The Development Of Gold Label Assay Kits For Four Kinds Of Transgenic Plants

With further research and rapid development of industrialization to the transgenic plant, it becomes necessary to set up fast, convenient, effective detection method. The commonly used detection methods at present can be divided into three kinds mainly: biology method(test in the laboratory and field), DNA level test ( PCR, Southern blotting) and protein level test (ELISA,Western blotting). But these routine methods are not suitable to detect transgenic plant quickly for their shortages of consuming time and strength and so on.This research uses the immune detection technique of colloidal gold lable for the first time,setting up CryIA(e),SCH ( Signal-Cry11Bal-HDEL) CpTI,Hpt four kinds of transgenic plant fast detection kits.Have worked as follows:1 .Recombinate plasmid pGEX-4T-3-Hpt has expressed high level fusion protein in E.coli,and purified protein was obtained by affinity chromatography with Glutathione Sepharose 4B.By immuning big white hare of New Zealand of purified Hpt albumen,we have gotten corresponding antibody.Measured by Indirect ELISA,the antibody can be combined with Hpt albumen peculiarly,its correlation coefficiency reaches the remarkable level.So,it can meet the measuring request whatever using the methods of ELISA or gold lable kit.2.Using the saturation sulphuric acid ammonium to purify antibody,we have the strengthes of four kinds of antibodies as follows: CryIA(e) :8643.42 g/ml,SCH: 9575.82 g/ml,CpTI: 9485.01 g/ml,Hpt: 9732.16 g/ml.Adopting indirect ELISA to examine the titer of antibodyafter purified,we have the results as follows: CrylA(e)>1 :10,000;SCH>1 :8000;CpTI>1 :7,000;Hpt>l : 16,000.3. The diameter of colloidal gold we have invited by adopting citric acid three sodium reducing system is 15nm.These colloidal gold particles are even and unanimous without oval and polygon existing by the electric mirror analysis.As we can see,the colour of the colloidal gold is wine red,and it is still very steady without the phenomenon of precipitating and changing colour after putting for half a year.In addition,the steady quantity of each antibody in the colloidal gold of each milliliter is as4. We have developed CryIA(e),SCH,CpTI,and Hpt four kinds of transgenic plant gold label detection kits.The comfirming time of testing results is as follows:the strong positive sample need 5 minutes to see red colloidal gold aggregated in measuring line,and the weak positive sample need about half an hour to see it.If there are two red lines in membrane, we can confirm the sample is positive.If there is only one red line in membrane, we can comfirm the sample is negative. What's more,if there is not red line in membrane,the detection strip is inefficiency.5. Regarding ELISA result as standard,the sensitivity of measuring CrylA(e) is 96.0%,the peculiarity is 100%,and the coincidence rate is 96.1%; the sensitivity of measuring SCH is 90.1%,the peculiarity is 91%,and the coincidence rate is 90%; the sensitivity of measuring CpTI is 92.4%,the peculiarity is 97%,and the coincidence rate is 94%. The measuring work of Hpt transgenic plant is at present still in prosess. Measured by Hpt pure albumen,the peculiarity is good.6.There is little difference of quality of gold lable kit whether put it in 37 C warm case or 4 C box of refrigerator .we can guarantee the quality of kits to store for half a year in the sealed dry place of the low temperature.Our research is to use the colloidal gold label technology to measure the transgenic plant for the first time,domestic and international not to see the document report yet at present.Compared with routine detection method,this method has advantages of single operation,simple,fast, pollution-freeing etc.And the revelant patent has been already in applying.