Monoclonal antibodies are known as mono-specific antibodies which are produced from a single B-lymphocyte clone and the monovalent antibodies which bind to the same epitope. Recently, the monoclonal antibody-based ELISA has become our emerging platform for the qualitative or quantitative analysis of foods or natural products. This method is a accurate, sensitive, and effective method without tedious pretreatment steps.
So that is the essential for biological samples and the future clinical applications. Preparation of the mABs of TCM is a vital step in studies on complex system characteristics of TCM formula. We have developed a protocol to generate mABs against the small molecular compounds, including artificial antigen synthesis, mouse immunization, and cell fusion.
The hapten is connected with the carrier protein to synthesize the artificial antigen. Artificial antigen and complete adjuvants are mixed and emulsified. The mouse was immunized by subcutaneous injection.
Take out the spleen of immunized mouse and make single-cell suspension. Mix the myeloma cells. Splenocytes are isolated and fused with HAT-sensitive mouse myeloma cell line by PEG method.
Select a cell line able to prepare antibodies by ELISA. Hybridomas secreting monoclonal antibodies that are reactive to antigen are cloned. Establish the hybridoma cell line is cryopreserved.
Periodate oxidation procedure was used for the synthesis of a narigin BSA conjugate. Firstly, narigin was dissolved at a final concentration of one milligram per milliliter at 100 microliters of freshly prepared sodium pariodate solution to five milliliters of the narigin solution. Stir the mixture at room temperature for one hour.
Secondly, add two milliliters of carrier protein to the narigin sodium periodate reaction mixture. Adjust the mixture to pH nine solution and continue to react for six to eight hours. Thirdly, the artificial antigen was purified with dialysis method in PDS for three days to estrange the CBS.
Freund's complete adjuvant and incomplete adjuvant were used for mouse immunization. For the vaccination, mix 50 micrograms of conjugate with an equal volume of Freund's complete adjuvant and emulsify completely. Administrate 100 micrograms of this mixture to each BALB/c mouse via dorsal subcutaneous injection.
Deliver a booster vaccination via subcutaneous injection two weeks later with the same amount of conjugate mixed with incomplete adjuvant. For primary immunization, Freund's complete adjuvant and immunogen were used via subdermal injection. For booster immunization, Freund's incomplete adjuvant and the immunogen were used via subdermal injection.
For impact immunization without adjuvant, only immunogen were used via subdermal injection or intraperineal injection. Feed layer cells mainly originated from abdominal epithelial cells or macrophage. RPMI 1640 FBS and HAT were used for preparing HAT screen medium.
HAT supplement was dissolved in 10 milliliters RPMI medium, then added into 500 milliliters RPMI 1640 containing 20%FBS. And ICR mouse was sterilized by 75%ethyl alcohol for five minutes. After removing the fur from the abdomen with hemostatic forceps, disinfect area with 75%ethanol.
Cut the outer skin to expose the abdominal cavity. Inject three milliliters of sterile RPMI 1640 medium into the abdomen. Then massage the abdomen to detach additional cells into the solution.
Aspirate the fetal cell suspension into a 15 milliliter centrifuge tube. After centrifuging the cell suspension for 10 minutes at 1, 000g, discard the supernatant and re-suspend the fetal cells to get single cell suspension. After this procedure, dissolve the cells by using HAT medium.
Count the cells to make it 100, 000 per milliliter from cell members. Transfer the cells into 96 well cell culture plates. Incubate the plates overnight at 37 degree, 5%carbon dioxide.
The chosen immunized mouse was sterilized by 75%ethyl alcohol for five minutes. Aspirate RPMI 1640 medium as the washing buffer. Remove the skin and muscle tissue using scissors to expose the spleen.
Isolate the spleen by the tweezers carefully. Then wash the spleen in the RPMI 1640. Isolate the spleen and cut into pieces.
Slowly pound the spleen. Then it was treated with an RMPI 1640 medium to dissolve the cells to prepare a spleen cell suspension. After that, the cells were filtered by 800 mesh cell strainer.
Nail the spleen carefully to remove the big tissues. Avoiding big tissues influence the fusion of cells. Was the spleen cell suspension with RPMI 1640 medium.
Then the cell suspension was added into the centrifuge tube. Harvest the spleen cells by centrifugation at 1, 000g for 10 minutes. And then discard the supernatant.
The spleen cells were supposed to be at the amount of one billion to 10 billion. Remove the cultivated and amplified myeloma cells from the incubator and gently shake the flask to obtain a cell suspension. Was the suspension with RPMI twice.
Mix the spleen cell suspension and the myeloma cells. Count the cells and adjust the concentration. The final ration of spleen cells to myeloma cells should be one to five to one to 10.
Centrifuge the cell mixture and then remove the supernatant. Add one milliliter of 50%polyethylene glycol solution to the cells and gently stir at 37 degree for one minute. Standing for 30 seconds, add two milliliters of RPMI 1640 medium and incubate at 37 degree for two minutes to terminate the reaction.
Add into two milliliters RPMI for another one minute. Then, add into 10 milliliters RPMI 1640. Centrifuge at 800g for 10 minutes.
Discarding the supernatant, add HAT solution and continue to cultivate the cells for seven days at 37 degree 5%carbon dioxide. After seven days, cell supernatants in 96 well plates were aspirated after cell fusion and added into ELISA plates pre-coated with coating antigen. Cultivated at 37 degree for one hour, secondary antibody was added and cultivated for half an hour.
After add 100 microliters of substrate solution. Stop the reaction. To the ELISA plates into the microplate reader, endpoint method was used read the data at 450 nanometer.
Measured absorbance at 450 nanometer and screen the positive hybridomas. Use the limiting dilution method to prepare the the monoclonal hybridomas. After removing the HD medium from the selected hybridomas, re-suspend the cells in RPMI 1640 and count them.
Dilute the cells of a concentration of one cell, two cells, and four cells per well by RPMI 1640 in a 96 well plate and culture. Transfer cells from the hybridomas that are positive to 24 well plates, 25 and 75 square centimeter flasks for cultivation. Store the myodomas in a gradient cooling box at minus 80 degree for 24 hours.
Then transfer to liquid nitrogen for longterm storage. Titer of antiserums were determined by indirect ELISA. Mouse one through four was immunized with narigin BSA conjugate was significantly higher than the control mouse without immunized.
The titer of one to 5, 000 is sufficient diffusion. The molecular weight of the hapten conjugate was confirmed by MALDI-TOF analysis. As shown in figure two, as a molecular weight of BSA standard and narigin have been known, we can calculate and determine that the molecules of narigin were conjugated with BSA.
Only the hybridoma can survive and grow in HAT selection media. After seven days of growth, the cell culture can be tested by ELISA. The positive hybridomas were re-cloned and expanded.
These images show the conventional outcome for stable monoclonal and polyclonal hybridoma cell lines. The critical point of this experiment is screening the mAbs specific for the hapten, but not carrier protein. The specificity of the mAb were then examined in the presence of other structurally related compounds.
The cross reactivity was calculated to evaluate the mAb specificity to the antigen. The calibration curve of naringin and the liner range were obtained. The antigen specific monoclonal hybridomas were expanded and cryopreserved in liquid nitrogen for longterm storage.
After watching this video, I hope you have a good understanding for how to generate monoclonal antibodies against a small moleculare compounds. So that's all. Thank you for your watching and good luck for your experiments.
