Monoclonal antibody identification test
Monoclonal antibody identification test
The identification of monoclonal antibodies is similar to that of polyclonal antibodies, and the methods used are common; however, the identification of some of the indicators is specific to McAb. Currently, there are 7 types of monoclonal antibody identification tests: antibody potency identification, antibody specificity identification, identification of Ig class, subclass and type of McAb, chromosome analysis of hybridoma cells, identification of McAb neutralizing activity, identification of McAb recognition epitopes and identification of McAb affinity.
Principle
The basic principle of the monoclonal antibody characterization assay is that after successful cell fusion, cloning and screening of hybridoma cells that can secrete specific McAb, and stable establishment of the line, a certain amount of McAb needs to be prepared for systematic characterization of its properties, so that it can be used for better application.
Operation method
Chromosome analysis experiments on hybridoma cells
Principle
The basic principle of the chromosome analysis experiment in hybridoma cells is that when cell division is in the middle stage, the chromosomes are at the right length and size, and it is the best stage to analyze the chromosomes. Therefore, in order to display the chromosomes, firstly, it is necessary to obtain as many mid-division images as possible; secondly, hybridoma cells have many chromosomes, densely packed in the cell, and they must be dispersed in order to see them clearly.
Materials and Instruments
Equipment: centrifuge tubes, centrifuge, thermostatic water bath, ultra-clean bench, microscope. Move The basic procedure of the chromosome analysis experiment of hybridoma cells can be divided into the following steps: Caveat 1, colchicine treatment time is too long, split cells more, short chromosomes; on the contrary, split cells less and chromosomes long, are not suitable for observation of morphology and counting, so the concentration of colchicine and the role of time should be accurately mastered. 2. The concentration and processing time of hypotonic solution should be appropriate, and the cells must be mixed gently after hypotonic treatment, otherwise it is easy to cause cell membrane rupture and chromosome dispersion. 3. The fixative should be prepared temporarily before use, otherwise the organic solvent will evaporate and will not be able to fix the cells effectively. 4. The slide must be washed thoroughly, do not leave any grease, otherwise the chromosomes are not well dispersed. For more product details, please visit Aladdin Scientific website.
Reagents:
① 20% FCS-RPMI 1640 culture solution.
② Colchicine solution: weigh 10 mg of colchicine, dissolve in 100 ml of saline (i.e., 100 μg/ml), filtered and sterilized, and freeze at -20℃.
③ Hypotonic solution: 0.075 mol/L KCl solution.
④ Fixation solution: take 3 parts of methanol and 1 part of glacial acetic acid, mix well to form (freshly prepared before use).
⑤ Giemsa dyeing solution: weigh 0.5 g of Giemsa powder, dissolve it in 33 ml of glycerol (add a small amount of glycerol into the mortar and mix with the Giemsa powder, grind it until there is no particles, and then pour in the rest of the glycerol), keep warm at 55-60℃ for 2 hours, add 33 ml of methanol and mix well, and keep it in a brown bottle.
(6) Giemsa working solution: take 1 portion of Giemsa staining solution, add 9 portions of 0.15 mol/L phosphate buffer (pH 6.8), and mix well to form the solution (freshly prepared before use).
(1) Cultured cells
A Passage of hybridoma cells 36 - 48 hours before the addition of colchicine (the hybridoma cells can be grown to the logarithmic phase at the time of the experiment).
(2) Colchicine treatment
A 4 - 6 hours before termination of culture, add colchicine to the cultured hybridoma cells to achieve a final concentration of 0.1 - 0.4 μg/ml, or 0.02 - 0.05 μg/ml if colchicine is used. B At the end of the culture, collect the cells in centrifuge tubes, and all of the above steps need to be aseptically operated.
(3) Chromosome preparation
A Centrifugation: centrifuge at 1000 r/min for 10 minutes, discard the supernatant. B Hypotonic treatment: add 5 ml of 0.075 mol/L KC1 hypotonic solution preheated to 37℃, gently suspend the cell precipitates and mix, and place it in a constant temperature water bath at 37℃ for 15-20 minutes. C Pre-fixation: add 1 ml of freshly prepared fixative to the suspension and mix well (make the surface of the cells fixate slightly, to prevent the cells from sticking after fixation). C Pre-fixation: add 1 ml of freshly prepared fixative into the suspension, mix well (make the cell surface slightly fixed first, which can prevent the cells from sticking together after fixation), centrifuge at 1000 r/min for 10 minutes, and discard the supernatant.D Fixation: add 5 ml of fixative, resuspend the cell precipitate and mix well, and let the cells stand at room temperature for 20-30 minutes, centrifuge at 1000 r/min for 10 minutes, and discard the supernatant.E Re-fixation: the procedure is the same as the above. G. Drops: Pipette 1-2 drops of cell suspension onto a slide freshly removed from ice water, blow it out immediately and pass it over the flame of an alcohol lamp several times, so that the cells will lay flat on the slide and dry naturally in the air.
(4) Staining
A Stain with freshly prepared Giemsa's working solution for 10 - 20 minutes, then wash off the stain with tap water and dry naturally in air.
(5) Microscopic observation
A Look for well-dispersed and moderately stained schizonts under low magnification, observe the chromosome morphology under oil microscope and count them. Each specimen should be counted with 100 complete intermediate nuclei, and the number of chromosomes should be recorded and tabulated to analyze the distribution of chromosome numbers and to observe whether there are marker chromosomes.