to CNBr-activated Sepharose 4B. It was found that the coupling efficiency and performance of the immunoaffinity column were greatly improved by prolonging the coupling reaction time from 3 h at 20 degrees C with shaking to incubation overnight at 4 degrees C after the 3 h shaking reaction. The pH and ionic strength were observed to be the most important factors that influence the binding of papaverine to the immunoaffinity column. Using 0.01 mol/L phosphate buffered saline (PBS, pH 8.3) and methanol-water (80:20, v/v) as the loading and eluting solutions, respectively, papaverine was first retained on the column and then quantitatively eluted out with a mean recovery of 86% at a loading concentration of 1 microg/mL. When applied to real samples of pericarpium papaveris and food products, the established immunoaffinity column showed high efficiency in removing the matrix interferences in the samples and satisfactory recovery results were obtained. The method was useful for extraction and purification of papaverine from related samples.
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http://dx.doi.org/10.1002/jssc.200500063DOI Listing
July 2005
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