The preparation process of monoclonal antibodies, super detailed!

Monoclonal antibody is the secretion of a hybrid tumor cell line that identifies a specific antigen cluster, which has the characteristics of strong specificity, high sensitivity, stable source and excellent performance, and is the premise and key of immunoanalysis technology.
monoclonal antibody drugs have experienced the stage of human-rat ninth-based monotoantisis and human-derived monotoantisis.
subsequent phage display library technology and genetically modified mouse technology, making the production of whole human-derived monotogenic possible, in 2002 the first all-human antibody Adamu monogenic market.
monoclonal antibodies are now promising in medical treatment, and are used to treat a variety of diseases such as tumors, autoimmune diseases, infectious diseases and transplant rejection.
the following small compilation to introduce for you the preparation process of monoclonal antibodies, the preparation process screening methods and significance, in clinical medicine application.
the preparation process of monoclonal antibodies, animal immunity according to the characteristics of antigens to choose the appropriate immune program, for soluble antigen immunogen weak, generally add adjucents.
commonly used adjorratives are Forsyth complete adjorratives and Forsyth incomplete adjorratives.
requires volumes such as antigens and adjectose to be mixed together and ground into a milky shape of oil-wrapped water.
1, initial immunity, Ag50ug/only, Gaford complete adjorpes subcutaneous multi-point injection, generally 1.5 ml, 3 weeks apart.
2, the second immunity, the dose path is the same, the gaford incomplete adjulate, 3 weeks apart.
3, 3, the third immunity, the dose is the same, no adjhoras, celiac injection, 7 days later blood collection to measure its efficacy, detection of immune effects, 3 weeks apart.
4, enhanced immunity, dose 50ug, abdominal injection. After 5 or 3 days of
, the spleen is combined.
, cell fusion 1, the preparation of cell-fed cells in the selective culture process after cell fusion, due to a large number of myeloma cells and spleen cells have died one after another, at this time a single or a few scattered hybrid tumor cells are mostly difficult to survive, usually must be added to other living cells to reproduce, this joined living cells called feeding cell mouse abdominal cell macrophage preparation: (1) with 6-10 weeks old BALB/c mouse.
(2) pull the neck, soak in 75% alcohol, disinfect 3 min, cut the skin with sterile scissors, exposing the peritoneal membrane.
6 ml of culture fluid is injected with a straw, rinsed repeatedly, and draws out the flushing fluid.
(3) put in a 10 ml centrifuge tube, 1200rpm centrifugal 5 min.(4) with 20% calf serum culture dispensing, adjust the number of cells to 1 x 105/ml. Add 96 well plate, 100ul/hole.
in a 37-degree incubator culture.
2, myeloma cell preparation (1) 48-36 hours before fusion, myeloma cell expansion culture (2) fusion day, with elbow dropper gently blowing the cell from the bottle wall, collected in 50 ml centrifuge tube or fusion tube.
(3) 1000r/min centrifugation 5-10 minutes, discarded.
(4) add 30 ml incomplete media and centrifuge once.
the cells are then resuspended on 10 ml incomplete media and mixed.
(5) take myeloma cell suspension, add 0.4% telsephenol blue dye liquid as a spare after living cell count.
3, spleen cells prepared to take the immune BALB/c mice, remove the eye blood, and isolate the serum as antibody test positive control serum.
at the same time through neck dislocation of dead mice, soaked in 75% alcohol for 5 minutes, fixed on the petri dish after lifting the left abdominal skin, can see the spleen, change the eye cut, in the ultra-clean table with sterile surgery to cut open the peritone, remove the spleen placed in a flat dish already filled with 10 ml of incomplete culture medium, gently wash, and carefully peel off the surrounding connective tissue.
stainless steel sieve in a flat dish and count edited into cell suspension with a syringe needle core.
, allowing spleen cells into an incomplete medium in a flat dish.
blowed several times with a straw to make a single-cell suspension.
usually 1 x 108-2.5 x 108 spleen cells per mouse.
4, cell fusion (1) the 1 x 10 x 8 spleen cells and 1 x 10 x 7 myeloma cells SP2/0 mixed in a 50 ml fusion tube, adding incomplete media to 30 ml, fully mixed.
(2) 1000r/min centrifugation for 5-10 minutes and will clean up as much as possible.
(3) tap the bottom of the fusion tube in the palm of the hand to make the sediment cells loose and uniform, and (4) add a preheated 50% PEG1 ml in 30s with a 1 ml straw and stir gently on the edges.
(5) the suction straw sits still for 1 min.
(6) add the warm-up incomplete culture liquid, terminate the PEG action, add 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml.(7) 800rpm, 5 minutes;
(8) add a 5 ml full media, gently blow the precipitated cells, allow them to suspend and mix, then add the full medium to 40-50 ml.
is divided into 96-well cell culture plates at 100ul per hole, and then the culture plates are placed at 37 degrees C, 5% CO2 culture box.
(9)6 h plus select the medium.
50ul. change the liquid with the selectmedium after 3 days.
(10) often observe the growth of hybrid tumor cells, until it grows to the bottom of the hole area of more than 1/10 to suck out the upper clean antibody test.
5, hybrid tumor cell selection (1) antigen package diluted to 10ug/ml.
(2) add the enzyme plate holes at 100ul/well and snap overnight at 4 degrees C or 37 degrees C for 2 hours.
(3) discard the liquid from the hole and wash it 3 times with washing liquid for 3 minutes each time.
(4) 100ul closed fluid per hole 37 oC closed for 1 hour; (5) washing liquid 3 times, (6) adding 100ul per hole to be tested for hybrid tumor cell culture, with positive, negative and blank controls, 37 degrees C incubation 1 hour; washing, patting dry.
(7) add enzyme label second antibody, 100ul per hole, 37 degrees C incubate for 1 hour, wash, pat dry.
(8) the substrate fluid, with a freshly formulated substrate with 100ul per hole, 37 degrees C for 20 minutes.
(9) terminates the reaction at 2mol/L H2SO4 and reads the OD value on the enzyme-linked immune reader.
(10) results determined that p/N 2.1, or P-N 3SD are positive.
if the negative control hole is colorless or close to colorless, the positive control hole clear color rendering, then can be directly with the naked eye observation results 6, hybrid tumor cell cloning (limited dilution method) (1) to prepare mouse spleen cells for feeding cells.
(2) prepare the hybrid tumor cell suspension to be cloned and dilute to 3 different dilutions per milliliter with HT media containing 20% serum.
(3) add 5 x 104-1 x 105 cells per milliliter, and add abdominal macrophages to the above-mentioned hybrid tumor cell suspension.
(4) each hybrid tumor cell is packed with 96-well plate, each hole is 100ul.(5) 37 degrees C, 5% CO2 culture 6 days, the appearance of visible clones can detect antibodies;
(6) take antibody-tested positive holes of the cell expansion culture, and frozen.
, monoclonal antibodies of the Ig and subclass identification (1) with a concentration of 10ug/ml of antigen package by enzyme plate, 50ul/hole, 4 degrees C overnight. After washing
(2), add the mono-resistant sample to be tested, 100ul/hole, 37 oC 1 hour; After
(3) washing, add HRP-labeled anti-mouse and sub-i-IG antibody reagents, 100ul/hole, 37 oC to avoid light for 20 minutes;
. The production and purification of monoclonal antibodies 1, the production of monoantinos (1) adult BALB/c mouse abdominal cavity inoculated with de-planteanor or liquid paraffin, 0.3-0.5 ml per mouse.
(2) 7-10 days after abdominal cavity inoculation with PBS or serum-free media diluted hybrid tumor cells, 5 x 105/0.2 ml per mouse. after 5 days
(3) interval, daily observation of the production of abdominal water in mice, such as the abdomen significantly expanded, with the hand touch, the skin has a nervous ness, you can collect ascites.
usually can pick up 3 ml of ascites per mouse, (4) centrifugation (2000r/min 5 minutes), remove cell composition and other sediments, collect, determine antibody price, subload, -70 degrees C frozen reserve.
2, monoclonal antibody purification (octanotic acid - ammonium sulfate precipitation method) (1) ascites 4 oC 12000 rpm centrifugation 15 min, to remove impurities.
(2) take 1 serving of ascites plus 2 servings 0.06mol/L PH5.0 acetic acid buffer, according to the proportion of diluted ascites per milliliter plus 33ul citric acid, at room temperature stir to add the acid, room temperature mixed 30 min (3) 4 degrees C to stand for 2 hours, remove 12000 g centrifugal for 30 minutes, discarded precipitation, (4) on-top cleared by nylon sieve filtration, at 50 times the volume of 0.01MPH7.4 PBS in 4 oC dialysis 6 h.(5) in the upper clearing after dialysis to add an iso-saturated ammonium sulfate solution.
(6) 4 degrees C to sit at more than 1 h, 10000 g centrifugation for 30 minutes, discarded.
(7) precipitation is dissolved in an appropriate amount of PBS (including 137 mmol/L NaCl, 2.6mol/L KCl, 0.2 mmol/L EDTA) and is dialysis overnight in 50-100 times volume PBS.
(8) take a small amount of dialysis samples after proper dilution, with ultraviolet photophotometer to detect protein content, SDS-PAGE, WB detection of antibody purity.
the selective culture of hybrid tumor cells is the key to the first screening after the first screening method and meaning of the screening cell fusion in the process of monoclonal antibody preparation. the commonly used HAT selective culture solution in
is the addition of hypothalamus (H), hyperaminophnics (A) and thymusnucleotides (T) to the common animal cell culture solution.
is based on two pathways of DNA synthesis in cells: (1) one path is the biosynthesis pathway ("D pathway"), that is, the synthesis of nucleotides by amino acids and other small molecular compounds, which provide raw materials for the synthesis of DNA molecules.
in this synthesis process, folic acid is involved as an important coenzyme, and ampicillin in HAT culture is an antagonist of folic acid, which can block the "D pathway" of DNA synthesis.
(2) Another way is the emergency or remedial pathway ("S path"), which is the use of hypothalamus-bird-phosphate nucleoside transferase (HGPRT) and thymus nucleotide kinase (TK) to catalyze sub-jaundice and thymus nucleotide to produce the corresponding nucleotides, two enzymes are indispensable.
therefore, in HAT culture liquid, unfused effect B cells and two effect B cells fusion of the "D path" is blocked by ammonia, although the "S-way" is normal, but due to lack of ability to multiply in vitro culture, generally about 10d will die.
for myeloma cells and self-fusion cells, because the usual use of myeloma cells is hypothalamus-oschaephosphate nucleotide defective (HGPRT) cells, so there is no "S-way" of themselves, and the "D path" is blocked by ammonia, so in HAT culture can not multiply and die quickly.
only the hybrid tumor cells formed by the fusion of myeloma cells and effect B cells, not only have the "S-way" of effect B cells, but also have the characteristics of myeloma cells in vitro culture liquid in the long-term proliferation, so it can selectively survive in THE HAT culture liquid, and continue to multiply.
the second screening in the actual immune process, because of the use of continuous injection of antigens, and an antigen determines cluster stimulation of the body to form a corresponding effect B lymphocytes, therefore, the effect B lymphocytes removed from the mouse spleen specificity is different, the hybrid tumor cell specificity of the HAT culture filter also has differences, so it is necessary to be screened from the hybrid tumor cell group can produce specific hybrid cells for a predetermined antigen cluster.
usually use a limited dilution of cloned cells, the hybrid cell multi-diluted, inoculated in porous cell culture board, so that each hole contains one or several hybrid tumor cells (theoretically 30% of the number of cells in the hole 0, to ensure that some holes are single cells), and then by these single-cell clone growth, and finally selectthet sized hybrid strains secreted to the special antibody for expansion culture.
monoclonal antibody in clinical medicine application 1, diagnosis of various types of pathogens this is the most common application of monoanti-resistance field, there are a large number of diagnostic reagents for selection.
for the diagnosis of hepatitis B virus, hepatitis C virus, herpes virus, cytomegalovirus, EB virus and various microorganisms, parasite infectionres.
the characteristics of high sensitivity and specificity of monoantigen, making it uniquely advantageous in identifying the type and subtype of the bacteria, the virus variant strain, and the antigen of the parasite in different life cycles.
2, tumor-specific antigens and tumor-related antigens are used in tumor diagnosis, typeandal and positioning.
Although monoantigens for tumor-specific antigens have not yet been prepared, monoantithesis for tumor-related antigens such as metaprotein, tumor alkaline protein, and cancer embryo antigen has long been used in clinical testing.
with the application of lymphocyte hybrid tumor technology, many hybrid tumor cell lines of anti-human tumor markers have been established, which lays the foundation for the early diagnosis of tumors and their clarification of tumor occurrence and development, understanding the biological activity of tumor cells and their quantitative research.
testing pathology specimens with anti-tumor monoantidsis can help determine the origin of metastatic tumors.
to radiation.