P24 Ultrasensitive Assay Protocol

Transcription

1 PerkinElmer Life Sciences, Inc. 549 Albany Street Boston, MA PROTOCOL Intended for Research Use Only. Not for Use in Human Diagnosis. P24 Ultrasensitive Assay Protocol PRINCIPLES OF THE PROCEDURE The Ultrasensitive HIV-1 p24 Antigen Assay is accomplished using the PerkinElmer Life Science, Inc., NEK 050/050A/050B HIV-1 p24 ELISA, PerkinElmer NEP116VL ELAST Amplification System, and Quanti-Kin Detection System Software. Immune complex disruption of serum/plasma samples is done by using a 1:6 sample dilution in a dissociation buffer followed by boiling for 5 minutes at 100 C. Samples are cooled and transferred to microplate wells that are coated with a highly specific mouse monoclonal antibody to HIV-1 p24. The immobilized monoclonal antibody captures both free HIV-1 p24 and that which has been released upon disruption of immune complexes in the serum/plasma samples. The captured antigen is complexed with biotinylated polyclonal antibodies to HIV-1 p24, followed by a streptavidin-hrp (horseradish peroxidase) conjugate. Signal amplification is then done via incubation with the NEP116 VL biotin tyramide solution, followed by NEP116 VL streptavidin-hrp (horseradish peroxidase) conjugate. Signal is detected by incubation with orthophenylenediamine-hcl (OPD) which produces a yellow color that is directly proportional to the amount of HIV-1 p24 core antigen captured. The absorbance of each well is determined using a microplate reader and calibrated against the absorbance of an HIV-1 p24 core antigen standard curve. Sample absorbance is measured in both kinetic and endpoint mode using the Quanti-Kin Detection System Software. The software allows automatic computer-driven data collection from the platereader and for quantitation of sample measurements. REAGENTS NEK 050/050A/050B HIV-1 p24 ELISA Reagents are supplied for one, two (050A) or five (050B) 96-well microplate(s). The following reagents supplied in the NEK050/A/B kits are used in the p24 Ultrasensitive Assay: 1 (2) (5) 96-well Antibody-coated Microplate(s) - coated with monoclonal antibody to HIV-1 p24. Preservative: 0.01% Proclin (1) (2) vial(s) Positive Control, 200ng/mL 0.4 ml/tube. Contains 200 ng/ml as HIV-1 p24 (approx. 800 ng/ml as total HIV-1 protein), in Phosphate Buffered Saline (PBS plus BSA and Triton X-100. Preservative: < 0.1% Sodium Azide. 1 of 8 PC

2 1 (2) (5) bottle(s) Negative Control - 13 ml/bottle. Recalcified human serum. Preservative: 0.5% 2-chloroacetamide. Non-reactive for Hepatitis B surface antigen, and antibodies to HIV-1, HIV-2, and HCV. 1 (2) (5) bottle(s) Detector Antibody - 11 ml/ bottle. Rabbit polyclonal anti-p24 antibody in PBS containing animal sera, casein, and human serum non-reactive for hepatitis B surface antigen and antibodies to HIV-1, HIV-2, and HCV. Preservative: 0.2% Proclin-300 and < 0.1% Sodium Azide. 1 (2) (4) vials(s) Streptavidin-HRP Concentrate (Labeled as 77607R)- 0.4 ml/ vial. A concentrate of SA-HRP in Citrate buffer with BSA and detergent. Preservative: 0.5% 2-chloroacetamide. T 1 (2) (5) bottle(s) Streptavidin-HRP Diluent 14 ml/ bottle. PBS with BSA and 0.05% Tween-20. Preservative: 0.5% 2-chloroacetamide. 1 (1) (2) bottle(s) Substrate Diluent - 60 ml/ bottle. Citrate buffer containing 0.03% Hydrogen Peroxide. Stabilizer: 0.002% sodium stannate. 1 (1) (2) bottle(s) 5% Triton X ml/ bottle. 5% Triton X-100 in Phosphate buffer plus an inert blue dye. Preservative: 0.02% Sodium Azide. 1 (1) (2) strip(s) OPD Tablets - 5 tablets per strip. Foil-wrapped OPD tablets. 1 (2) (5) bottle(s) Stop Solution - 12 ml/bottle. 4N Sulfuric Acid. 2 (3) (5) Plate Wash Concentrate, 20X - 100ml/bottle. Concentrated phosphate buffer plus 1% Tween-20. Preservative: 2% 2-chloroacetamide. 12 (24) (36) plate covers. The following reagents are supplied in the NEK050/A/B kits but are NOT used in the p24 Ultrasensitive assay: 1 (1) (2) bottle(s) Glycine Reagent - 25 ml/bottle. 1.5M Glycine. 1 (1) (2) bottle(s) Tris Reagent - 25 ml/bottle. 1.5M Tris. 1 (1) (1) bottle Immune Complex Control 1.5 ml/bottle. Human serum plus HIV-1 p24 antibody and inactivated HIV-1 p24 antigen. Non-reactive for Hepatitis B surface antigen, and antibodies to HIV-1, HIV-2, and HCV. Preservative: 0.5% 2-chloroacetamide 2 of 8 PC

3 NEP116VL ELAST ELISA Amplification System Reagents are supplied for five 96-well microplate(s). A maximum of 15 assays can be run using reagents supplied. 1 Bottle Biotin Tyramide Solution 2 ml/ bottle. Biotinyl-tyramide in ethanol 1 Bottle Amplification Diluent Concentrate, 2X 70 ml/ bottle. Borate buffer with hydrogen peroxide. 1 Vial Streptavidin HRP Concentrate (Labeled as FP105) 0.22 ml/vial. Streptavidin-horseradish peroxidase concentrate in citrate buffer with added protein and preservative. Lot specific dilution instructions are supplied on accompanying TD sheet. 1 Bottle Standard/Sample Diluent ml PBS containing added protein, detergent, and 0.1% Sodium Azide as preservative. 1 Bottle 5% Triton X ml/ bottle. 5% Triton X-100 in Phosphate buffer plus an inert blue dye. Preservative: 0.02% Sodium Azide. Same as in the NEK050 kit. 5 Bottles Streptavidin-HRP Diluent 14 ml/ bottle. PBS with BSA and 0.05% Tween- 20. Preservative: 0.5% 2-chloroacetamide. Same as in the NEK050 kit. 1 strip OPD Tablets - 5 tablets/ strip. Foil-wrapped OPD tablets. Same as in NEK050 kit. 1 Bottle Substrate Diluent - 60 ml/ bottle. Citrate buffer containing 0.03% Hydrogen Peroxide. Stabilizer: 0.002% sodium stannate. Same as in the NEK050 kit. Storage of Components Stop Solution, Plate Wash Concentrate, 20X, and plate covers (from NEK050/A/B kits) may be stored at room temperature. All other kit components should be kept refrigerated at 2-8 C. Equipment and Reagents Required but not Supplied Dry heat block (100 C) with thermometer Microtiter plate shaker (optional) Microplate reader with filters at 450, 490 or 492, and >600nm filters Plate Washer 37 C Incubator Quanti-Kin Detection System Software Precision pipettors and tips Polypropylene tubes Disposable gloves Disposable reagent reservoirs Vortex mix 3 of 8 PC

4 Warnings and Precautions Follow all Warnings contained in the NEK050 manual on pages 4 and 5. NEP116VL reagent pack contains: 1. OPD tablets. OPD is toxic, an irritant, a sensitizer, and is also classified as a potential carcinogen. Avoid inhalation and contact with the skin. Do not allow OPD Tablets to come into contact with metal or other oxidizing agents. If skin is contacted, flush with water. Solutions containing OPD should be disposed of in compliance with local regulations. 2. Biotinyl-Tyramide Solution contains ethanol. Ethyl Alcohol may cause respiratory tract, skin, and eye irritation. Central nervous system depressant. Highly Flammable. Vapors are heavier than air and may travel to a source of ignition and flash back. 3. Some of the reagents contain sodium azide as a preservative and may react with lead or copper in drain lines to form explosive metal azides. Upon disposal, flush with a large volume of water to prevent azide buildup in drains. ULTRASENSITIVE P24 ANTIGEN DETECTION PROTOCOL 1. Bring all the reagents to room temperature (15-30 C) prior to use. Standard Curve Preparation 2. Pre-dilute 1:16 the NEK 050/A/B Positive Control, 200ng/mL with NEP116VL Standard/Sample Diluent (10uL positive control + 150uL std/sample diluent =160uL total volume) (Pre-Dilution Tube). Prepare the Reference Standard Curve diluting 1:2 as indicated in the table using the same NEP116VL Standard/Sample Diluent. Mix well by pipetting several times, do not use Vortex to avoid the formation of foam. Standard pg/ml Standard fg/ml 6,250 6,250,00 0 Vial ID A Volume (μl) 100 of 1:16 prediluted Positive Control Std/Sam. Diluent (μl) Final Volume (μl) ,125 3,125,000 B 100 Vial A , C 100 Vial B ,000 D 100 Vial C E 100 Vial D ,000 F 100 Vial E ,00 G 100 Vial F H 100 Vial G I 100 Vial H J 100 Vial I K 100 Vial J ,000 L 100 Vial K ,5 1,500 M 100 Vial L Only A-C-E-G-I-J-K and M dilution vials should be used as indicated below in the microplate scheme (step 9). 4 of 8 PC

5 Reagent Preparation 3. Bring all the reagents to room temperature (15-30 C) prior to use. 4. Prepare an adequate volume of Dissociation Solution. Dilute NEK050/A/B kit 5% Triton X- 100 to 0.5 % in PBS or distilled, deionized water 5. # of Strips # of Tests Dissociation Solution Volume (ml) Dilute NEK 050/A/B kit Plate Wash Concentrate 20X to 1X by adding one volume plate wash concentrate to 19 volumes distilled deionized H 2 O. Crystals may form in the Plate Wash Concentrate 20X if refrigerated. These should re-dissolved by gentle warming prior to use. Approximately 1,500 ml of diluted 1X wash buffer is needed per plate. When using an automatic washer, prepare 200 ml more (Total Volume: 1700 ml). Diluted (1X) wash buffer should be prepared fresh prior to assay. Prepare all other working reagents within 15 minutes of use. While preparing the reagents, pre-heat thermal (dry heat) block to 100 C. Pre-Treatment of Controls, Standards and Serum/Plasma Samples 6. All the Controls, Reference Standards, and serum/plasma samples must be diluted 1:6 prior to the heat treatment. 7. Add 250μL of prepared Dissociation Solution (TRITON X %) to each tube. Add 50μL of each control, reference standard and serum/plasma sample into designated tube to obtain a 1:6 final dilution. Mix well by pipetting several times. Do NOT use vortex to avoid the formation of foam. Heat Treatment of Plasma Samples 8. Transfer all the tubes into the thermal block. Screw-capped polypropylene tubes are optimal. Boil at 100 C for 5 minutes, then remove the tubes and let them cool down for at least 5 minutes at room temperature. 5 of 8 PC

6 Transfer Into Microplate and Incubation 9. Take care not to transfer coagulated particles Mix samples by pipetting. Transfer 250μL of the heat-denatured materials into designated NEK050/A/B antibody- coated plate microwells (as indicated below). It is important to change tip after each transfer. Well A SB 390,000 (E) S6 S14 B NC 1,562,000 (C) S7 S15 C NC 6,250,000 (A) S8 S16 D 1500 (M) S1 S9 S17 E 6,000 (K) S2 S10 S18 F 12,000 (J) S3 S11 S19 G 24,000 (I) S4 S12 S20 H 98,000 (G) S5 S13 S21 SB:Substrate Blank; NC:Negative Control; 1500 to 6,250,000:Reference Standards fg/ml; S1; S2: serum/ plasma samples Cover the microplate and incubate 2 h ± 5 min. at room temperature on a microtiter shaker (i.e. MICROMIX 5 - Diagnostic Product Corporation - Form20/AMP 5). Alternatively, it is possible to incubate: 1 h ± 5 min at 37 C in a dry incubator Overnight (12-20 hrs) in refrigerator (2-8 C). Detector Antibody 11. Prior to washing, manually remove the samples from the wells by vacuum aspiration or with a multichannel pipette. 12. Wash microplate 10 times with 1X wash buffer. Washing can be done automatically or manually; two cycles of 5 washes with 300μL of washing buffer for each well are recommended. 13. Add 100μL of the NEK050/A/B kit Biotinylated Detector Antibody to all wells except that designated for the Substrate Blank. 14. Cover the microplate and incubate 60 ± 5 minutes at 37± 1 C. ATTENTION: In the following steps a Streptavidin-HRP Concentrate from the NEK050/A/B and a Streptavidin-HRP Concentrate from NEP116VL Kit are used. The Vials are similar but they have two different concentrations: be sure to use the correct Streptavidin Vial according to protocol instructions. 6 of 8 PC

7 Streptavidin-HRP minutes prior to use, dilute 1:50 the NEK050/A/B kit Streptavidin-HRP Concentrate (77607R) with the NEK050/A/B kit Streptavidin-HRP diluent. Prepare a sufficient volume as indicated in the following table: # of Strips # of Tests Streptavidin-HRP Concentrate (μl) Streptavidin-HRP Diluent (μl) , , , , , , Wash the microplate as described at step Add 100μL of diluted Streptavidin-HRP in all wells except Substrate Blank. Incubate 15 min at 37 C. Signal Amplification 18. Dilute 1:2 the NEP116VL Amplification Diluent Concentrate, 2X with distilled, deionized H 2 O in a proper volume. 19. Biotinyl Tyramide Working Solution: Add 10 μl of NEP116VL Biotinyl-Tyramide per each ml of prepared working solution (i.e. for a 96 microplate, use 100μL of Biotinyl-Tyramide in 10mL.) # of Strips # of Tests Amplification Diluent Concentrate, 2X (μl) Distilled Water (μl) Biotinyl-Tyramide (μl) ,000 2, ,000 3, ,000 4, ,000 5, Wash the microplate as described at step Add 100μl of Biotinyl-Tyramide Working Solution in all wells except Substrate Blank. Incubate 15 minutes at room temperature. 22. Dilute the NEP116VL Streptavidin-HRP (FP105) to make the SA-HRP Working Solution. Prepare working solution using the NEK050 Streptavidin-HRP Diluent (either that supplied in NEK050 kit or a supplemental bottle supplied in NEP116VL reagent pack). Use lot-specific dilution instructions supplied in the accompanying TD sheet. 23. Wash the microplate as described at step 11 7 of 8 PC

8 24. Add 100μL of NEP116VL SA-HRP Working Solution to all wells except Substrate Blank. Incubate 15 minutes at room temperature. Substrate Adding and Reading 25. Prepare sufficient OPD Substrate Solution within 15 minutes of use. With non-metallic forceps, add one NEK050/A/B kit OPD Tablet to 11 ml of NEK050/A/B kit Substrate Diluent. Vortex vigorously to assure complete dissolution. Protect from light. Store at 2-8 C or on ice prior to use. (Prepared substrate solution MUST be chilled prior to adding to the microplate.) The OPD substrate solution should be colorless. A yellow-orange color indicates that the reagent is contaminated and must be discarded. 26. Wash the microplate as described at step Add 100μL of refrigerated, diluted Substrate Solution to ALL wells, including Substrate Blank. 28. Insert the microplate into the plate reader and perform kinetic reading using the QUANTI- KIN DETECTION SYSTEM software for HIV-1 p24 ELISA kit. 29. After 30 minutes of incubation, stop the reaction by adding 100 μl of NEK050/A/B kit Stop Solution to all wells. Perform the end-point reading as indicated by the QUANTI-KIN software. REFERENCES 1. Schüpbach J, Böni J. Quantitative and sensitive detection of immune-complexed and free HIV antigen after boiling of serum. Journal of Virological Methods 43: , Schüpbach J, Flepp M, Pontelli D, Tomasik Z, Lüthy R, Böni J. Heat-mediated immune complex dissociation and enzyme-linked immunosorbent assay signal amplification render p24 antigen detection in plasma as sensitive as HIV-1 RNA detection by polymerase chain reaction. AIDS 10: , Giacomini M, McDermott JL, Giri AA, Martini I, Lillo FB, Varnier OE. A novel and innovative quantitative kinetic software for virological colorimetric assays. Journal of Virological Methods, 73: , Schupbach J. and Varnier O.E. HIV-1 p24 antigen a sensitive and precise, yet inexpensive alternative to PCR for viral DNA or RNA. Newsletter. International AIDS Society, 15:9-11, TSA and the use of TSA are protected by U.S. patents issued to PerkinElmer Life Sciences, Inc., #5,731,158; 5,583,001; and 5,196,306 and patents pending, and foreign equivalents thereof. By purchasing this product the end user is granted a limited license to use TSA for research purposes only. No license or right is granted to use TSA for diagnostic or therapeutic purposes. Purchase does not include or carry any right or license to use, develop or otherwise exploit this product commercially. Any commercial use, development or exploitation of this product without the express prior authorization of PerkinElmer Life Sciences, Inc. is strictly prohibited and constitutes an infringement of the intellectual property rights of PerkinElmer Life Sciences, Inc. under the aforementioned patents. TSA is a trademark of PerkinElmer Life Sciences, Inc. ELAST (ELISA Amplification System) is a registered trademark of PerkinElmer Life Sciences, Inc. All Quanti Kin softwares are produced by RILAB srl, Research and Computer Science for the Environmental and Biomedical Laboratory. 8 of 8 PC