HIV-1 p24 ELISA Kit: Overview
Contents
1. 7 50n1 A 50n1l B 15 8 50 1 Poz lt 9 450nm 650nm Q 15 ia ae XM KI gt Abs 450 650 nm 1 10 100 1 000 P24 std pg ml gt 2 8 gt gt TEL 098 943 3740 FAX 098 943 3740 E mail info rimco jp2. HRP JOH HITV 1p24 1 EDTA LRT Vary av Ly pie HRP HIV 1p24 1 p24 HRP p24 HRP Ab p24 Ab HRP
3. lt 3 60 a 4 5 30 60 5 50n1 50nl B 15 6 50 1 7 Q o 450nm 650nm 15 gt ag m m gt gt 2
4. HIV 1 p24 D 2 HIV 1 p24 1mlx1 HIV 1 p24 2000pg ml ELISA 1 ae E P 0 Imlx1 Ei 1 HTIV 1 p24 HIV 2 p26 x 100 4 100mlxi 5 X20 50mlxl VR BEEN HIV 1 p24 ELISA gt p EF Paza 6 A 8mlx1 HIV 1 p24 7 B 8mlxl p24 8 8mlx1 2N 3 EDTA
5. 2 2 3 SR 4 5 2 8 3 20C 6 gt 1 ELISA ARICA 350 400 1 5 gt 1 EDTA 1 HIV 1 p24 50n1
6. HIV 1 p24 gt 1 2 3 IEN gt on W WwW NO gt 25 1000n1 25 1000m1 1 450nm 2 450nm 650nm WP MLE 1 EDTA 2
7. 6 7 8 HRP 9 1 1 20 2 3 HRP HIV 1 p24 HRP 1 2
8. azide and EDTA or citric acid are measured 1 Standard HIV 1 p24 solution is diluted stepwise with Ag Ab dilution buffer and 50 uL is added to each well The measurement sample is also added to each well in 50 uL aliquots For the blank 50 uL of the Ag Ab dilution buffer is added to a well HRP conjugated antibody prepared by 100 fold dilution with Ag Ab dilution buffer is added to each well in 50 uL aliquots The wells are covered with a plate sealer and allowed to react at room temperature for 60 minutes Each well is washed five times with diluted wash buffer If the washing is manual the micro wells are filled for 30 to 60 s in each cycle and after the final washing cycle the plate is inverted over highly absorbent tissue paper and tapped lightly to remove remaining liquid To each well including the blanks 50 uL each of chromogen solutions A and B are added and the plate is covered with a plate sealer and gently agitated followed by incubation for 15 minutes at room temperature with protection from light To each well 50 uL of stop solution is added following by gentle agitation Optical absorbance is measured using a plate reader with the measurement wavelength being 450 nm and the reference wavelength being 650nm The absorbance should be measured within 15 minutes after stopping the reaction Measurement Method 2 This is used when samples containi
9. 2000pg mL 3 HRP conjugated Liquid 01mLx1 HRP antibody x100 conjugated HIV 1 p24 antibody 4 Antigen antibody Liquid 100mLx1 Phosphate Ag Ab dilution buffer buffer 5 Concentrated Liquid 50 mL x 1 Phosphate wash buffer buffer x20 6 Chromogen Liquid 8mLx1 Citrate buffer solution A 7 Chromogen Liquid 8 mL x 1 Citrate buffer solution B 8 Stop solution Liquid 8 mL x 1 2 N sulfuric acid Additional item included Three sheets of adhesive film Materials and equipment required other than included in Kit 1 Purified water 2 Micro pipette 25 to 1000 uL 3 Measuring cylinder 25 to 1000 mL 4 Microplate washer 5 Microplate reader single wavelength of 450 nm or two wavelengths with main wavelength of 450nm and_ reference wavelength of 650 nm 6 Paper towels Disposable gloves 8 Agents and or devices for treatment of liquid waste Precautions relating to operations Samples for measurement include cell culture supernatants cell lysates serum and plasma 1 When measurements are carried out with samples containing sodium nitride and EDTA or citric acid the reactions should be carried out by the two step method Measurement Method 2 2 Care should be taken to not allow microbial contamination of serum and plasma samples 3 There is the potential for incorrect results with chyle containing plasma samples samples from jaundice patients and hemolytic samples s
10. EDTA 1 HIV 1 p24 100ul 100ul 100ul 2 60 3 5 30 60 4 200 HRP 100n1l 5 60 6 5 30 60
11. HIV 1 p24 ELISA Kit Overview The HIV 1 p24 ELISA Kit is used to measure the amount of HIV 1 p24 antigen in samples by means of the enzyme linked immunosorbant assay ELISA HIV 1 p24 antigen measurement is included in the fourth generation ELISA in the human immunodeficiency virus HIV tests This Kit is used in research for quantification of the HIV 1 p24 antigen Because a detection antibody conjugated with horseradish peroxidase HRP is used the process can be completed by carrying the reaction out once in contrast with other ELISA kits in which biotin conjugated antibodies are used although this depends upon the antibody type With this Kit there is cross reactivity with HIV 2 p26 which corresponds to HIV 1 p24 reducing the sensitivity yet quantification is still sometimes possible The HIV 1 p24 ELISA Kit is a sandwich enzyme linked immunological detection kit involving the use of polystyrene micro wells coated with anti HIV 1 p24 antibody In the first step the p24 antigen standard and antibody treated with a surface active agent are introduced to the wells For measurement of samples not General precautions 1 This product is a reagent for use in research and should not be used for other purposes 2 This product should be used in accordance with the methods stipulated in the Package Insert 3 This product should be used after carefully reading the leaflets and the explanatory documents about handling rela
12. be taken to not allow the microplates to dry 5 Any samples collected from humans have the potential to be infective and the appropriate precautions should therefore be taken 6 Pipette tips and tubes that have been used for measurement should be treated appropriately with respect to wastewater etc and should then be disposed of 7 Ifany of the stop solution is spilt it should be wiped up immediately If it comes into contact with the skin or eyes these should be washed with water 8 The enzymatic activities of HRP conjugated compounds can be affected by materials such as dust reactive drugs sodium hypochlorite acids and alkalis and measurements should therefore be carried out in places where these are not present 9 The relevant Material Safety Data Sheets will be provided as requested Preparation Preparation stage 1 Wash buffer is prepared by mixing the concentrate with distilled or de ionized water in a 1 20 ratio Preparation stage 2 The required number of strips is placed in the strip holder Unused strips are returned to the pack Preparation stage 3 HRP conjugated anti HIV 1 p24 antibody is prepared The amounts of HRP conjugated antibody to be used are measured as portions into tubes and then diluted to the dilution concentrations required by Measurement Method 1 or 2 using the Ag Ab dilution buffer Measurement Method 1 This is used when samples not containing sodium
13. l 50n1 50u 2 100 HRP 50unl 2 5 350 400 1 5 gt 1 2 3 18 30C 4 5
14. ng sodium azide and EDTA or citric acid are measured 1 Standard HIV 1 p24 solution is diluted 2 stepwise with Ag Ab dilution buffer and 100 uL is added to each well The measurement sample is also added to each well in 100 uL aliquots For the blank 100 uL of the Ag Ab dilution buffer is used The wells are covered with a plate sealer and HRP conjugated allowed to react at room temperature for 60 minutes Each well is washed five times with diluted wash buffer If the washing is manual the micro wells are filled for 30 to 60 s in each cycle and after the final washing cycle the plate is inverted over highly absorbent tissue paper and tapped lightly to remove remaining liquid antibody prepared by 200 fold dilution with Ag Ab dilution buffer is added to each well in 100 wL aliquots The wells are covered with a plate sealer and allowed to react at room temperature for 60 minutes Each well is washed five times with diluted wash buffer If the washing is manual the micro wells are filled for 30 to 60 s in each cycle and after the final washing cycle the plate is inverted over highly absorbent tissue paper and tapped lightly to remove remaining liquid To each well including the blanks 50 uL each of chromogen solutions A and B are added and the plate is covered with a plate sealer and gently agitated followed by incubation for 15 minutes at room
15. o the Kit should not be used with these 4 Measurements can be carried out even if samples have been inactivated 5 Samples should be stored at 2 to 8 C Samples with which tests are not needed within 3 days should be stored frozen at 20 C or lower 6 Reagents and samples should not be repeatedly frozen and thawed Precautions relating to washing 1 A high quality ELISA microplate washing device is recommended so that a high level of washing performance can be maintained In general in order to avoid false positive reactions and high background levels automated washing is recommended with at least five cycles each at 350 to 400 nuL well 2 If washing is carried out manually it should involve five cycles with 350 to 400 pL well of wash buffer being added in each cycle following by removal by aspiration If the results are poor with high background levels for example either the number of washing cycles or the time for which the wash buffer is left in the well in each cycle should be increased Safety and precautions 1 The reagents should not be mixed between kits of different manufacturer s lots At the time of the test the reagents in this Kit should be prepared carefully so that the optimal test results can be obtained 2 Reagents should not be used when the use expiry date has passed 3 Reagents and samples should be returned to room temperature 18 to 30 C before use 4 After washing care should
16. temperature with protection from light To each well 50 uL of stop solution is added following by gentle agitation Optical absorbance is measured using a plate reader with the measurement wavelength being 450 nm and the reference wavelength being 650nm The absorbance should be measured within 15 minutes after stopping the reaction Calibration curve Abs 450 650 nm 1 10 100 1 000 P24 std pg ml Storage and stability The kit is stored at 2 to 8 C without freezing Precautions relating to use This kit is for research use and should not be used for diagnoses etc Manufacturing amp Technical Support RYUKYU IMMUNOLOGY CORPORATION Tel 098 943 3740 fax 098 943 3740 E mail info rimco jp HIV 1 p24 ELISA gt HIV 1 p24 ELISA gt l P Ma RK ELISA HIV 1 p24 HIV 4 ELISA 1 96 92h HTV 1 p24 92 nm
17. ting to the equipment used Details of Kit composition containing sodium nitride and EDTA or citric acid HRP conjugated anti HIV 1 p24 antibody is added as the capture antibody followed by incubation for 1 hour On the other hand for measurement of antibodies containing sodium nitride and EDTA or citric acid just the antigen and antibody are added beforehand followed by incubation for 1 hour washing and then addition of HRP conjugated anti HIV 1 p24 capture antibody and incubation for another hour If the sample contains p24 antigen it is captured in the wells and at the same time a sandwich is formed with HRP conjugated p24 antibody In order to exclude unbound HRP conjugated antibody the micro wells are washed and an enzyme substrate chromogen solution is added In the case of wells containing the antibody p24 antibody HRP sandwich immunocomplex the solution undergoes blue coloration but if the reaction is stopped by addition of sulfuric acid the color of the solution changes from blue to yellow The coloration intensity is correlated to the antigen content of the sample and wells containing samples without HIV 1 p24 remain colorless Specifica Principal No Reagent Properties tions components 1 Solid phase Plate 96 wells Anti HIV 1 microplate p24 murine mono clonal antibody 2 HIV 1 p24 Liquid 1 mL x 1 Recombinant standard bulk HIV 1 p24 solution Antigen
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