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Polyclonal antibody protocols:
Polyclonal Antibody
Polyclonal antibodies are antibodies derived from many B-cells or B-cell lines, similar to the mixture of antibodies found in blood sera.
Polyclonal antibodies are therefore a mixture of many different antibody specificities.
Polyclonal antibody preparations purchased are mixtures of antibodies of many different specificities to the same antigen.
Immunization of a rabbit, mouse, goat, chicken, or other animal produces polyclonal antibodies. Animal sera can be used directly, however it is better to purify polyclonal antibodies for laboratory usage.
Polyclonal Antibody Preparation
Based on the protocol by Dr. David Bowtell
Immunization of Mice:
Injections to immunize mice for the generation of polyclonal antibodies should be made at intervals of at least two weeks apart.
The following 2 adjuvants have been successful:
- Freund adjuvant and MPL+TDM adjuvant ( RIBI ImmunoChem Research, Inc. ). MPL is less hazardous than the first adjuvant.
- For the first immunization injection: inject 15 - 50 ug if Antigen in 200 ul adjuvant, injected subcutaneously.
- The second injection should occur 2 weeks later; You must then boost with 15 - 50 ug of Antigen in adjuvant. (If time permits, boost again a month later).
- Seven to 10 days later bleed the mouse and test serum using the assay which will be used for screening. If titre is not high enough, boost again two weeks after previous boost.
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Serum Preparation:
- After collecting, blood should be allowed to clot for 60 min . at 37°C or O.N at 4°C.
- Separate the clot from the sides of the tube (ringing) using a pasteur pipette. Place clot at 4°C O.N.
- Spin at 10000 x g for 10 min. at 4°C to separate the serum.
- Serum can be stored at -20°C after adding Glycerol to 50%.
If monoclonal antibodies are desired, once you have a "good" polyclonal and a reliable screening method proceed to the next step.
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Rabbits:
Rabbits are immunized essentially as described above. Injections:
- First in Freund's complete 100-200ug/1ml injection, two sites (over each shoulder).
- Second 1 month later in Freund's INCOMPLETE as above.
- Bleed 2 weeks later. Boost 2 weeks later again (2 + 2 week cycle).
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PRODUCTION OF POLYCLONAL ANTIBODIES IN RABBITS (NEUSEELAND WHITE)
Based on the protocol by Dr.Walter Steffen.
The rabbits should be 3-4 kg in weight and about 3 months old. About a third of the rabbit have endogenous anti-keratin antibodies and immunoactivity for various other cellular components. This emphasizes the importance of taking pre-immune serum from any given rabbit.
Buffers and Solutions:
- Saturated ammonium sulfate solution, 2 mM EDTA, pH 8.0
- 50 mM ammonium acetate, pH 8.0
- 70% ethanol
- Freund's adjuvant (complete & incomplete)
- Phosphate buffered saline (PBS) (see below)
- Vaseline
- Xylene
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- Bring the rabbit from its cage and place it securely in a restraining box leaving the ear accessible. Keep the rabbit relatively calm. Volumes of blood up to 50 ml can easily be collected from the main artery running down the center of the dorsal surface of the ear.
- Shave left ear around left vein with a fresh razor blade.
- Swab ear with 70% ethanol or isopropanol.
- Swab ear with xylene around artery-vein loop to dilate.
- Hold the tip of the ear (firmly) and the collection tube in one hand and with the free hand insert a 19G hypodermic needle into the dilated artery. The blood should begin to flow immediately into the collection tube. For best results use a portion of artery mid-way along the ear.
- Collect up to 50 ml blood. Apply pressure just anterior to point of entry of needle to stop bleeding.
- Apply Vaseline to ear to soothe xylene irritation.
- Put extra Vaseline on cut.
Obtaining Serum:
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- Swirl tube with collected blood to coat surface and to prevent clot attachment to tube.
- Incubate at 37 oC for 1 hour.
- Centrifuge at 2,500 g (setting #7 in a clinical table top) for 10 min.
- Remove clot and spin serum at 3,000 g for 10 min.
- Serum should be stored frozen.
Ammonium Sulfate Precipitation of IgG from Serum (4 oC, carried out in cold room):
Ammonium sulfate precipitation is an easy method to obtain a fairly pure IgG fraction.
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- Pool serum from one rabbit
- Weight serum
W1 = _____ g
- Wash tubes with min. PBS and weigh again.
W2 = _____ g
- Add 2/3 volume of W2 saturated ammonium sulfate, 2 mM EDTA, pH 8.0
Note: add very slowly, dropwise while stirring at 4 oC or use peristaltic pump.
- Stir for 15 min.
- Transfer to 40 ml centrifuge tubes; wash beaker with 3 parts PBS : 2 part ammonium sulfate and add to tubes.
- Spin at 12,000 g (Beckman JA-20: 10,000 rpm) for 10 min.
- Discard supernatant.
- Resuspend pellet in PBS [1/2 W1 = 1/2 vol.].
- Spin at 12,000 g for 10 min, discard pellet.
- Collect supernatant in beaker, wash walls with PBS.
Determine W3 = _____
- Add 2/3 vol. W3 saturated ammonium sulfate, while stirring. Continue stirring for 15 min.
- Transfer suspension to 40 ml centrifuge tubes, wash tube with PBS/ammonium sulfate solution.
- Spin at 12,000 g for 10 min.
- Discard supernatant.
- Resuspend pellets in 1/4 W1 PBS.
- Dialyse against PBS, 3-times 6 hours each.
- Put serum in a graduated tube, rinse dialysis bag well with PBS.
- Bring up to 1/2 W1 = 1/2 vol. in PBS.
- Determine protein concentration and aliquot for storage. Measure the absorption at 280 nm. The absorption of 1 mg/ml IgG solution is approximately 1.5.
Immunization:
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- Dissolve ~400-600 g antigen (~200-300 g per injection) in 2.2 ml 50 mM ammonium acetate, pH 8.0 or other appropriate buffer. Concentration depends on antigenicity of protein to be injected.
- The amount of antigen needed to immunize an animal depends on it antigenicity and the method of immunization. If proteins with low antigenicity are used, it will be helpful to denature the protein with a low concentration of SDS (up to 0.2%). Only highly purified antigen should be used for the production of polyclonal antibodies. An easy method to obtain a pure antigen is by SDS-PAGE. Proteins can be transferred to a sheet of nitrocellulose, stained with Ponceau S, cut out with a razor blade, and used as antigen (look for standard protocols in SDS-PAGE).
- Draw up 1.1.ml Freund's complete adjuvant with a 20G hypodermic needle, then draw up 1.1 ml of antigen solution. Freeze remaining 1.1 ml for boost.
- Keeping the plunger immobile, remove the needle and attach a sterile 3-way stopcock.
- Mix the antigen with the adjuvant by attaching a second glass syringe to the stopcock and injecting back and forth. (Caution: some plastic syringes might react with the mineral oil.)
- A complete emulsion will be formed after about 20-30 min; indicated by a rather sudden increase in viscosity of the solution.
Caution: be sure that the mounting is secure!
- After mixing wait about 20 min before injection to control that mixture is complete (If incomplete, oil will separate).
- Shave back of rabbit from shoulder blades to pelvis.
- Clean back with 70% ethanol or isopropanol
- Inject under the skin into about 10-20 sides along the spine between shoulder blade and pelvis using a 21G hypodermic needle. (Note: First injection one week after last preimmune bleeding).
- Wait one month and boost with a mixture of antigen and incomplete adjuvant. Avoid scars from previous injection.
- Bleed after one week to check for immune-response. The boost should be repeated, if immune response was too weak; however, a different rabbit should be chosen, if rabbit did not react at all. /LI>
References: Garvey, J.S., Cremer, N.E., & Sussdorf, D.H. (1977) In: Methods in Immunology. Pub. The Benjamin/Cummings Publishing Company. Readings Mass. pp 545
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Screening of Polyclonal Antibody using ELISA Assay
ELISA Protocol for Antibody Screen:
Materials for ELISA:
° Plates - 96 well Dynatech Immulon, type 2. (Fisher 17-0221-199).
° PBS, TBS
° PBS + 0.1% Tween 20 or TBS + 0.1% Tween 20.
° Blocking solution = 2% BSA (type V ) in PBS. (Add 0.02% azide for longer storage.)
° Elisa buffer = 2% BSA + 0.1% Tween 20 in PBS ( azide optional ).
° Enzyme linked antibody = Horseradish peroxidase 1
° Substrate = ABTS - 100X (from Zymed 00-2001)1
° HRP buffer: 100mM Na Citrate pH 4.2( 490 mg citric acid + 720 mg Na citrate
dihydrate + 50 ml H2O, pH 4.2 ).
° Hydrogen peroxide 30% ( 1000X )
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ELISA Protocol for Polyclonal Antibody
1) ADSORBTION OF ANTIGEN
a) Dilute Ag to 10 ug/ml in PBS.
b) Add 100 ul of Ag solution to each well.
c) Leave O.N covered with saran wrap, at 4°C.
2) BLOCKING
a) Wash unbound Ag by inverting the plates and flicking the wells dry.
b) Rinse by adding PBS to each well and inverting it again (use squirt bottle).
c) Repeat the rinse twice.
d) Add 100 ul of blocking solution to every well, leave 1 hr at room Temp or O.N at 4°C.
3) PRIMARY ANTIBODY
a) Add the antibody to be tested :
Sup of cells = 25 ul , mix well by pipeting up and down (10 times).
serum, ascites = 1:100 and a serie of 1/5 dilutions. Do dilutions in blocking solution.
b) Leave 1 hr at room temp or O.N at 4°C.
4) SECONDARY ANTIBODY
a) Wash unbound antibody 4 times with PBS + 0.1% Tween 20.
b) Add 100 ul of enzyme linked antibody to all wells. Do the appropriate dilutions in the Elisa buffer. (ex: HRP is 2000X).
c) Leave 1 hr at room temp or O.N at 4°C.
5) SUBSTRATE
a) Disolve substrate in water.
b) Wash plate 4 times with PBS + 0.1% Tween 20 (use TBS instead of PBS for AP).
c) Add 100 ul of substrate to every well.
d) Watch color development. This could take from a few seconds to 20 min.
e) If needed, stop the reaction by adding 50 ul of 4M NaOH.
f) Read absorption in Elisa reader at correct wavelength (for HRP system 416 nm, for AP - 405 nm).
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