Preparation Of Antigen And Antibody Aginst Traditional Chinese Medicine-Giant Knotweed Rhizome And Their Specificity Identification

Objective: Immunological approaches were used to prepare all kinds of crude antigen from Giant Knotweed Rhizome (GKR) and anti-crude antigen antibody, and then detect their specificity.Method: The crude materials of various GKR were separated. And then emodin-carrier conjugates were synthesized by three kinds of modification of the procedure including Succinyl Oxide Law (SOL),Diazonium Para-aminobenzoic Acid Law (DPAL) and Improvement Diazonium Para-aminobenzoic Acid Law (IDPAL). Kunming mice were immunized with three kinds of modification emodin-carrier conjugates or crude materials from GKR and the serum was obtained.Results:1 The conjunctional result of emodin to BSA: Scan emodin, BSA, PBS, emodin - BSA1, emodin - BSA2 and emodin - BSA3 separately by UV spectrophotometer (Figure 3, Figure 7).The figure showed that emodin and BSA were conjugated successfully. According to the result of UV spectrophotometer the conjugation ratio was calculated (Table 1).2 The comparison of the titer of antibodies and immunogenicity: Double immunodiffusion in acidum aceticum fiber filter paper was performed to determine the titer of the antibody and immunogenicity of the crude materials of various GKR by sero-reaction between sera and the 1:10 crude antigen of various GKR. In each group of the GKR's CTY, the antibody titer of the GKR's STCCW group was the highest ,which was 1:16, among the GKR's STY group with titer of 1:4,the GKR's STCCSQ group which with titer of 1:2. In the each group of purely raised emodin, the antibody's titer is as follows: the emodin - BSA3 group is 1:8, the emodin - BSA2 group is 1:4 and the emodin - BSA1 group is 1:2. Therefore, it's proved that the immunogenicity of the emodin - BSA3 group is the strongest, the emodin - BSA1 group is weaker, and the pure emodin did not have the immunogenic at all (Table 2).3 The influence to specificity of different method conjugated Emodin with BSA: Choice the different bases to conjugate to the BSA, It's inevitably influence the expressed structure of the hapten, thus came to the different antigen epitope, and certainly affect its immunogenicity and the antibody's specificity.Dot blot, which antigen including emodin, rhein, physcion and chrysophanol was used in, was used to determine the specificity or the cross reaction reactivity of antibody produced in different modification of the procedure used in synthesized emodin - BSA conjugates. The antibody in emodin - BSA1 SOL group might react not only with emodin but also the physcion and chrysophanol. But the antibody in the emodin - BSA2 DPAL group might only react specifically with emodin. The antibody specifically with emodin, but had the dubious cross reaction with the physcion and chrysophanol. But antibody in three groups didn't have the cross reaction to the rhein (Table 3).4 Anti-small molecular ingredient antibody production: The TCM macro-molecule ingredient (such as traditional Chinese medicine protein, polysaccharide and so on), could stimulate the animal to produce the antibody directly, But theoretically, hapten in small molecular couldn't stimulate the animal to produce the antibody directly. The result showed anti-emodin antibody, anti-rhein antibody, anti- physcion antibody and anti- chrysophanol antibody were produced by the thickly raised GKR (including STY, STCCW, STCCSQ) to immunize the mice. The most comprehensive and the highest titer were produced in the STCCW group. But any antibodies was not detected in pure emodin group( Table 4).5 The comparison of antibody specificity by thickly raised GKR: The thickly raised GKR and the Od's STY which is far from its heredity and does not included identical component were used to prepare antibody. Double immunodiffusion in acidum aceticum fiber filter paper was performed to determine their antibodies (Table 5). Each group of antiserum produced by the thickly raised GKR only response to GKR's STY, and there was only mild cross reaction to Rhubarb's STY but didn't response to OD's STY; the antiserum of OD's STY only response to OD, but didn't response to GKR's STY.Conclusion:1 The conjugate result of emodin and BSA: the antibody produced by the compound of emodin– BSA conjugated by the SOL not only combine with emodin, but also had the cross reaction to the physcion and chrysophanol. The emodin - BSA compound conjugated with the DPAL and the IDPAL retain rhein's 6-OH, that's namely retained rhein's characteristic structure. The antibody (antiserum) produced by the immunized mice mainly combine with the emodin, but if in high concentration, there is also cross reaction to physcion.2 The comparison of the titer of antibodies and immunogenicity: The quantity conjugated to the hatpen, namely combine ratio, in certain scope, is higher and the hatpen immunogenic is bigger under the same conjugation method. Affects to the immunogenic not only determined by the combine ratio but also by conjugated method. It possible depend on the immunogenicity of emodin's antigen epitope structure by the different method of conjugated and also depend on the kinds and quantity of antigen epitope made by the emodin - BSA conjugates.3 The influence to specificity of different method conjugated Emodin with BSA: The GKR's STCCW group mainly contained the macro-molecule protein and the polysaccharide in every crude GKR groups, thus immunogenicity is the strongest. The antiserum prepared by crude GKR groups only responded to the GKR's STY group, specificity in antibodies. In that, there is mild cross reaction to the same branch plant such as rhubarb STY. Maybe they have certain homology, including the common protein and the common small molecular ingredients.4 Anti-small molecular ingredient antibody production: Use crude GKR to immunize mice then produced the anti-small molecular ingredient antibodies, (such as anti-emodin antibody, anti-rhein antibody, anti-physcion, and anti-chrysophanol antibody). And the highest titer and the most anti-emodin antibody content is the STCCW group which mainly contain protein and polysaccharide macro-molecule ingredients. It revels that certain TCM small molecule ingredient antibody may be directly obtained by immunizing animal with STCCW.