Membrane Strip Affinity Purification of Autoantibodies Protocol

Introduction of Membrane Strip Affinity Purification Protocol

Anti-nuclear antibodies (ANA), directed against a variety of nuclear antigens, are a common characteristic of autoimmune diseases. Its detection depends on the evaluation of immunofluorescence of tissue culture cells. ANA positive sera, typically, exhibit nuclear homogeneous, nuclear speckled or nucleolar immunofluorescence patterns on Hep-2 cells. However, in certain instances a distinct set of fluorescent spots called multiple nuclear dot (MND) ANA occur all over the nucleus except in the nucleoli. The MND ANA is similar to the anti-centromere pattern but differs in that the discrete fluorescent dots are larger, fewer in number and seen only in the interphase cell, whereas the anti-centromere antibodies (ACA) give a speckled or punctate staining of the chromosomes in metaphase.

Figure 1. Demonstration of immunoglobulin purifi ed from the 70 kDa band binding back to the 70 kDa band on immunoblot.

Scientists undertook a study to determine the antigen bound by sera from a group of 21 patients with MND ANA but no ACA. They found that the crude sera from these patients showed a MND ANA pattern on Hep-2 cells. These sera were also found to bind to a 70,000 molecular weight antigen on a HeLa cell extract immunoblot. This study was undertaken to see whether autoantibodies affinity purified off this 70,000 molecular weight protein could reproduce the pattern that they found using the crude sera.

Purified antibodies are essential for a number of techniques, such as immunoblots, immunoassays, or cell staining (e.g., Hep-2 cells). There are several methods for purifying antibodies, e.g., precipitation with ammonium sulfate, use of hydroxyapatite column, gel filtration, protein A/B beads and column affinity chromatography. Ammonium sulfate precipitation is advantageous in that it is cheap and convenient to work with large volumes. However, the antibody yields are impure and therefore this procedure has to be coupled with other methods to obtain pure antibodies. Gel filtration is appropriate for IgM derived from all sources, since it can efficiently separate IgM from other antibody in polyclonal sera. Sample dilution, impure antibody yields, low sample capacity are some of the disadvantages of this method. Protein A beads are useful for IgG that bind to it from various sources and results in pure antibody providing high yield in a single step. It is disadvantageous in that it is expensive and also because it is not suitable for all species and classes. Affinity column chromatography has been commonly used for purifying antibodies from polyclonal sera. This procedure requires pure antigen for coupling to the column, is expensive and involves multiple steps. However, it is possible to obtain pure and specific antibody. The main disadvantage of this procedure is that it requires large amounts of pure antigen.

However, when the antigen of interest is of low abundance in a mixture of proteins (like in HeLa cell extract) and antibodies to that antigen is to be characterized from limited amounts of patient sera, affinity purification using a western blot strip containing that antigen becomes very useful. The protein mixture is electrophoresed on a SDS polyacrylamide gel, transferred to nitrocellulose membrane and stained with fast green. Two thin strips, one from each side of the blotted membrane, are excised and immunoblotted with specific antisera to identify the target band. Then the target band is excised horizontally and used for affinity purification of autoantibodies from patient sera. A sham band of the nitrocellulose, approximately similar in size to the excised target band, was also cut from an area away from the 70 kDa band. We have used affinity purification using nitrocellulose membrane strips to purify antibodies to the 70,000 molecular weight protein derived from HeLa cell extract to reproduce binding to the 70 kDa protein on immunoblot as well as the multiple nuclear dot antinuclear antibody pattern detected using crude sera from 21 patients without primary biliary cirrhosis or anti-mitochondrial antibody.

Materials of Membrane Strip Affinity Purification Protocol

Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 MΩ-cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. We do not add sodium azide to reagents.
Resolving gel buffer: 1.5 M Tris–HCl, pH 8.8. Add about 100 mL water to a 1 L graduated cylinder or a glass beaker. Weigh 181.7 g Tris and transfer to the cylinder. Add water to a volume of 900 mL. Mix and adjust pH with HCl. Make up to 1 L with water.
Stacking gel buffer: 0.5 M Tris–HCl, pH 6.8. Weigh 60.6 g Tris and prepare a 1 L solution as in previous step.
Thirty percent acrylamide/bis solution (29.2:0.8) acrylamide:bis: Weigh 29.2 g of acrylamide monomer and 0.8 g bis (cross-linker) and transfer to a 100 mL graduated cylinder containing about 40 mL of water. Add a spatula of mixed-resin beads and mix for about 30 min. Make up to 100 mL with water and filter through a 0.45 μm Corning filter. Store at 4 °C, with bottle wrapped with aluminum foil.
Ammonium persulfate: 10 % solution in water.
N, N, N ′, N ′-Tetramethyl-ethylenediamine. Store at 4 °C.
SDS-PAGE running buffer: 0.025 M Tris–HCl, pH 8.3, 0.192 M glycine, 0.1 % SDS.
SDS lysis buffer (5×): 0.3 M Tris–HCl (pH 6.8), 10 % SDS, 25 % β-mercaptoethanol, 0.1 % bromophenol blue, 45 % glycerol. Leave one aliquot at 4 °C for current use and store remaining aliquots at −20 °C.
Bromophenol blue (BPB) solution: Dissolve 0.1 g BPB in 100 mL water.
Phosphate buffered saline (PBS), pH 7.4. Dissolve five PBS tablets in 1 L water to obtain PBS solution, pH 7.4 at 25 °C (0.01 M phosphate, 0.00027 potassium chloride, 0.137 M sodium chloride).
Human lymphocytes: Purify lymphocytes from normal human peripheral blood using Lymphoprep according to manufacturer's instruction.
BenchMark pre-stained molecular weight standards.
Nitrocellulose membranes.
Western blot transfer buffer: 0.025 M Tris, 0.192 M glycine, 20 % methanol.
Western blot transfer apparatus.
Tris buffered saline (TBS; 10×): 1.5 M NaCl, 0.1 M Tris–HCl, pH 7.4.
Blocking solution: 5 % milk in TBS. Store at 4 °C.
Diluent solution: 5 % milk in TBST. Store at 4 °C.
Alkaline phosphatase (AP) buffer: Weigh 6.1 g of Tris, 2.9 g sodium chloride, and 0.51 g magnesium chloride-6H₂O and make it to 500 mL with water after adjusting pH to 9.3 with HCl. Store at 4 °C.
Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP): Add 33 μL of BCIP and 66 μL of NBT to 10 mL of AP buffer at the time of assay. Alternatively, use 1-Step^TM premixed NBT/BCIP solution.
Glycine elution buffer (Glycine Buffered Saline-Tween 20; GBST): 0.2 M glycine, 0.15 M NaCl, 0.05 % Tween 20, pH 2.7 (GBST). Store at 4 °C.
Electrophoresis system: Mighty small vertical slab gel unit.
HEp-2 slides.
Centricon 30 micro-concentrators.
Alkaline phosphatase conjugated affinity purified goat antihuman IgG.
Nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP): Dissolve 1 g NBT in 20 mL of 70 % dimethylformamide (DMF). Dissolve 1 g BCIP in 20 mL of 100 % DMF. Add 33 μL of BCIP and 66 μL of NBT to 10 mL of alkaline phosphatase buffer just before adding to membrane. Alternatively, use 1-Step^TM premixed NBT/BCIP solution.
Fluorescent isothiocyanate conjugated affinity purified goat anti-human IgG.
3.5 mL tubes.
0.009″ single edge razor blades–Smith Brand.
Membrane strip mini-incubation container (Bio-Rad).
Helium.

Methods of Membrane Strip Affinity Purification Protocol

All procedures are carried out at room temperature unless otherwise specified.

Preparation of Human Lymphocyte Extract

Lyse the lymphocytes by sonication in PBS using a Branson sonicator (setting # 4) and spin at 10,000 × g for 10 min. Analyze an aliquot of the supernatant by SDS-PAGE.

Preparative SDS Polyacrylamide Gel

Mix 2.5 mL of resolving buffer, 3.33 mL of acrylamide mixture, and 4 mL water in a 50 mL conical flask. Degas with helium for 10 min. Add 100 μL of SDS, 80 μL of ammonium persulfate, and 10 μL of TEMED and cast gel within a 7.25 cm × 10 cm × 1.5 mm gel cassette. Allow space for stacking gel and gently overlay with isobutanol or water.
Prepare the stacking gel by mixing 1.25 mL of resolving buffer, 0.67 mL of acrylamide mixture, and 3 mL water in a 50 mL conical flask. Degas with helium for 10 min. Add 100μL of SDS, 40 μL of ammonium persulfate, and 5μL of TEMED. Insert a preparative gel comb immediately without introducing air bubbles.
Heat an aliquot of the lymphocyte extract at 95 °C for 5 min. Do not add lysis buffer to the prestained protein standard or subject it to heat (manufacturer instructions). The heated lymphocyte extract was centrifuged at 3,000 × g for 30 s to bring down the condensate. Load the sample and the protein standard (10μL/well) on the gel. Electrophorese at 15 mA until the sample enters the gel and then continue at 25 mA till the dye front (from the BPB dye in the samples) reached the bottom of the gel.

Immunoblotting

Transfer proteins resolved on SDS PAGE to nitrocellulose membrane by Towbin's electrophoretic transfer method.
Stain with fast green to visualize proteins transferred to the membrane.
Trim the edges of the membrane. Wrap the fast green stained membrane in cling wrap and make a photocopy for record keeping purposes.
Excise the lane with molecular weight standards from the main nitrocellulose sheet containing the transferred protein. Excise a tiny wedge from the bottom left side of the marker lane and the main membrane sheet for orientation purposes.
Excise two strips, each about 2 mm in width, from either ends of the main nitrocellulose membrane sheet. Place them in a 12-lane mini-incubation container.
Destain strips with TBST.
Block for 1 h with 1 mL each of blocking solution.
Add 1 mL primary sera at 1:100 dilution and incubate for 2 h. Wash 5× with TBST, 5 min each time. Dilute anti-human IgG conjugated to alkaline phosphatase 1:5,000 with diluent and add 1 mL to each strip. Incubate for 1 h. Wash 5× with TBST, 5 min each time. Add 0.5 mL substrate and develop bands.
Align the two strips (with developed bands) on either side of the main nitrocellulose membrane and excise the desired antigen strip (horizontally this time). Use this strip, containing the antigen of interest, for the affinity purification of autoantibodies.
Cut a sham strip away from the target antigen strip and also similar to the size of the target antigen strip. This is used as a sham purification control.

Purification of Autoantibodies

Block the tubes for collecting purified antibodies, the tubes for incubating the membrane strips with sera and the Centriprep 30 concentrator with 1 % milk/TBST solution for 30 min at 4 °C.
Wash the tubes adequately with TBST to get rid of unbound milk protein.
Cut the nitrocellulose membrane strip with the desired antigen into smaller pieces and transfer to Sarstedt 3.5 mL 'B' tube (for blocking). Incubate these pieces with 2.5 mL 5 % milk/TBST for 30 min on an orbital shaker.
Wash well with several changes of TBST.
Transfer pieces from the 'Blocking tube' ('B') to a serum Incubation ('I') previously blocked with 1 % milk/TBST.
Incubate nitrocellulose pieces with 2.5 mL of a 1:50 sera dilution in 5 % milk/TBST for 1 h on an orbital shaker.
At the end of the incubation, pipet off and save the diluted sera (sera diluted 1:50 in 5 % milk/TBST) into another tube for use in later repeat sera incubations with the membrane pieces.
Transfer the membrane pieces to a TBST 'Wash' tube ('W') and wash fi ve times (10 min each time) on the orbital shaker.
After the wash, transfer the membrane pieces to a 'Glycine Elution tube' ('E').
Add 2.5 mL glycine elution buffer (GBST) and shake vigorously on an orbital shaker for 2 min. Then pipette the solution to a previously blocked collection tube.
Repeat with 2.5 mL of GBST and pipette into collection tube.
Add 2.5 mL of TBST and shake for approximately 1 min and pipette this solution into the collection tube.
Neutralize GBST with 500 μL of 1 M Tris.
Transfer pieces back to Blocking tube ('B'). Block for 30 min in 5 % milk/TBST. Wash with TBST.
Repeat steps 4 – 10 for two more elution cycles. However, care need to be taken to use a 1:100 dilution of sera in 5 % milk/TBST saved in step 7.
Upon completion of three elution cycles, transfer nitrocellulose pieces back to Blocking tube ('B'). Block in 5 % milk/TBST for 30 min. The nitrocellulose can be stored at 4 °C for 1–2 weeks if further elutions are needed.
Concentrate the eluted antibody solution to required volume. Antibody is now ready for western blot assay carried out as in Subheading immunoblotting or ANA testing.
Arrange the immunoblotted strips on card board inserts.

Notes of Membrane Strip Affinity Purification Protocol

Having water at the bottom of the cylinder helps to dissolve the Tris much faster, allowing the magnetic stirrer to go to work immediately. If using a glass beaker, the Tris can also be dissolved faster if the water is warmed to about 37 °C. However, the downside is that care should be taken to bring the solution to room temperature before adjusting pH.
Concentrated HCl (12N) can be used at first to narrow the gap from the starting pH to the required pH. From then on it would be better to use a series of HCl (e.g., 6N and 1N) with lower ionic strengths to avoid a sudden drop in pH below the required pH.
Wear a mask when weighing acrylamide. To avoid exposing acrylamide to co-workers, cover the weigh boat containing the weighed acrylamide with another weigh boat (similar size to the original weigh boat containing the weighed acrylamide) when transporting it to the fume hood. Transfer the weighed acrylamide to the cylinder inside the fume hood and mix on a mixer inside the hood. Unpolymerized acrylamide is a neurotoxin and care should be exercised to avoid skin contact.
The acrylamide solution can be stored at 4 °C for 1 month. The acrylamide mixture, buffer and water can be prepared in large batches, frozen in aliquots (for greater day-to-day reproducibility) and used indefinitely. Remove the required amount, bring to room temperature and add the other ingredients for polymerization.
It is best to prepare this fresh each time.
Storing at 4 °C reduces its pungent smell.
Simple method of preparing running buffer: Prepare 10× native buffer (0.25 M Tris, 1.92 M glycine). Weigh 30.3 g Tris and 144 g glycine, mix and make it to 1 L with water. Dilute 100 mL of 10× native buffer to 990 mL with water and add 10 mL of 10 % SDS. Care should be taken to add SDS solution last, since it makes bubbles.
SDS precipitates at 4 °C. Therefore, the lysis buffer needs to be warmed prior to use.
Dilute 100 mL of 10╳native buffer to 800 mL with water and add 200 mL of methanol. Avoid adding methanol directly to the 10× buffer, since it precipitates its ingredients. Even in such a scenario the precipitate can be redissolved by the addition of 800 mL water.
Add 100 mL of 10× TBS to a 1 L graduated cylinder and make it to about 800 mL with water. Transfer 50 g skim milk powder into the cylinder and mix stir until dissolved. Make to 1 L with water. Separate 500 mL as the blocking solution. To the remaining 500 mL add 250 μL of Tween 20 (cut end of blue tip to aspirate Tween easily), dissolve and use it as diluent.
Cells were lysed four times, 15 s each time. The tube with the lysate is chilled on ice in between sonication steps to prevent the contents from heating up. Do this in a fume hood with an ear protector.
The gel cassette was sealed at the base using 1 % agarose. Overlay the resolving gel with water for gels having acrylamide concentration lower than 8 % and use isobutanol (or isobutanol saturated with water) for gels of 10 % or greater. This overlay prevents contact with atmospheric oxygen, (which inhibits acrylamide polymerization) in addition to helping to level the resolving gel solution.
Centrifuging the samples prior to the run helps remove insoluble debris, which could produce streaks in the protein lanes (revealed when stained with Coomassie blue). Add a drop of 0.1 % BPB to the upper chamber buffer. This helps to form a much stronger dye front during the electrophoretic run.
Using a dilute fast green solution help to prevent overstaining of the membrane. Dilute stock fast green solution in gel destaining solution (25 % methanol, 10 % acetic acid). Use TBST to destain the membrane if it is overstained with fast green.
Strips can be excised very nicely using a razor blade. A glass plate is placed at an angle on the nitrocellulose at a distance of 2 mm from the edge and the razor is used to cut the strip (pull the razor blade along the sides of the glass plate to cut).
The fast green stained proteins bands in the main nitrocellulose membrane should be still visible. After aligning the two strips on either side of the membrane the developed band on the strips will line up with one of the bands on the membrane. This will permit the easy excision of the desired band from the main membrane.
This will prevent antibody loss as a consequence of adherence to the tubes.
Exercise universal serum handling precaution when handling human sera. Treat each serum sample as potentially hazardous.
It is possible to concentrate up to 500 μL with Centri-Prep 30 concentrator. If there is a need to reduce the volume further, use a 10,000 or 30,000 molecular weight cut off microcentrifuge tube.
If assaying for antibody purity (of the purified antibody) on western Blot, best results are obtained by incubating on nitrocellulose strips overnight at room temperature on the orbital shaker. This allows better binding of low titer antibody recovery and also washes should be longer (10 min instead of 5 min) and more frequent (five versus normally four washes).
Use paper boards placed in between stacks of ELISA plates in packages of ELISA plates for this purpose.