출처: 프랜시스 V. 자아스타드1,2,휘트니 스완슨2,3,토마스 에스 그리피스1,2,3,4
1 미생물학, 면역학 및 암 생물학 대학원 프로그램, 미네소타 대학교, 미니애폴리스, MN 55455
2 면역학 센터, 미네소타 대학, 미니애폴리스, MN 55455
3 미네소타 대학교 비뇨기과, 미니애폴리스, MN 55455
4 Masonic 암 센터, 미네소타 대학, 미니애폴리스, MN 55455
다각성 항체는 항원 또는 여러 항원(1)의 상이한 항원 결정제에 대하여 지시된 항체의 집합으로 정의됩니다. 다각성 항체는 생물학적 분자를 식별하기위한 강력한 도구이지만, 한 가지 중요한 한계가 있습니다 – 항원 결정제를 공유하는 항원을 구별 할 수 없습니다. 예를 들어, 소 혈청 알부민이 동물을 예방 접종하는 데 사용될 때, 다른 표면이 있는 B 세포는 소 세럼 알부민에 대한 상이한 항원 결정제에 반응하게 된다. 그 결과 항체의 혼합물이 항혈구에 혼합된다. 소 세럼 알부민은 단백질의 진화적으로 보존된 영역에서 인간 혈청 알부민과 일부 에피토페를 공유하기 때문에, 이 안티소빈 세럼 알부민 안티세럼은 또한 인간 혈청 알부민과 반응할 것이다. 따라서,이 항세럼은 소와 인간의 혈청 알부민을 구별하는 데 유용하지 않습니다.
다발성 항세라의 특이성 문제를 극복하기 위해 몇 가지 접근법이 취해졌습니다. 하나는 고정된 항원의 크로마토그래피 컬럼을 통해 항혈을 전달함으로써 원치 않는 항체를 흡수함으로써(2)이다. 이 방법은 지루하고 자주 완전히 원치 않는 항체를 제거 할 수 없습니다. 또 다른 접근법은 개별 항체 생산 B 세포를 분리하고 배양에서 확장하는 것입니다. 그러나, 대부분의 일반적인 변환되지 않은 세포같이, B 세포는 장기 배양에서 살아남지 않습니다.
배양에서 살아남기 위해 B 세포의 무능력을 극복하기 위해, 한 가지 방법은 골수종-B 세포 혼종을 준비하는 것이다. 1847년, 헨리 Bence-Jones는 림프성 종양인 다발성 골수종을 가진 환자가 다량의 항체(3)를 생산한다는 것을 발견했습니다. 이 환자에 있는 B 세포는 악성되고 통제할 수 없는 성장합니다. 악성 B 세포는 단일 클론으로부터 파생되기 때문에, 그들은 동일하고 항체의 단일유형(즉,단일 클론 항체 또는 mAb)만 생성한다. 그러나, 이러한 골수종 세포의 대부분은 알 수 없는 특이성의 항체를 생산. 1975년, 골수종 세포를 B 세포에 융합시킴으로써, 세자르 밀스타인과 조지 콜러는 시험관내에서 무기한 배양될 수 있는 혼종 생성에 성공하여 알려진 항원 특이성의 무제한 의 단일클론 항체를 생산한다(4). 그들의 접근의 뒤에 근거는 골수종 세포의 불멸의 속성 및 B 세포의 생성 하는 항체를 결합 하는. 그들의 기술은 항체 생산을 혁명화하고 단일 클론 항체를 사용하여 생물학적 분자의 식별 및 정화를위한 강력한 수단을 제공합니다.
일반적으로 단일 클론 항체를 준비하는 것은 몇 달이 필요합니다. 일반 절차에는 다음 단계가 포함됩니다.
이 프로토콜은 마지막 단계에 초점을 맞추고 – 혼성종의 성장과 단일 클론 항체의 준비. 항체는 암모늄 황산염 강수량(종종 염화아웃이라고도 함)에 의해 배양 상수로부터 정제된다 – 용액에서 단백질을 제거하는 일반적으로 사용되는 방법. 용액의 단백질은 노출된 극지 및 이온 그룹을 통해 물과 함께 다른 친수성 상호 작용과 함께 수소 결합을 형성합니다. 작고 고압이 많은 이온(예: 암모늄 또는 황산염)의 농도가 추가되면, 이들 그룹은 물에 결합하기 위한 단백질과 경쟁한다. 이것은 단백질에서 물 분자를 제거하고 그것의 용해도를 감소시켜 단백질의 강수량을 초래합니다.
Using this protocol, we have obtained the following results with several different hybridomas:
Hybridoma: RB6-BC5 (rat anti-mouse Ly6C/Ly6G (Gr1) IgG2b, κ mAb)
OD280 – 1.103
(1.103/1.43)(20) = 15.42 mg/mL
Hybridoma: GK1.5 (rat anti-mouse CD4 IgG2b, κ mAb)
OD280 – 0.485
(0.485/1.43)(20) = 6.78 mg/mL
Hybridoma: 2.43 (rat anti-mouse CD8 IgG2b, κ mAb)
OD280 – 0.209
(0.209/1.43)(20) = 2.92 mg/mL
These are all example results, and it is important to note that each production run with the same hybridoma can be slightly different in the amount of mAb available at the end.
The procedure outlined above is a simple, straight-forward way to purify monoclonal antibodies from hybridoma culture supernatant. It is important to remember, though, that the ammonium sulfate will precipitate other proteins that may be in the culture supernatant. Consequently, the antibody concentrations determined from the absorbance measurements are estimates. The user may wish to assess the purity of the dialyzed sample by running a small amount on an SDS-polyacrylamide gel. The mAb produced by a hybridoma, once purified using this methodology, can be used in a variety of ways. The above-described RB6-BC5, GK1.5, and 2.43 mAb are commonly used for in vivo depletion of neutrophils, CD4 T cell, and CD8 T cells, respectively, in mice. Other mAb produced and purified using this protocol can be used for flow cytometry (when conjugated to a fluorophore or in conjunction with a secondary Ab), ELISA, or Western blotting.
Antibodies are a powerful tool for research and diagnosis, which means producing them in large quantities is often necessary.
The first step to generating antibodies is to inject the antigen of interest into a host animal. The antigen activates the host’s B-cells which then produce and release antibodies specific to that antigen. Then, regular screening of the host animal’s antisera for the presence of the target antibody is carried out, using ELISA or another detection method. Once it’s detected, the host animal’s spleen, which contains the B-cells, is removed. If all of the B-cells from the spleen are now isolated, this should include a population which are secreting antibodies to the antigen of interest. We refer to this population as polyclonal, because each cell likely bound to a different epitope of the antigen, and therefore, generated its own individual and unique antibody.
To generate monoclonal antibodies, antibodies raised to recognize one specific epitope, the individual B-cell that produces the desired antibody must first be isolated and cultured. Unfortunately, B-cells do not survive well in culture. So to overcome this hurdle, scientists fuse B-cells with immortal myeloma cells, resulting in hybridomas. These cells are then grown in a selective medium that only allows the hybridomas to grow and release antibodies. Again, the medium is screened using a method such as ELISA for the desired antibody. Once it is detected, the hybridomas are cloned via a process called limiting dilution, a serial dilution of the parent culture, which should result in single cells being seeded into the wells of a screening plate. This allows growth of hybridomas from a single parent cell, yielding a monoclonal cell line that only releases the desired antibody. These monoclonal lines can be expanded in tissue culture flasks to produce large quantities of monoclonal antibody. After this, as the cells begin to die off, the antibodies can be precipitated from the medium with ammonium sulfate. Normally, in solution, antibodies interact with water through hydrophilic interactions. However, ammonium and sulfate are highly-charged ions that separate the water molecules from the antibodies, decreasing the solubility of the antibodies and causing them to precipitate.
To begin, first check the list of materials and prepare all the media, supplies, and work surfaces for the protocol.
Then, turn on a water bath and set it to 37 degrees Celsius. Next, add 10 milliliters of complete RPMI to a 15-milliliter conical tube and 15 milliliters of complete RPMI to a T75 cell culture flask and set them aside. Using caution and wearing the appropriate personal protective equipment, remove the frozen vial containing hybridoma cells from the liquid nitrogen storage. To release the pressure inside the vial, loosen the cap slightly. Now, carefully incubate the vial in the water bath, making sure that the cap remains above the water surface to minimize the chances of contamination. When the cells are almost thawed, which typically takes around two minutes, move the vial to the tissue culture hood.
Then, wipe the outside of the vial with 70% ethanol before removing the cap. Using a sterile pipette, transfer the cells into the conical tube that contains 10 milliliters of complete RPMI medium. Then, centrifuge the tube for five minutes at 1200 RPM. After centrifugation, move the tube back to the tissue hood and wipe the outside of the tube with ethanol. Without disturbing the pellet, discard the supernatant and then add five milliliters of fresh complete RPMI medium and gently pipette up and down to resuspend. Next, transfer the cells to the T75 cell culture flask and place the flask inside a 5% carbon dioxide incubator at 37 degrees Celsius. Allow the cells to reach approximately 80% confluency, which usually takes about three days. Notice that hybridoma cells are nonadherent and will grow suspended in the medium. The time to reach sufficient confluency may vary based on the starting number of live cells and the type of hybridoma cell used.
Once the cells are sufficiently confluent, use a sterile 25-milliliter pipette to transfer them from the culture flask into a conical centrifuge tube. Pellet the cells by centrifugation at 1200 RPM for five minutes. While the cells are in the centrifuge, add 18 milliliters of complete RPMI into each of three new T75 cell culture flasks and set these aside. After centrifugation, remove the supernatant and gently resuspend the cell pellet in six milliliters of complete RPMI. Next, add two milliliters of the cell suspension into each of the three new cell culture flasks. Finally, place the flasks into an incubator set to 5% carbon dioxide and 37 degrees Celsius and incubate until the flasks are around 80% confluent, approximately three days.
At this point, the cells are ready to continue their growth in the serum-free medium designed for hybridoma cell lines, such as commercially-available HB Basal Liquid medium containing the HB101 supplement. Transfer the cells from each cell culture flask into conical centrifuge tubes and then pellet the cells by centrifugation at 1200 RPM for five minutes. Now, add 230 milliliters of supplemented HB101 serum-free medium into each of six 225-centimeter-squared cell culture flasks and set them aside. When centrifugation is complete, remove the supernatant and resuspend each pellet in 10 milliliters of supplemented HB101 medium. Then, into each cell culture flask, add five milliliters of the cell suspension. Place the flasks in the 5% carbon dioxide incubator at 37 degrees Celsius and continue growing the cells for about three weeks. During this time, the cells will produce and release the monoclonal antibody of interest into the culture medium and the antibody will be ready for purification when the cells start to die.
To remove the cellular debris from the antibody-containing culture media, pour the contents of the culture flasks into tubes for a fixed angle rotor. Place the tubes in the rotor and make sure it is properly balanced prior to centrifugation. Spin the tubes at 10,000 RPM for eight minutes. While the samples are centrifuging, place a two-liter plastic beaker with a stir bar into an ice bucket and then put the ice bucket on a stir plate.
Next, attach a 500-milliliter filter top to a one-liter bottle. Attach this bottle top filter unit to a house vacuum using the appropriate tubing. Then, pour the supernatant that contains the antibody into the filter top. Centrifuge the remaining media to separate the cell debris from the antibody-containing supernatant. When the filter top is full of supernatant, start the vacuum. Then, when the one-liter collection bottle is close to full, remove the filter top and pour the filtered supernatant into the two-liter beaker on ice. Repeat the filtration steps until all of the supernatant is processed.
When all of the sample has been processed, weigh 295 grams of ammonium sulfate per one liter of filtered supernatant. Start the stir plate and slowly add the ammonium sulfate to the supernatant over the next couple of hours. This prevents a localized high concentration of ammonium sulfate salt that may cause unwanted proteins to precipitate. Once all of the ammonium sulfate has been added, cover the beaker with foil and move it, along with the stir plate, to a cold room at four degrees Celsius and set it to stir the antibody solution overnight.
The next morning, pour the ammonium sulfate-containing antibody solution from the two-liter beaker into clean tubes for the fixed angle rotor. Centrifuge the tubes at 6500 RPM for 20 minutes without break to pellet the antibody at the bottom of the tubes. Next, vacuum aspirate the supernatant, using caution not to suck up the soft pellet. Continue using the same set of tubes to collect the pelleted antibody from the remainder of the ammonium sulfate-containing supernatant. After the last aspiration, re suspend each antibody pellet in approximately one milliliter of PBS.
To remove the ammonium sulfate from the antibody solution, first cut approximately one inch of dialysis tubing for each milliliter of antibody solution. Next, wipe the tubing with distilled water and tie a knot on one end of the tubing. Fill the tubing with distilled water to check for leakage from the knot. If there is no leakage after a few minutes, empty the water out of the tubing.
Next, pipette the antibody solution into the tubing. To recover as much antibody as possible, rinse the tubes with an additional 0.25 milliliters of PBS and transfer this to the tubing also. Secure the top of the tubing as close to the solution as possible with a dialysis clip. Then, tape the top of the tubing to the outside top of a four-liter beaker with the filled portion of the tubing hanging into the beaker. Now, take the beaker to the four degree Celsius cold room and place it onto a stir plate. Fill the beaker to the top with PBS and add a stir bar. Allow the tube and solution to stir overnight for approximately eight hours. The next morning, replace the PBS in the beaker with fresh PBS and then leave the beaker to stir again for approximately eight hours. Later that evening, repeat the process one final time. In the morning, open up the dialysis tube and then transfer the antibody solution from the tubing to 15-milliliter conical tubes. To remove any precipitant that may have formed during dialysis, centrifuge the tubes for five minutes at 1200 RPM. Finally, transfer the supernatant to fresh tubes.
To quantify the antibody concentration, first make a 20-fold dilution by adding five microliters from an antibody aliquot to 95 microliters of PBS. Then, pipette the diluted antibody into a cuvette and use a spectrophotometer to record the concentration at 280 nanometers. Next, calculate the antibody concentration using the formula shown. Finally, label screw cap vials with the antibody name, concentration, date of preparation, and, if applicable, batch number and experimenter name, and then aliquot the antibody into the labeled screw cap vials. These can be stored at minus 80 degrees Celsius until needed.
Example yields using the 120G8 anti-mouse CD317 or PDCA-1 hybridoma line ranged between 44 and 99.6 milligrams, which typically yields, on average, 67.3 milligrams amount. It is important to note that each production run with the same hybridoma cell line can be slightly different in the amount of monoclonal antibody available at the end.
