| The rabies is a common amphixenosis caused by rabies virus. It has strong latent ability, many animals carried these virus would not express the symptoms, and this causes an obstacle at the detection and precaution. This experiment aimed at preparing a convenient and specific colloidal gold test paper for rabies virus, in order to making the detection more comfortable and taking the controls at the sources of infection. Some preliminary work on the preparation of colloidal gold test paper has been done at the first half of this paper.Because of the culture tradition, increase of passages and freezing and defrosting, the antibody secretion of some hybridoma would be weaken, even lost. The methods to making it up would be replacing with new hybridoma. This would cause the extending of experiments duration and discontinuation. However, immune cells with antibody secretion ability could be obtained through in vitro immune. If it is possible that making the hybridoma reacquiring the secretion ability that restimulating the one lost through in vitro immune, the duration will be shorten greatly. Some basic research was placed at the last half of this paper.1 The selection of hybridoma and antibody purificationThe affinity and epitopes of seven hybridomas were tested through ELISA, the results shows that the hybridoma 17E3 has the strongest affinity and longest distances are placed between 17F3 and 17B2,17E3,17E4, the shortest distances are placed between 17E3 and 17B2,17E4. Ascites with immunopotency has been prepared throughout injection of hybridoma 17E3,17B2 and 17F3 to matured Balb/c mice treated by wax, and purifying the IgG antibodies with octanoic acid-ammonium sulfate method. The concentrations of 17E3 monoclonal antibody and mixed antibody (17B2 and 17F3) are 1.88mg/mL and 1.51mg/mL respectively. There are two lines appeared at the places of 50kD and 25kD respectively. This indicates that the protein purified is antibodies, and they are high-purity, meeting the requirements of experiments subsequently.2 Preparation of colloidal goldSodium citrate reduction method has been taken to preparing the colloidal gold with 20-30nm particles. The colloidal gold obtained is wine red, and has not precipitated within a month at 4℃. This indicates that the diameters of gold particle are between 20nm and 30nm, the colloidal gold are stable.3 Coating antibodies with colloidal goldCoat the antibodies with colloidal gold to preparing the combining pad of test paper. The minimum concentration of monoclonal antibody to stabilizing the colloid gold must be found. 200μL colloidal gold with 4μL 17E3 antibodies diluted 4 folds would not precipitated after 40μL 10%NaCl was added. And it could be conclude that 24μL 17E3 antibodies diluted 4 folds should be added to 1 mL colloidal gold.4 The preparation of colloidal gold test paperThere are 4 parts of colloidal gold test paper:the sample pad, combining pad, test pad and absorbent pad. The materials for sample pad, combining pad and test pad were selected, the Ahlstrom 8964 glass fiber is most suitable to coat the colloidal gold, and Millipore 135, Immunopore FP and Prima 40 are suitable to be the test pad.The Tris-HCl buffer which contained 0.2% Tween-20 at pH9.0 is the ideal buffer to coat the sample pad, the combining pad will be most perfect with 1:2 diluted colloidal gold coated. There are still some defects on these test paper that the test line show light color in the positive tests and the control line show no color either in the positive or negative tests. What makes this happen may be the goat-anti-mouse IgG which is used to coated the control line has low concentration or pure titer.5 The in vitro immune of hybridomaThree different systems were taken at this experiment:hybridomas, hybridomas+spleen cells and spleen cells were cultured with gradient antigens for 4 days respectively. Thymus cells-conditioned medium was added to the system of in vitro immune. And ELISA was taken to test whether it happened that antibodies increasing or not. The results indicated that these systems could not make the antibodies secretion improving, and this would cause by:1 the hybridomas cannot be resensitized; 2 the in vitro immune systems cannot meet the requirements. After all, the reasons must be proofed with additional work. |
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