A Method For Rapid Detection Of Active Ingredients In Vitro And In Vivo Based On Immunochromatography

BackgroundImmunochromatographic assay represented by pregnancy test strip has characteristics such as cheap,instant,simple,self-service operation,on-site detection,and does not require large-scale equipment and professionals,which is very suitable for quickly quality information obtaining of traditional Chinese medicines(TCM).To monitor the quality of TCM in the whole process from picking,processing and production to sales,the simple and fast method is.required.Based on the previous results,this study established various kinds of immunochromatographic test strips for different needs.Purpose:The immunochromatography assay is applied in the quality control of TCM.Some representative active ingredients such as saikosaponin D,puerarin and rhein were used as model subjects to develop an immunochromatographic method for the index components of TCM,which characteristic by self-service,instant,and rapid.We believe it can provide an entirely new approach to quality control and material basis research of TCM.Method:I.The obtained positive monoclonal hybridoma in previous work was adopted for Production of mab.The hybridoma was thawed and induced mouse ascites by intraperitoneal injection.The mabs secreted by hybridoma were by purified by ammonium sulphate method and Protein G column and used as the probe of the test strips.II.Preparation of saikosaponin D(SSD)colloidal gold immunochromatographic strips1.Preparation of colloidal gold:Colloidal gold of different particle sizes was prepared by citric acid oxidation method.An appropriate particle size was selected as the IgG marker.2.Preparation of Colloidal Gold-IgG conjugate:the colloidal gold was conjugated with the mAbs by electrostatic adsorption3.Select the suitable pH and the minimum amount of labelled IgG.Optimize the preparation process of colloidal gold test strips.4.According to the optimized concentration,T line and C line were sprayed on the nitrocellulose membrane and labelled antibody was sprayed the on the conjugate pad.The NC membrane,absorbent pad,conjugate pad and the sample pad are sequentially adhered to the corresponding positions of the PVC plate.And the assembled test is cut into test strips with cutting machine.5.Evaluate the sensitivity,specificity,stability,repeatability of the test strip,and the correlation with existing methods.Then a variety of TCM preparations were used for test the immunochromatographic strips?.As the limitations of colloidal gold in quantitative detection,a new type of nanoparticles quantum dots(QDs)were selected as the label,which has high sensitivity and good stability.A QDs-IgG immunochromatographic paper was prepared for PUE detection.1.Select suitable QDs and detection modes for the strip.And characterize the properties of quantum dots2.Preparation of QDs-IgG conjugate:the QDs was conjugated with the mabs by carbodiimide method.3.Select the suitable material for sample pad,conjugate pad and NC membrane.Optimize the preparation process of the QDs test strips,including buffer solution system,surface active agent,T/C line concentration and other conditions.4.According to the optimized concentration,T line and C line were sprayed on the NC membrane and QDs-IgG was sprayed the on the conjugate pad.Then assemble the strip.5.Evaluate the sensitivity,specificity,stability,repeatability of the test strip,and correlation with HPLC.Then a variety of TCM preparations were used for test the immunochromatographic stripsIV.A QDs-IgY based immunochromatographic paper was prepared for RHE detection.As IgY is low cost and easy to produce from the yolk,it is used as a label instead of IgG1.Production of IgY:An artificial immunogen of RHE was prepared,and the female Roman chicken was immunized under the wing.The eggs were collected,and IgY was extracted and purified from the egg yolk.2.Titer and sensitivity of IgY against RHE were evaluated.3.Preparation of QDs-IgY conjugate:the QDs was conjugated with the IgY by carbodiimide method.4.Preparation of test strips:The preparation process of the QDs-IgY test strips was optimized.T line and C line were sprayed on the NC membrane and QDs-IgY was sprayed the on the conjugate pad.Then assemble the strip.5.Evaluate the sensitivity,specificity,stability and repeatability of QDs-IgY immunochromatographic method.V.Preliminary exploration of in vivo detectionThis section serves as an exploratory study to test the feasibility of QDs-IgG in vivo detection.1.The biological toxicity of QDs was evaluated by MTT method and growth curve.2.The behaviour and distribution of QDs within four different cell lines were investigated.3.The distribution characteristics of QDs in animals was investigated.The feasibility of QDs-IgG for in vivo detection of TCM compounds was investigated.Results:?.A colloidal gold immunochromatographic strip for SSD was successfully prepared and a rapid detection method for SSD was developed.1.The specific mabs against PUE and SSD were produced,and the purity was over 90%after purified by protein G column.2.Colloidal gold with different particle sizes were prepared and identified by UV and TEM.And a colloidal gold immunochromatographic strip for SSD was developed using 22.3 nm colloidal gold.3.The linear regression equation of the colloidal gold strip was:y =-0.1131n(x)+1.5451,R2=0.983.The linear curve has a detection range from 96 ng/mL to 150 ?g/mL and a detection limit of 96 ng/mL.The methodological investigation results show that the strip has good specificity and stable performance,which can be applied to the qualitative and quantitative detection of Chinese herbal medicines.?.A QDs immunochromatographic strip for PUE was successfully prepared and a rapid detection method for PUE was developed.1.A dry fluorescence method was selected for the detection,and QDs with an emission wavelength of 605 nm(QD2605)were selected as marker.2.QDs-IgG conjugate were prepared.To optimize preparation process of the QDs strips and improve the anti-interference ability,we screened the suitable material and aperture of sample pads,conjugate pads.And we extended the effective length of the sample pad to increase the ability to pre-treat the samples.As a result,the Ahlstrom8964 was used as conjugate pad,the XQ-Y7 was used as sample pad,and millpore 135s was chosen for NC membrane.A high buffer capacity,high ionic strength,and high blocking protein buffer system based Tris-HCl was used for the material pretreatment.3.The linear regression equation of the QDs strip was:y =-0.11 ln(x)+ 0.979,R2 ?0.9816.The linear curve has a detection range from 96 ng/mL to 150 ?gg/mL and a detection limit of 96 ng/mL.1 ng/mL-10 ?g/mL.The limit of detection is 5.8 ng/mL.4.The methodological investigation results show that the QDs immunochromatographic strip has good specificity and stable performance,which can be applied for the qualitative and quantitative detection of Chinese herbal medicines.?.We obtained high affinity IgY against RHE.A immunochromatographic strip based on QDs-IgY for RHE was successfully prepared and a rapid detection method for PUE was developed.1.In this study,artificial antigen of RHE was prepared and Roman chicken was immunized.The IgY against RHE was extracted and purified from egg yolk.The ELISA regression equation was y =-0.0941n(x)+ 1.0224,R2 = 0.9944,and the linear range was 5-3125 ng/mL.2.The linear regression equation of the QDs-IgY strip was:y =-0.1281n(x)+ 1.7627,R2 = 0.9792.The linear curve has a detection range from 80 ng/mL to 50 ?g/mL and a detection limit of 98.2 ng/mL.The methodological investigation results show that the QDs-IgY immunochromatographic strip has good specificity and stable performance,which can be applied to the qualitative and quantitative detection of traditional Chinese medicine components.IV.Preliminary exploration of in vivo detection1.The results showed that QDs are safe enough to apply in test strips.2.The results showed that QDs can be uptaken by cells and distributed in cytoplasm near the nucleus,which suspected in endoplasmic reticulum.3.QDs were found to accumulate in lymph nodes.However,the distribution of QDs-IgG was consistent with the distribution of QDs,which did not reflect the distribution of TCM components in vivo.ConclusionIn this study,the concept of POCT was introduced into TCM compounds detection.The results showed that the immunochromatographic assay has significance and broad application prospects in the analysis of TCM active ingredients.The development of colloidal gold immunochromatographic assay shows that the rapid and self-service detection meets the needs of fragmented mass detection.The development of QDs immunochromatographic assay shows the value of satisfying the need for high sensitivity and accurate quantitative detection.The IgY-based QDs immunochromatographic assay shows the value of inexpensive detection.In addition,the investigation of the toxicity of QDs and their distribution in organs,tissues,and cells will be good for the further application of QDs in vivo and in vitro testing.In summary,the development of several immunochromatographic assays in this study provides a powerful tool and a new technology for instant-self-testing of TCM compounds,and provides a reference for the development of new methods of TCM quality control.