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Objective:To establish a quantitative detection method for the main components of dust mite allergens Der p 1, Der p 2 specific immunoglobulin E (sIgE) by using the nano-magnetic particle chemiluminescence immunoassay.Methods:The performance indexes of the established method were evaluated after setting up and optimizing the chemiluminescence detection system and immune reaction conditions of sIgE for dust mite allergen. Serum sIgE levels of 50 suspected allergic patients with dust mite were determined by this chemiluminescence method. At the same time, this method was compared with the Phadia kit and the consistency was analyzed by Kappa test. Results:The optimal amount of magnetic beads was 25 μg, the optimal reaction buffer (pH=7.4) contained 0.1 mol/L Tris-HCl and 0.25%( W/ W) casein, the optimal coating solution contatined 20 mmol/L phosphate buffer (PB) and 1%( W/ W) bovine serum albumin (BSA), and the luminescence enhancement solution contained 0.05%( V/ V) Triton X-100. The two-step immunoreaction was adopted, and the detection could be completed with 20 μl sample at the optimal reaction temperature of 37℃. The limit of detection (LOD) of the established nano-magnetic particle chemiluminescence system in detecting Der p 1 and Der p 2 sIgE antibodies were both less than 0.01 kU/L, with the linear range of 0.2-100.0 kU/L, the precision of less than 7%, and the cross contamination rate of 0.19% and 0.21%. Compared with the Phadia system, the positive and negative coincidence rate of Der p 1 were 78.0%(32/41) and 9/9 with good consistency ( Kappa=0.65, P=0.008), and the positive and negative coincidence rate of Der P 2 were 93.3%(28/30) and 85.0%(17/20) with good consistency ( Kappa=0.79, P=0.003). Conclusion:The nano-magnetic particle chemiluminescence immunoassay is successfully established for detecting dust mite allergen sIgE, which has good detection performance and good consistency with Phadia system.
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Bronchial asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and recurrent episodes of reversible airway obstruction. Due to different pathophysiological mechanisms, the disease is very heterogeneous in clinical manifestation, course of disease, response to treatment, phenotype etc. There is a strong need for biomarkers to assess the characteration and severity of the disease. Recently, lymphocyte and blood cells, antibodies, cytokines, chemokines, noncoding RNA and other protein markers have been studied as blood biomarkers of asthma. The present article summarized these biomarkers in diagnosis, phenotyping and treatment efficacy.
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Objective To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen.Methods According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method. Results In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml. In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen.Conclusion A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen.
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Objective@#To establish a nanometer magnetic particle chemiluminescence methodfor the detection of specific IgE antibodies to Tyrophagus putrefaciens allergen.@*Methods@#According to the routine operation steps of chemiluminescence, chemiluminescence reaction system and suitable immune reaction conditions for detection of specific IgE of Tyrophagus putrescentiae allergen are established. Considering the performance of the chemiluminescence method established in this paper, the American Somerfly Phadia method wasused as the gold standard to evaluate the test performance of the established chemiluminescence method.@*Results@#In thechemiluminescence reaction system, Luminol concentration in luminescent substrate A solution is 0.4 mg/ml, Urea hydrogen peroxidein luminescent substrate B solutionis 0.2 mg/ml.The sensitivity of this luminescent reaction system to horseradish peroxidase (HRP) is very high, the minimum detectable HRP is 0.01 ng/ml.In the immune response, the room temperature light-shielding reaction was detected for 5 min after the addition of substrates A and B, and the detection values were effective within 5-30 min. 37 ℃ is optimum luminescence reaction temperature. Choose the range of 0.35-100 IU/ml as the standard curve. According to the results of 120 clinical trials, using Phadia (immunofluorescence method) as the gold standard, the chemiluminescence method established in this study has significant significance for the detection of Tyrophagus putrescentiae allergen.@*Conclusion@#A nanometer magnetic particle chemiluminescence method was successfully established for the detection of specific IgE antibodies to Tyrophagus putrescentiae allergen.
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Objective:To clone and express the Der f 7 recombinant protein from Dermatophagoides farinae and prepare the Der f 7 monoclonal antibody.Methods: The Der f 7 recombinant protein was expressed in prokaryotic expression system of pET28a(+)-Der f 7.BALB /c mice were immunized with the recombinant protein.Myeloma cells and spleen cells were fused,and hybridoma cells were screened by ELISA.Hybridoma cells were injected into the mice abdominal cavity to obtain ascites.It was purified by protein A agarose medium ascites,and then to dentified the titer and purity of the antibody.The specificity of the antibody was identified by Western blot.Results: Three hybridoma cells which stably secret recombinant Der f 7 monoclonal antibody were obtained.The monoclonal antibody had high purity,the titer was higher than 1∶243 000.Western blot showed that Der f 7 recombinant protein could be recognized well.Conclusion: We successfully obtained Der f 7 monoclonal antibody,which can be used for the quantification and localization of Der f 7 allergen and the diagnosis and treatment of allergic diseases.
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Objective:To obtain the prokaryotic expression product for the group 6 allergen of Dermatophagoides farine ( Der f 6) and detect its IgE-binding rates with sera from asthmatic children. Methods: By enzyme digestion of pET28a (+)-Der f 6 with BamHⅠ plus XhoⅠ,the target gene Der f 6 was obtained and linked into the vector pET32a (+) to construct the recombinant plasmid pET32a(+)-Der f 6, which was then transfected into E. coli BL21 cells for expression, induced with isopropyl-β-D-thiogalactoside ( IPTG) ,purified by affinity chromatography and identified by SDS-PAGE,Western blot and AMLDI-TOF,and tested by ELISA for IgE reactivity with sera from asthmatic children. Results:The plasmids pET32a(+)-Der f 6 were constructed,transformed into E. coli BL21 and expressed successfully. SDS-PAGE of the purification product showed a specific band,Western blot showed the successful binding between the purification product and the His-tag in the plasmids,and MALDI-TOF/TOF identified the identical structure to the allergen Der f 6. Using the ELISA method developed with the recombinant proteins as coating antigen,the positive rate was 41. 3% (19/46) in asthmatic children allergic to dust mite. Conclusion: The plasmids pET32a (+)-Der f 6 were constructed successfully,expressed in E. coli BL 21 (DE3). The recombinant fusion protein has a good reactivity with sera from asthmatic children.
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OBJECTIVE@#To explore the eff ect of artenisiae scopariae and poriae powder (ASPD) on calpain-2 expression in liver tissue from rats with obstructive jaundice.@*METHODS@#The rat model of obstructive jaundice was established. SD rats was divided into the control group, the obstructive jaundice group, the obstructive jaundice model plus ASPD group, the obstructive jaundice model plus saline group. Th e serum levels of TBIL, ALT, AST and other biochemical indexes were detected. The pathological changes of liver tissue were evaluated by HE staining. The calpain-2 mRNA and protein expression in liver was measured by Real-time PCR and immunohistochemistry or Western blot, respectively.@*RESULTS@#The calpain-2 mRNA and protein expression levels were significantly up-regulated in live tissues from the rats with obstructive jaundice in a time-dependent manner. The ASPD could inhibit the calpain-2 expression in rats with obstructive jaundice concomitant with the decreased liver damage and the improved liver function, suggesting that calpain-2 was involved in endoplasmic reticulum stress-mediated cellular apoptosis and the occurrence of obstructive jaundice.@*CONCLUSION@#ASPD could be used for patients with obstructive jaundice to promote the recovery of liver function after operation and to reduce the incidence of complications, which provide a theoretical basis for the reasonable application of traditional Chinese medicine in the peroperative period.
Subject(s)
Animals , Rats , Apoptosis , Artemisia , Chemistry , Calpain , Metabolism , Disease Models, Animal , Drugs, Chinese Herbal , Jaundice, Obstructive , Liver , Metabolism , RNA, Messenger , Rats, Sprague-Dawley , Real-Time Polymerase Chain ReactionABSTRACT
Objective:To construct the plant expression vector of Der f1 allergen of Dermatophagoides pteronyssinu and expression in tobacco lamina.Methods:The Der f1 gene was amplified from the glycerin bacterium which contained pET28a(+)-Der f1 plasmid,cloned into the pMD 19-T plasmid,and then sequenced.The Der f1 gene was digested by ClaⅠand SalⅠ,and cloned into potato virus X (PVX) to construct plant expression vector PVX-Der f1,and then was transformed agrobacterium tumefaciens.The positive one was selected to infect tobacco lamina for expressing target protein.The protein was identified and analysed by SDS-PAGEand Western blot.Results:Digestion and sequence analysis confirmed that the plant expression vector was correct,and the SDS-PAGE and Western blot results showed that the molecular weight of the protein was about 34M_r and it could specific binding with positive serum.Conclusion:The plant expression vector of Der f1 is successfully constructed and the recombinant protein is also produced.
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Objective To obtain the gene coding for the group 5 allergens from Dermatophagoides farinae ( Derf5 ) and predict its molecular characteristics. Methods The total RNA of D. farinae were extracted, and the gene Derf5 was amplified by RT-PCR with the primers designed according to previous sequence published in GenBank. The target gene was linked into pMD19-T Simple plasmid, sequenced and analyzed by bioinformatics software. Results The sequence homology reached to 97.8% between our sequenced result with one complete open reading fragment (ORF) and the reference. The gene encode an extracellular hydrophobic protein with 132 amino acid resides, one signal peptide from 1 to 19 position and one transmembrane domain from 1 to 19 position. The secondary structure was composed of extended strand (1. 52% ), random coil (7.58%) and alpha helix (90.91%). The encoded protein was deduced to have two Casein kinase Ⅱ phosphorylation sites. The similarity of the amino acid sequence of the group 5 allergens were 78% between D. farinae and D. pteronyssinus. Conclusion The gene Derf5 was cloned successfully, and its characteristics was primarily predicted.
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Objectve To find out an effective method for treatment of postoperative wounds of the patients with anal and rectal diseases.Methods Cryoprecipitate was smeared over postoperative wounds of the patients with anal and bowel diseases(the experimental group),and the mean healing time,infection rates and hemostatic effect were observed and compared to the patients who were treated by routine medicine(the control group).Results The mean healing time of patients with hemorrhoid,anal fistula and perianal abscess was shorter than the control group by 5.68 days,6.01 days and 1.87 days respectively.And the infection rates of the experimental and control group were 9.18%(9/98)and 44.68%(42/94)respectively,significant difference between them was found( P
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Objective To investigate the situation of mites breeding in student dormitories. Methods Dusts samples from beds, clothes and houses were collected every month in the same campus, separated with direcmioroscopy and identified for mites. Results A total of 1 385 specimens were collected, and the total detectable rate was 56.46%. The detectable rates of mites in dust samples collected from beds, clothes and houses were 63.73%?41.72% and 47.78% respectively, which were significant different (?2=44.43,P
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The authors reviewed the breeding situations of mites in homes,including rates,distribution,seasonal variation and common species,and the diagnosis and treatment of the allergic diseases caused by mites,as well as their parasitizing in gastrointestinal tract,lungs,urinary tract and other organs.In the end,the measurements for control of mites in homes were demonstrated.