Procedure: Western Blot Technique for Detection of HIV-1/2 and HTLV-I/II Antibodies. Prepared by Date Adopted Supersedes Procedure # Dana Gallo 7/1/89

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1 Procedure: Western Blot Technique for Detection of HIV-1/2 and HTLV-I/II Antibodies Prepared by Date Adopted Supersedes Procedure # Dana Gallo 7/1/89 Review Date Revision Date Signature 7/1/89 R. W. Emmons 10/31/90 R. W. Emmons 10/30/91 R. W. Emmons 9/28/92 R. W. Emmons 10/27/94 R. W. Emmons 11/28/95 M.S. Ascher 10/16/96 M.S. Ascher 2/10/99 M.S. Ascher 7/28/00 C. V. Hanson 8/1/01 C. V. Hanson 10/3/02 C. Glaser 10/18/04 C. Glaser 10/13/06 C. V. Hanson 10/21/09 C. V. Hanson 9/21/10 D. P. Schnurr 2/11/11 C. V. Hanson 3/4/13 Dongxiang Xia # of Distributed to Copies Medical Records 1 Retrovirus Diagnostic Section 1 Distributed to # of Copies PRINCIPLE: The antigen is prepared from supernatant fluids from infected cell cultures which are concentrated by ultracentrifugation and purified by sucrose density gradient. The antigen is disrupted and the proteins are separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The paper is cut into strips and each strip is incubated with a patient s serum. The antigen-antibody complex is detected by EIA. 1

2 SPECIMEN: Type: Human serum or plasma Handling Conditions: Store at 2-8 C. EQUIPMENT AND MATERIALS: Equipment: A. Electrophoresis gel apparatus and gel casting apparatus B. Power pack 500/200 C. Electrophoresis transblot apparatus D. Power pack 250/2.5 E. Incubation trays Materials: A. Western blot strips containing HIV or HTLV protein bands B. Control serum or plasma possessing Western blot antibody to: HIV-1 Include strongly positive, weakly positive, and negative control sera in the first run from a set of nitrocellulose strips cut from one sheet (one gel). In subsequent runs, include at least the weakly reactive and negative control sera. HIV-2 Include positive and negative control sera in each run. HTLV-I/II Include positives (HTLV-I and HTLV-II) and negative control sera in each run. C. Working dilution of GAH-IgG HRP conjugate D. Working concentration of Diaminobenzidene substrate 2

3 QUALITY CONTROL: Gel electrophoresis run parameters should be within normal range With amperage set at 10 milliamps, voltage should = volts Transblot electrophoresis With voltage set at 60 volts, amperage should = amps Protein molecular weight standards should be included with every gel run to determine relative molecular weights of viral proteins. Serum controls (see Materials B) PROCEDURE - STEPWISE: A. Gel electrophoresis (See Preparation of Reagents below) 1. Degas distilled water for 15 minutes in a 250 ml side-arm flask. Mix together in a 125 ml side-arm flask and bring to RT: Deionized water ml Lower gel buffer ml Resolving acrylamide gel solution ml 2. Place casting stand on level platform. Check gaskets to assure they are clean and seated correctly. 3. Wipe the plates clean using 95% ethanol soaked kimwipes. Wipe dry with kimwipes. Assemble plates. (See Bio-Rad Mini-Protean 3 cell instructions for more detail) Slide glass plates into the casting frame keeping the short plate facing front. Insure both plates are flush along the bottom. 4. Lock the pressure cams to secure the glass plates. 5. Secure the casting frame to the casting stand by engaging the spring loaded lever. 6. Temporarily insert the comb and mark on glass plate 1 cm below the bottom of the comb. Remove comb. 7. Degas resolving gel solution for 15 minutes. 8. Prepare AP and TEMED solutions. (Also label tubes for disruption mixture and antigen.) a. Weigh out 30 mg AP into a 15 ml plastic tube. Add 2 ml degassed water. Mix well. b. Add 75 µl TEMED to ml degassed water in a glass tube. 3

4 9. Add 0.25 ml TEMED solution and 0.25 ml AP solution (in that order) to degassed gel solution. Swirl gently to mix. 10. Pipette acrylamide solution between plates to slightly above the mark on the plate (about 3ml). Overlay with degassed water using a one ml syringe with a 23 gauge, 1½ inch needle. Put remaining solution in a large tube to check for polymerization. Allow to polymerize for 45 minutes. 11. Prepare antigen: (See below for Preparation of Virus for Electrophoresis) a. Use a pipettor to add 50µl of β- mercaptoethanol (stored at 4ºC) to 0.95ml disruption mixture (stored at -20º C). b. Add appropriate volume of antigen to 70µl disruption mixture with a pipettor. c. Disrupt for 30 minutes at 65ºC (HIV-1) or by boiling for 3 minutes (HIV-2, HTLV). 12. Prepare and cast stacking gel a. Dilute TEMED 1:4 by adding 0.5 ml TEMED solution to 1.5 ml degassed water. Dilute AP 1:8 by adding 1ml of AP solution to 7ml of degassed water. b. Remove water of polymerization from resolving gel with syringe. Rinse with degassed water and dry the top of the gel by blotting with filter paper. c. Insert comb between plates at an angle (10º) d. In a small beaker mix the following ingredients in the order listed: Upper gel buffer... 1ml Stacking gel acrylamide... 1ml TEMED (1:4 dilution)... 1ml AP (1:8 dilution)... 1ml Swirl to mix. Using a 3ml syringe with a 23-guage, 1½ inch needle attached or a pipetor with a narrow tip attached, inject the stacking gel mixture at the edge of the spacer. Fill to the top of the casting apparatus to allow for seepage of the stacking gel along the edge of the resolving gel. Reposition the comb. Allow to polymerize for 15 minutes. e. Before removing the combs, mark the location of the wells with a felt tip marker. Slowly pull the combs out (pulling up on one side helps break the suction). Use a syringe to remove excess fluid from the wells and then rinse the wells with upper chamber buffer. Blot the wells with filter paper. 13. Remove gel assemblies from casting frame. a. Wipe excess acrylamide from plates with kimwipes. b. Place gel cassette into electrode assembly with the short plate facing inward. Slide gel cassettes and electrode assembly into the clamping frame. Press down the electrode assembly while closing the cam levers on the clamping frame. 14. Lower the inner chamber into the tank. Fill the upper chamber with 125 ml cold upper chamber buffer. Observe for leaks. Fill the bottom chamber with 190 ml of lower chamber buffer. Check for bubbles under the plates. Upper and lower buffers must not mix. 15. Using a microcapillary pipet tip, add all of the antigen preparation evenly over the wide lane of the stacking gel and 6µl of the protein standards (See Treatment of Protein 4

5 Standards below) to the small lane. These materials contain glycerol and will settle into the wells. 16. Put the lid on the chamber and connect to the power supply. Turn power on and set the voltage to 500 volts. Set current at 10 ma until the proteins have moved through the stacking gel and are about to enter the resolving gel (about 15 minutes). At that point, increase the current to 20 ma. Run time is approximately 45 minutes. When blue tracking dye reaches the bottom of the plate and just begins to run out of the bottom, turn off the power supply. 17. While gel is running, prepare for transfer of proteins. (See Bio-Rad Mini Transfer cell instructions for more detail) a. Place about 250ml of blot buffer in each of two shallow dishes. b. Draw a line ½ cm from the top edge of two nitrocellulose sheets measuring 7x8 cm. Wear gloves while handling nitrocellulose. In one of the dishes wet the nitrocellulose sheets, first one side, then the other. c. In the other dish wet the fiber pads and place them on the plastic presses. Place two 7x8 cm pieces of filter paper in the dish. B. Transfer (See below for Reagent Preparation) 1. Pour off and discard the chamber buffers. Open the cams of the clamping frame. Pull the electrode assembly out of the frame and remove the gel cassettes. 2. Use one of the spacers to gently pry the plates apart. The gel will adhere to one of the glass plates. Cut a corner or two off the gel if necessary to distinguish the gels. 3. Remove the stacking gel by scraping with one of the spacers. Immerse the top portion of the plate into the blot buffer over the filter square. Ease the gel from the plate at the gel bottom and let it fall onto the filter square. Position the gel squarely over the filter square. With a single smooth motion, while gently holding the gel and filter square together with thumb and forefinger, slide the gel and filter both out of the buffer. 4. Place the filter square and gel onto the fiber pad with the gel facing upwards. 5. Wet the surface of the gel with 5-7ml of blot buffer. 6. Ease the wet nitrocellulose sheet (ink side down) onto the gel by bending it slightly and positioning the middle. Line up the ink line with the top of the gel. First lower one edge, then the other. 7. Use a 10ml pipette and beginning on the edge containing the protein standards, roll the pipette over the nitrocellulose to express all the liquid and air bubbles. This will ensure good contact between gel and nitrocellulose. 8. Place another piece of wet filter paper on top of the nitrocellulose and roll with a pipette again, going in the opposite direction. 9. Place the second fiber pad on top. 10. Close and secure the presses with the clamp. 11. Slowly push the press into the core of the blot cell, making sure the nitrocellulose is toward the positive (red) electrode. Insert bio-ice unit. Add buffer until it reaches the level of the top of the top row of red circles. Place chamber on magnestirrer and connect to power supply. 5

6 12. Turn on power supply and set to 100 volts and record the amperage. Run for 30 minutes. (May run at 60 volts for 60 minutes, change bio-ice unit, and then turn up to 100 volts and run for 30 minutes.) 13. Prepare PBS and PBS-Tween. (See Western blot EIA below) 14. At end of run, record amperage and turn off power. Do not reuse buffer for transfer. 15. Disassemble sandwich, leaving only nitrocellulose, gel, and filter squares in contact with each other. Remove the filter paper from the nitrocellulose. Place the remainder of the sandwich, nitrocellulose side down, on a thick glass plate. Remove the filter paper from the gel. Using a ballpoint pen, mark the outer edges of the gel and the location of the wells. Remove the gel from the nitrocellulose. Using a scalpel and ruler, cut off the strip containing the protein standards. Stain these strips. (See below for Amido Black Stain) 16. Wash the nitrocellulose sheets 4 times in 100ml PBS-Tween per wash at room temperature for 5 minutes on the platform rocker. After use rinse all equipment in hot water followed by deionized water. 17. If the strips are to be used immediately for enzyme immunoassay, place on a thick glass plate and cut into 4 mm wide strips. 18. If blot strips or sheets are to be stored, they should be washed in PBS (without Tween). They may be stored between sheets of filter paper, wrapped in plastic wrap at -20ºC or -70ºC indefinitely. FOR ENZYME IMMUNOASSAY SEE WESTERN BLOT EIA BELOW 6

7 REAGENTS REQUIRED FOR GEL ELECTROPHORESIS NOTE: Water used to make all reagents in this section must be deionized. Lower gel buffer: Weigh g of Tris into a 1 liter beaker. Add approximately 800 ml of water. Mix on stirrer until in solution. Add 10 ml concentrated HCL and ph. Don t adjust ph. PH should be Add water to bring final volume to 1 liter. Filter through a prefilter and then a 0.2-µm filter. Store at 4 C. Lower chamber buffer: Lower gel buffer diluted 1:4 with water. Store at 4 C. Upper gel buffer: Titrate 2.62 g of Tris with 2M H 2 SO 4 to exactly ph When the ph has reached 7, dilute the 2M H 2 SO 4 1:100 and continue titrating to ph Add water to bring the final volume to 100 ml. Store at 4 C. Upper chamber buffer: Add 2.47 g boric acid and 4.92 g Tris to a 1 liter graduate. Add ml of water. Swirl to dissolve. Add water to bring volume to 950 ml. Add 10 ml of 10% SDS. Add water to bring final volume to 1000 ml. (The ph of this solution usually is 8.64; however, it will vary with different electrodes. The actual weight of the components is more important.) Store at 4 C. Resolving acrylamide gel solution (40% T:1% C [ratio of acrylamide to bis]): CAUTION: Unpolymerized acrylamide is a neurotoxin. Handle accordingly. Weigh 40 g acrylamide monomers and 1 g N,N -methylene-bisacrylamide (bis) into a 100 ml beaker. Add boiling deionized water to ~90 ml. Stir to dissolve. After the solution cools to room temperature, bring the final volume to 100 ml with deionized water. Filter through a 0.2µm filter. This solution must be used within one month, but do not use it on the day it is prepared. Store in a GLASS container at 4 C. Stacking acrylamide gel solution (12% T:1.2%C): Weigh 3.0 g acrylamide monomers and 0.3 g N,N -methylene-bisacrylamide (bis) into a beaker. Add approximately 15 ml of boiling deionized water. Stir to dissolve. After the solution cools to room temperature, bring the final volume to 25 ml with deionized water. Filter through a 0.2µm filter. Do not use until the day after the solution is prepared. Store in a GLASS container at 4 C. The solution is stable for 1 month. Catalysts: Ammonium persulfate solutions: Weigh out and add 30 mg of ammonium persulfate to a PLASTIC test tube. Add 2.0 ml degassed, deionized water to make a final concentration of 10 mg per ml. Mix well. Prepare this solution fresh daily for the RESOLVING GEL. This solution should be diluted 1:8 with water for use in the STACKING GEL. TEMED (N,N,N,N -tetramethylethylenediamine) solutions: NOTE: Dilute TEMED in a GLASS tube (concentrated TEMED reacts with plastic). a. Pipette ml of degassed, deionized water into a glass tube. 7

8 b. Add 75µl of TEMED and mix, rinsing the pipette tip with the solution. Prepare fresh daily for the RESOLVING GEL. This solution should be diluted 1:4 with water for use in the STACKING GEL. NOTE: Store TEMED and ammonium persulfate in an airtight container with silica gel dessicant at 4 C. Allow the contents to come to room temperature before opening. Replace TEMED and ammonium persulfate every 3 months. HIV/HTLV antigens: See PREPARATION OF VIRUS FOR ELECTROPHORESIS Molucular weight standards: See TREATMENT OF PROTEIN STANDARDS 8

9 PREPARATION OF VIRUS FOR ELECTROPHORESIS Disrupt antigen in 0.01 M Tris containing sodium dodecyl sulfate (SDS), 5% V/V 2- mercaptoethanol, 10% glycerol, and 0.25% mg/ml bromphenol blue and then heat. DISRUPTION MIXTURE HIV-1 0.1% SDS, ph8 Tris To make 100 ml: Weigh 0.12 g Tris Qs to 80 ml with distilled water Adjust ph to 8.0 using dilute HCl Add: 0.1 g SDS 10.0 ml glycerol g bromphenol blue Qs to 95 ml with distilled water ALIQUOT AND STORE AT -20ºc ADD 5% 2-MERCAPTOETHANOL JUST BEFORE USE ANTIGEN DISRUPTION HIV-1 Use 0.1µg of protein per mm of lane width For a 74 mm wide lane, add the appropriate amount of the current lot of HIV-1 viral lysate to 70µl disruption mixture (containing 2-ME) and heat at 65ºC for 30 minutes ANTIGEN DISRUPTION HTLV-I/II Use 1% SDS to prepare disruption mixture as above. Add the appropriate amounts of HTLV-I viral lysate and recombinant p21e to 70µl disruption mixture (containing 2-ME). Disrupt by boiling for 3 minutes. ANTIGEN DISRUPTION HIV-2 Adjust ph of buffer to 6.8. Use 1% SDS in the disruption mixture. Add the appropriate amount of HIV-2 lysate to 70µl disruption mixture (containing 2-ME). Disrupt by boiling for 3 minutes. 9

10 TREATMENT OF PROTEIN STANDARDS Bio-Rad Low Molecular Weight Markers in glycerol Mix the following: 50 µl standards 250 µl 10% SDS 100 µl 1.0M DTT 250 µl 0.05M Tris/HCL with 50% glycerol ph µl deionized water Heat at 100 C for 5 minutes Add 50 µl tracking dye (see below) Final concentration of each protein = 0.1 µg/µl Dispense 20 µl per vial Store at -70 C Tracking dye 1. Combine the following: 50 mg bromphenol blue dye 8 ml glycerol 1 ml 0.5M Tris/HCl ph8 1 ml distilled water 2. Mix until all dye is dissolved (~ 3 hours) 3. Store at -20 C 1.0M DTT g DTT in 0.25 ml distilled water MAKE FRESH 0.05M Tris/HCl + 50% glycerol ph8 0.3 g Tris base 20 ml distilled water adjust to ph8 with distilled water qs to 25 ml with distilled water add 25 ml glycerol store at -20 C 0.5M Tris HCl 3.0 g Tris base 40 ml distilled water adjust to ph8 with 1N HCl qs to 50 ml with distilled water store at -20 C 10

11 REAGENTS REQUIRED FOR TRANSBLOT NOTE: Water used to make all reagents in this section must be deionized. Blot buffer: Make 2 liters of blot buffer, as follows: To 1 liter of lower chamber buffer (see appendix E), add 400 ml of absolute methanol. Add 600 ml of water, mix and use without further adjustment. Store at 4 C. Phosphate-buffered saline (PBS): 0.01M, ph 7.2, 10X stock Procedure: Weigh into an Erlenmeyer flask: 1 liter of 10X stock Sodium chloride (NaCl) g Sodium phosphate, dibasic, anhydrous (Na 2 HPO 4 ) g Sodium phosphate, monobasic, monohydrate (NaH 2 PO 4.H 2 O) g Add distilled water to ml Stir until dissolved and qs with distilled water ml 1. Check ph of a 1:10 dilution using the following: 5 ml PBS + 45 ml water = 50 ml 0.01M PBS (discard this sample after testing) 2. Adjust as necessary to ph Millipore filter for sterilization 4. Store at room temperature 5. For use: Dilute 1:10 with distilled water 6. Label: 10X STK FOR 0.01M PBS ph 7.2 date PBS-Tween: Add 3ml of Tween 20 (polyoxyethylene sorbitan monolaurate) per liter of PBS. Add Tween slowly, using a syringe fitted with a 14 gauge cannula. Stir the PBS while adding the Tween. Prepare fresh daily. 11

12 AMIDO BLACK STAIN 0.1% amido black 45% methanol 10% acetic acid To make 100 ml: 0.1 g amido black 45.0 ml methanol 10.0 ml acetic acid dissolve thoroughly (~ 1 hour) qs to 100 ml with water store at room temperature DESTAIN FOR AMIDO BLACK 10% methanol 7.5% acetic acid To make 100 ml: 10.0 ml methanol 7.5 ml acetic acid qs to 100 ml with water store at room temperature TO STAIN STRIPS 1. Add amido black stain to strip containing the protein standards for 10 minutes 2. Pour off the stain and rinse the strip in water 3. Add destain for 2 minutes 4. Pour off and add more destain 5. Wait until can see bands well 6. Rinse in water and allow strip to air dry WESTERN BLOT EIA 12

13 1. Prepare runsheet. 2. Calculate volumes needed and prepare PBS, PBS-Tween, and Blotto. 3. Place one strip in each trough of the incubation tray. 4. Add 1ml of Blotto to each strip. 5. Add 10µl of control or patient sample to the appropriate strip. 6. Cover the tray and incubate overnight (14-20 hours) at room temperature (18-28 C) on a platform rocker. 7. Heat PBS-Tween to 56 C for the first wash. For all subsequent steps PBS-Tween may be allowed to cool to room temperature. 8. Aspirate the sample dilutions from the strips and wash four times with 2 ml of PBS-Tween for 5 minutes each time on the platform rocker. 9. To each strip, add 1 ml of conjugate, optimally diluted in Blotto. 10. Incubate for 1 hour at room temperature on the platform rocker. 11. Wash the strips four times for 5 minutes each time with 2 ml of PBS-Tween on the platform rocker. 12. Wash the strips one time for 5 minutes with 2 ml of PBS on the platform rocker. 13. Add 1 ml of DAB substrate solution prepared just before use (see below). 14. Incubate 10 minutes on the platform rocker. 15. Remove the substrate and wash the strips three times in distilled water. 16. Thoroughly clean the slotted incubation tray by filling the troughs with undiluted bleach (3% sodium hypochlorite) for one hour. Aspirate and soak trays overnight in distilled water. Add1% 7X cleaning solution to troughs and clean with a cotton swab. Rinse with distilled water and allow to dry. REAGENTS PBS ph 7.2, 0.01M PBS-Tween Blotto Conjugate Substrate 3,3 -Diaminobenzidine (3,3 - Diaminobenzidine tetrahydrochloride dihydrate [DAB] REPORTING RESULTS: Prepare 1X from 10X stock (See Reagents Required for Transblot above) in distilled water Add 3 ml of Tween-20 per liter of 1X PBS Add 5 grams of nonfat dry milk (Bio-Rad Blocker) plus gram of thimersol per 100 ml PBS Dilute Bio-Rad Blotting Grade GAH-IgG HRP in Blotto to the optimal dilution. (Recommended dilution is usually 1:3000) 1X DAB = 50 mg DAB per 100 ml PBS plus hydrogen peroxide. DAB is prepared as a 10X stock solution, aliquoted and stored at -20 C. Just before use, dilute 10X stock to 1X in PBS and add 10µl of 30% Hydrogen Peroxide per 100 ml of 1X DAB 13

14 Reference Ranges: HIV-1 Negative control no bands present Weak positive control bands present at 24, 120, 160 Strong positive control bands present at 17, 24, 31, 41, 51, 55, 66, 120, 160 HIV-2 Negative control no bands present Positive control bands present at 26, 36, 55, 68 HTLV-I/II Negative control no bands present HTLV-1 positive control bands present at 19, 24, p21e HTLV-II positive control bands present at 19, 24, p21e Procedures for Abnormal Results: Repeat run if above criteria are not met. Reporting Format: A. HIV-1 B. HIV-2 1. POSITIVE: at least two of the following three bands: p24, gp41, gp120/ INDETERMINATE: one of the following three bands: p24, gp41, gp120/ NEGATIVE: any pattern that does not meet the criteria for positive or indeterminate 1. POSITIVE: p26 plus at least one envelope band (gp36, gp125, gp140) or two envelope bands 2. INDETERMINATE: any band pattern containing p26, gp36, p55, p68, gp125, gp140 and not meeting the criteria for a 14

15 C. HTLV State of California positive 3. NEGATIVE: none of the reported bands (p26, gp36, p55, p68, gp125, gp140) 1. POSITIVE : p19 and/or p24 plus p21e 2. INDETERMINATE: p21e only 3. NEGATIVE: no p21e (with or without core bands) REFERENCES: Enzyme-linked immunoelectrotransfer blot technique (Western Blot) for Human T- Lymphocytic virus type III/Lymphadenopathy-associated virus (HTLV- III/LAV). Centers for Disease Control, U.S. Department of Health and Human Services, immunology series no. 15. Centers for Disease Control, Atlanta Tsang, V.C., K. Hancock, M. Wilson, D.F. Palmer, S. Whaley, J.S. McDougal, S. Kennedy 15