Design And Synthesis Of Modified Geosmin Hapten And Study On The Relationship Between Structure And Antibody

Geosmin(GSM)is a metabolite of overgrown actinomycetes and blue-green algae in eutrophic water bodies.It has a strong earthy smell and is easily adsorbed by aquatic organisms in water bodies,resulting in unpleasant odors.The human sense of smell is so sensitive to GSM that it can be detected even at very low levels in water.The permissible limit of GSM in Chinese "Sanitation Standard for Drinking Water"(GB5749-2006)is 10 ng·L-1.In order to ensure the safety of water quality and people’s health,it is urgent to establish an analytical method with high sensitivity,strong selectivity,rapidity and simplicity for the determination of trace GSM in water samples and aquatic products.At present,the main method for detecting GSM is gas chromatography-mass spectrometry.Since the content of GSM in the sample is extremely low,the sample needs to be separated and extracted by solid-phase extraction or solid-phase microextraction.However,above method has disadvantages of expensive instrument,complicated operation,high detection cost,and cannot be used for on-site rapid detection.Immunoassay is an analytical method based on the specific reaction between antigen and antibody.Currently,enzyme-linked immunosorbent assay(ELISA),which is widely used in clinical,biological,food and environmental analysis,has the advantages of high sensitivity,strong specificity,simplicity,rapidity,and simultaneous detection of a large number of samples.A prerequisite for establishing an immunoassay method is to obtain antibodies that specifically bind to the analyte.Macromolecular compounds such as proteins can be directly used as immunogens to immunize animals to produce antibodies.Small-molecule compounds with a molecular weight less than 1000Da(usually called haptens)have no sufficient immunogenicity and cannot directly immunize animals to produce antibodies.However,the active groups such as carboxyl groups on haptens(self-existing or modified)can link to carrier proteins(such as bovine serum albumin)to form hapten-protein conjugate,which can be used as the immunogen to immunize animals to obtain antibodies.The principle of hapten modification is that the molecular structure in obtained neo-hapten should be similar to original molecular structure as much as possible.The molecular weight of GSM is only 182,and the structure is simple.GSM contains a hydroxyl group,and it seems that the hydroxyl group can be esterified by succinic anhydride so that the modified GSM contains a spacer arm with a carboxylic group at the end.Nevertheless,due to too expensive of GSM(about ￥1000μg-1),the modification cost will be too high.On the other hand,the steric hindrance of the hydroxyl group is too large,and the modification is difficult.More importance,the loss of hydroxyl group will likely result in the great decrease in antibody specificity.Some scholars have synthesized the modified GSM hapten using cheap 2-methylcyclohexanone as the starting material.As there was a relative big difference between the structure of the modified product and the original molecular structure of GSM,the generated antibody exhibited low sensitivity and poor specificity.In order to obtain high sensitive and specific antibodies against GSM,the main researches of this thesis are:①Design and synthesis of various GSM hapten modifiers;②Connect active groups(mainly carboxyl groups)to carrier proteins to obtain immunogens and coating antigens;③Immune mice with immunogens to obtain a variety of mouse anti-GSM polyclonal antibodies;④Establish indirect competitive ELISA(ic-ELISA)for evaluation of antibody properities based on the prepared coating antigens.In addition,study the effect of molecular structure of modified GSM hapten on antibody properities.The details are as follows:①In synthesis of hapten,a new method,namely nucleophilic substitutionintramolecular ring-forming tandem reaction instead of the former Robinson cyclization method,was created,which reduced the synthesis steps and risks.By this new method,the modified GSM haptens obtain the same GSM skeleton and a suitable length of a spacer arms with an active group at the end.②In terms of hapten structure,through different synthetic routes,several modified GSM haptens with hydroxyl retention,hydroxyl ortho-methyl retention or no retention,and the spacer arms with structural changes were synthesized.③In preparation of immunogens and animal experiments,five hapten-BSA conjugates used as the immunogens for immunizing animals were synthesized.③In evaluating Ab properties,four coated antigens used to establish an ic-ELISA for evaluating antibody properties,e.g.the binding ability and specificity of Ab with GSM,were synthesized.Using P-C3 as coating antigen and the serum from the animals immunized with immunogen of P-B4 as the polyclonal antibodies,an ic-ELISA was initially constructed.The detection limit of the ELISA for GSM(±)was found to be 42 pg·L-1.