| Honey is a natural sweet food that is fully brewed by bees after combining the nectar,secretions or honeydew of the plant with their secretions.As a nutrient-rich natural functional food,honey is deeply loved by consumers around the world,but it is very easy to be adulterated.In recent years,honey adulteration is becoming progressively serious,and the level of adulteration is also becoming higher and higher,making it one of the most seriously faked food around the world.Honey adulteration not only impairs the fair competition in the market,but also endangers consumers health.Ensuring the authenticity of honey is still a research focus,because so far no reliable method has been developed to quickly detect all types of honey adulteration In this paper,major royal jelly protein-1(MRJP-1)was extracted from royal jelly(RJ)and immunized mice to produce the specific monoclonal antibody(mAb)ascites against MRJP-1.At the same time,rapid cDNA end amplification technology(RACE)was used to clone 3'and 5'sequence of mAbs genes.By splicing 3'and 5'fragments,this study obtained the full-length cDNA of mAbs genes and then constructed recombinant plasmids to transfect Chinese hamster ovary cells(CHO-S)which successfully produced specific mAbs against MRJP-1.This study established rapid detection methods of honey authenticity based on an indirect ELISA method and a colloidal gold immunochromatographic strip(CGIS).The main findings are as follows:(1)Isolation of MRJP-1 and preparation of mAb ascitesRJ was separated by three times ultracentrifugation,obtaining MRJP-1 with a purity of 91.6%?94.7%.Using MRJP-1 as an antigen to immunize mice,seven hybridoma cell lines were obtained:1H74D3,8H84E8,2G51E6,1F81D7,4G102B5,5H21D7,2B11B7.Through preliminary test of CGIS,three candidate cell lines were selected:5H21D7(named MAM-1H)cell line with epitope 1,4G102B5(named MAM-1G)and 2B11B7(named MAM-1B)cell line with epitope 2.They were injected into mice to prepare a large amount of mAbs from ascites.The mAbs from ascites were purified by HiTrap Protein G HP protein column and obtained 4.2 mg/mL MAM-1H,3 mg/mL MAM-1G and 4 mg/mL MAM-1B.The titer of MAM-1H,MAM-1G and MAM-1B was about 1:32000,1:640000 and 1:1800000,respectively(2)Full-length cDNA cloning of mAbs genes and its CHO-S expressionThe full-length cDNA sequences of heavy chain genes and light chain genes of MAM-1H or MAM-1G were successfully amplified by RACE technology.The full-length cDNA sequence of the MAM-1H heavy chain genes were 1521 bp,which included the 5'-UTR with 54 bp,3'-UTR with 99 bp,and the ORF with 1368 bp,encoding 456 amino acids;the full-length cDNA sequence of the MAM-1H light chain genes were 942 bp,which included the 5'-UTR with 90 bp,3'-UTR with 204 bp,and the ORF with 648 bp,encoding 216 amino acids.The full-length cDNA sequence of the MAM-1G heavy chain genes were 1503 bp,which included the 5'-UTR with 54 bp,3'-UTR with 99 bp,and the ORF with 1350 bp,encoding 450 amino acids;the full-length cDNA sequence of the MAM-1G light chain genes were 924 bp,which included the 5'-UTR with 66 bp,3'-UTR with 210 bp,and the ORF with 648 bp,encoding 216 amino acidsThe study constructed recombinant plasmids,containing the full-length cDNA sequence of both heavy chain genes and light chain genes of MAM-1H and MAM-1G respectively,to transfect CHO-S and obtained MAM-1H-CHO and MAM-1G-CHO The results of the titer experiments showed that the titer of MAM-1G-CHO was about 1:320000-1:640000 and the titer of MAM-1H-CHO was about 1:16000-1:32000 Compared with mAbs from mice ascites,even though the concentration of mAbs produced by transfected CHO-S cells was about half of mAbs from ascites,its titer was similar to that of mAbs from ascites,which mean antibodies produced by CHO-S cells still had high reactivity as mAbs from ascitesSDS-PAGE results showed that MAM-1G-CHO and MAM-1H-CHO each had two mAbs heavy chain bands at 55 kDa and one light chain band at about 25 kDa.It was found that MAM-1G-CHO and MAM-1H-CHO could specifically recognized MRJP-1 and didn't have cross-reaction with glucose,corn syrup or rice syrup(3)Developement of an indirect ELISA method using MRJP-1 mAbsThe optimized condition of this indirect ELISA method using MAM-1G as the primary antibody and HRP-goat anti-mouse IgG as the secondary antibody was established as following:4? and overnight for 1.5 ?g/mL MRJP-1 coating,37? and 1 h for 2%skimmed milk blocking,37? and 1 h for 100000-fold diluted MAM-1G binding,37? and 1 h for 250-fold diluted HRP-goat anti-mouse IgG reacting and finally,37? and 15 minutes for TMB reactingIn this study,a standard curve was obtained by this established indirect ELISA method with MAM-1G as the primary antibody.The linear detection range was 46.9 ng/mL-3 ?g/mL.In this detection,the sample recovery rate was 92.17%-117.01%,the coefficient of variation was 0.09%-7.58%,and no cross-reaction happened with rice syrup or corn syrup.Moreover,when the corn syrup adulteration rate of commercial rapeseed honey or vitex honey ? 30%,the error between the calculated adulteration rate and the theoretical adulteration rate determined by the indirect ELISA method<10%.The linear detection range of this indirect ELISA method with MAM-1G-CHO,as the primary antibody under the optimized condition was 187.5 ng/mL-6 ?g/mL Compared with MAM-1G as the primary antibody,the sensitivity of MAM-1G-CHO as the primary antibody was lower than that of MAM-1G,but the detection range of MAM-1G-CHO as the primary antiboy was wider than that of MAM-1G.Therefore,these two mAbs could be replaced depending on different honey authenticity tests(4)Development of a CGIS using MRJP-1 mAbsIn this study,a CGIS using MAM-1G as gold-labeled antibody,MAM-1H as test line(TL)antibody,and goat anti-mouse IgG as control line(CL)antibody was developed.After optimization,the best pH of MAM-1G labeled colloidal gold solution was 9.0,the best concentration of colloidal gold-labeled MAM-1G was 0.6 mg/mL,the spray amount of colloidal gold-labeled MAM-1G was when its OD533=1.2,the optimum MAM-1H coating concentration of TL was 0.3 mg/mL,and the optimum goat anti-mouse IgG coating concentration of CL was 0.3 mg/mL.The CGIS had good repeatability and had no cross reaction with common adulterants like corn syrup or rice syrup.The prepared CGIS was used to detect the authenticity of seven commercially available honey from different flower.The results showed consistency with those obtained by elemental analysis-isotope ratio mass spectrometry,which verified the feasibility of using CGIS to detect honey adulteration. |
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