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1. 5 COSMO BIO Co LTD Inspiration for Life Science Beacle ver 1 02E Oss User s Manual HBs Pre S1 Quantitative Kit high sensitivity Background and Features of HBs Pre S1 Quantitative Kit Three types of hepatitis B virus surface antigen HBsAg L M and S proteins are known The L protein is composed of S Pre S2 and Pre S1 domains and the M protein is composed of S Pre S2 domains and the S protein S domain alone L protein plays an important role in HBV infection to human hepatic cells where Pre S1 region is the key for recognizing human hepatic cells The HBs Pre S1 Quantitative Kit high sensitivity enables to explore the human hepatic cell recognizing activity of HBsAg and the HBV infectivity by determining Pre S1 antigens Features The world only one detection system for Pre S1 antigen of HBV Determination of Pre S1 activity in human serum possible High sensitivity range 0 2 10 nUnit mL 1 nUnit is defined as the Pre S1 activity possessed by 1ng of standard antigen provided to the kit Related products The related products are listed in the below table We provide a variety of HBV related product Product Product name Content BCL AGS 01 HBsAg L protein ST Preci ane Pre S2 __ 100 pg recombinant HBsAg displaying Pre S1 and Pre S2 activities suitable as control antigen BCL AG 001 TBs L rotein Us recombinant HBsAg displaying all S Pre S1 and Pre S2 regions su2. be accurately 20 min Stop the reaction by adding 50 uL of stop solution note The yellow color generated by the chromogenic reaction diminishes rather quickly Thus to obtain accurate determinations it is important to measure the absorbance soon after the addition of stop solution lt Absorbance measurement and calculation gt 1 Measure the absorbance at 450 nm of each well by using a plate reader 2 Calculate the specific absorbance of each sample or standard by subtracting the blank value 3 Calculate the best fit curve between the specific absorbance values and the corresponding standard concentrations using 4 parameters logistic model 4 The concentration of samples can be calculated from the equation of the best fit curve 5 The calculated concentrations which are out of the range lt 0 2 or gt 10 nUnit mL are not reliable note Clean up the bottom of the plate before absorbance measurement to avoid inaccurate determination lt Example of standard curve gt Below is an example of measurements of standard solutions and resulting standard curve 3 5 Abs460nm Va a 3 0 nm STOO 3304 STOO 1 651 STOO 0 785 N f 10 E magnification 10 4 0 5 ia 0 0 Abs 450nm 0 0 0 5 1 0 a 2 4 6 8 10 12 STD concentration nUnit ml note The above data is an example and does not guarantee the performance of the kit The re
3. d by gentle tapping on the paper towel You can also use plate washer machine 3 In triplicate manner add 100 uL of sample standard solutions or PBS as the blank to each well Beacle ver 1 02E Oss 4 Cover the plate by plate sealer and let it stay at 4 C for overnight more than 15hr lt Reaction with detection IgG HRP and antigens gt 1 Dilute 10 pL of HRP labeled detection IgG solution with 10 mL of Dilution solution for Detection IgG About 10 mL of the diluted detection IgG solution is required to treat one 96 well plate 2 Remove the sample or standard solution from the plate by decantation followed by gentle tapping on the paper towel 3 Wash each well with 200 pL well of PBS T three times Then wash with 210 pL well of PBS three times The washing procedure is the same as described above 4 Add 100 pL of the diluted detection IgG to each well by using 8 channel pipette Incubate for 2 hr at 37 C after covering the plate by plate sealer lt Chromogenic reaction gt 1 Remove the detection IgG solution from each well by decantation followed by gentle tapping on the paper towel 2 Wash each well with 200 pL well of PBS T four times The washing procedure is the same as described above 3 Add 100 pL of the chromogenic solution to each well by using an 8 channel pipette and incubate for 20 min at room temperature in the light tight container To obtain reproducible result the incubation time with chromogenic solution must
4. e two plates User s manual Equipments required for the assay and Reagents not provided by the kit Microplate reader equipped to measure absorbance at 450 nm Micropipettes for the handling of standard antigen and samples we recommend to use tips of protein low bind type Micro tubes for the handling of standard antigen and samples we recommend to use tubes of protein low bind type Plastic tubes bottles plate sealers or plastic films Multi 8 channel pipette Distilled water or pure water Assay Procedure lt Preparation gt Dilute 20 x PBS and 20 x PBS T to 20 fold with distilled water To do so first return the bottles containing concentrated solution to room temperature for complete dissolution chilled concentrated buffers often contain depositions of salts Take 10 ml of the concentrated solutions and add 190 mL of distilled water The diluted buffer solution of 200 mL each is enough to treat one 96 well plate and can be stored at 4 C Before usage the diluted solutions should be returned to room temperature lt Coating of capture IgG to microplate gt 1 Add 50 uL of the capture IgG to 10 mL of Coating Buffer and mix well About 10 mL is required to coat one 96 well plate 2 By using an 8 channel pipette add 100 pL of the diluted capture IgG to each well 3 Let it stay at 37 C for 120 min or at 4 C for overnight after covering the plate by plate sealer lt Blocking gt 1 Disolve 0 2 g of Block
5. ing Agent powder by 20 mL of PBS and mix well This blocking solution of 20 mL is enough to treat one 96 well plate 2 Remove the diluted Capture IgG solution from the plate by decantation followed by gentle tapping on the paper towel 3 Add 200 pL of the blocking solution to each well by using an 8 channel pipette Incubate at 37 C for 1 hrs or at 4 C for overnight after covering the plate by plate sealer 4 It is recommended that preparation of standard antigen and sample solutions should be performed during this incubation time note Please remove the diluted Capture IgG solution completely and do not dry the well after the solution removal to avoid inconsistent measurement lt Preparation of standard solutions gt 1 Prepare ten low protein binding tubes The standard antigen adsorbs to plastic tubes so that the Beacle ver 1 02E Oss standard antigen solution must be diluted in the protein low bind tubes 2 The standard antigen is provided as a lyophilized form in a tube Add 100 pL of distilled water into the tube and mix well to dissolve the antigen completely Be careful when open the tube because the lyophilized power may be attached on the cap of the tube The completely dissolved antigen solution makes the concentration of 300 pg mL or 300 pUnit mL This concentrated standard antigen solution can be stored at 4 C for at least 3 months 3 Put 870 pL of PBS into one of the low protein bind tubes add 30 uL of the c
6. inition the detection range of the kit is from 0 2 to 10 nUnit mL To imagine the sensitivity of the kit and the unit system employed here we give you following information one ng mL of the recombinant HBsAg L protein BCL AG 001 shows Pre S1 activity of approximately 1 nUnit mL and S antigen activity of about 1 2 of standard human serum obtained from infected patients Thus suppose a human serum sample has Pre S1 activity of about 1 10 of S antigen activity the kit has great chance to detect the Pre S1 activity Storage condition and stability All components can be stored at 4 C At the condition all reagents are stable for at least 12 months Beacle ver 1 02E Oss Materials and Reagents Kit composition The kit contains following materials Please ensure that all materials are provided in the kit Capture IgG anti Pre S1 120 pL 50 pL 96 well plate Detection IgG HRP labeled anti Pre S1 25 uL 10 pL 96 well plate Because of small amount collect all liquid in the bottom of tube by centrifugation before use Standard antigen lyophilized form recombinant antigen 30 pg tube Coating Buffer 25 mL 10 mL 96 well plate Blocking Agent 0 2g x 3 powder 0 2 g dissolved in 20 mL PBS is enough for 96 well plate Dilution solution for Detection IgG 25 mL 10 mL 96 well plate 20 x PBS 25 mL 20 x PBS T 25 mL Chromogenic Reagent 25 mL light shield 10 mL 96 well plate Stop Solution 15 mL Microplates 96 wells split typ
7. itable as control antigen BCL AB 001 Anti Pre S1 antibody mouse mono 1 100 pg mouse monoclonal antibody ELISA can be with combination of Pre S1 antibody mouse mono 2 Anti Pre S1 antibody mouse mono 2 100 ug BCL AB 002 s 0 mouse monoclonal antibody suitable for western Blotting and ELISA Other Related products are also available visit our Website www beacle com The principle and outline of assay The key components of the kit are two anti Pre S1 mouse monoclonal antibodies and Pre S1 antigen So called sandwich system is employed where antibody A captures the antigen on the microplate surface and the captured antigen is detected by another antibody antibody B which is labeled with HRP Finally the amount of HRP attached was determined using chromatic substrate Approximate time required for an assay is as follows IgG coating 120 min or overnight Plate blocking 60 min or overnight Reaction with capture IgG Overnight Reaction with detection IgG 120 min gt Chromatic reaction 20 min You need additionally times for reagent preparing pipetting sampling washing measuring etc The definition of Pre S1 antigen activity There is not established definition to express Pre S1 antigen activity In this kit we expressed the Pre S1 antigen activity as follows the Pre S1 activity of 1 nUnit equals to that of 1 ng of standard antigen see below that is provided in the kit According to this def
8. ns Therefore we recommend to use low protein binding tips or not to dip tips too deep in the standard solution when the concentration is below 30 nUnit mL lt Preparation of sample solutions gt 1 Dilute the sample solution by PBS so that the expected activity is within the measurable range 0 2 10 nUnit mL 2 When Pre S1 activity of a sample is unknown please prepare multiple samples with different dilution factors 3 When human serum or plasma is assayed serum or plasma sample should be diluted more than 100 fold by PBS to prevent the effect of serum or plasma components note 1 Measurement of highly contaminated samples may not be accurate It is recommended to ensure the accuracy by using samples which is added by known amount of standard antigen 2 Please use the low binding tubes to dilute the sample in order to prevent attachment of samples to plastic tubes 3 We do not guarantee the successful determination of non human animal species The addition of rodent serum to standard antigen is known to give inaccurate measurement lt Reaction with capture IgG and antigens gt 1 Remove the blocking solution from the plate by decantation followed by gentle tapping on the paper towel 2 Wash each well by 200 pL well of PBS T three times Then wash with 210 pL well of PBS three times The one cycle of washing procedure is as follows first add the indicated volume of PBS T or PBS then remove the solution by decantation followe
9. oncentrated standard solution and mix well immediately using a vortex After mixing we recommended to wash the tip by repeated pipeting in the diluted solution to achieve complete transfer of the concentrated standard solution The final concentration of the standard solution is 10 pUnit mL 4 Put 475 uL of PBS into one of the low protein bind tubes add 25 pL of the above diluted standard solution at 10 pUnit mL and mix well immediately using a vortex After mixing we recommended to wash the tip by repeated pipeting in the diluted solution tube to achieve complete transfer of the standard solution Likewise prepare the series of standard solutions as indicated in below table Antigen solution for dilution STD LD 475 uL 10 u Unit mL 25 uL 500 nUnit mL 950 uL 500 nUnit mL 50 uL 25 nUnit mL 25 nUnit mL 400 uL 10 nUnit mL STD 10 nUnit mL 400 uL 5 nUnit mL STD 5 To draw the standard curve standard solution from STD to STD are used 6 Human serum components influence the assay When Pre S1 activity of human serum or plasma is determined it is recommended to add serum or plasma to the standard antigen solution to minimize the influence To do so dilute the standard solution 10 pUnit mL to 500 nUnit mL by using HBsAg negative serum or plasma instead of PBS Further dilution should be done by PBS note It is possible that the antigen adsorbs to plastic tips during pipetting procedure especially in diluted standard solutio
10. sults may differ due to the difference of the technique and other conditions Contact Information Beacle Inc 5303 Haga Kita ku Okayama 701 1221 Japan E mail technical support beacle com Website www beacle com
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