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1. 1 Background 1 1 Staphylococcal Protein A SpA SpA is an immunoglobulin IgG binding protein found in the bacterial cell wall of Staphylococcus aureus ref 1 SpA binds to most mammalian IgG and can be used for detection or purification of such antibodies ref 2 Affinity chromatography on SpA columns is widely used for the purification of monoclonal and polyclonal antibodies SoA may sometimes leak from the column and contaminate the preparation which may give false results in immunological assays In the in vivo setting SpA can react with IgG causing anaphylactic reactions ref 1 Thus it is important that antibody preparations are free from SpA before being used Page 2 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 This kit has been developed and is primarily tested with recombinant SpA from GE Healthcare which is also the source of the included SpA reference Native SpA and other sources of recombinant SpA generally give good recoveries but in some cases it may be necessary to use the same SpA source for the standard as for the samples being analysed 1 2 Non specific interactions between SpA and IgG Analysing for the presence of SpA in samples containing IgG involves two major problems which must be overcome in order to obtain reliable results 7 Fe binding SpA reacts with the Fc region of IgG from most animal species which makes its specific detection by immunoassay difficult2. Anti bodies specific for SpA will bind specifically as well as non specifically due to the Fc reactivity of SpA In this kit the problem is solved by using chicken anti SpA IgY Chicken antibody is one of the few immunoglobulins that does not bind SpA in the Fc region IgG IgY xX Y IgG binds SpA nonspecifically in the Fc region This problem is overcome using chicken IgY since it is not Fc reactive to SpA g Other non specific interactions In samples containing mammalian IgG immunologically active epitopes of SpA can be blocked by non specifically bound IgG To overcome this problem SpA can be analysed at low pH ref 4 since this should cause SpA and IgG to dissociate However high affinity antibodies e g human IgG and certain mouse monoclonals bind to SpA even below pH 3 Unfortunately the specific immunological detection of SpA is very difficult at such low pH and an assay at this pH would likely have an unsatisfactory analytical performance Instead we have chosen to establish a flexible assay system that can be tailored to each customer s situation The standard references are made with known amounts of IgG present The IgG solution which is added to the standard references should be as similar as possible to the IgG found in the Page 3 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 samples To dissociate SpA and IgG the samples and standard references are boiled for 4 minutes In oth
3. in lgG solution same concentration and isotype as samples Step 7 3 7 4 page 9 Boil standard IIl and samples Centrifuge and 4 min boil dilute supernatant 10x in Dilution buffer 2000g for 60 sec Step 7 5 page 9 10x dilution Add standards and samples to wells take from 20 to 0 3125 ng mL supernatant 100 uL well Step 8 1 1 8 1 4 page 10 Incubate 60 min in RT Wash x4 and add 1 100 diluted Biotinylated 100 uL well anti SpA IgY Incubate 60 min in RT Step 8 1 5 8 1 6 page 10 Wash x4 and add 1 100 diluted HRP 100 uL well conjugate Incubate 30 min in RT Step 8 1 7 page 11 Wash x4 and add substrate solution 100 pL well Step 8 1 8 page 11 Incubate 10 min dark Add stop solution and read plate at 450 nm 100 pL well Step 8 1 9 8 1 10 page 11 Page 19 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 16 Contact and order information IMMUNSYSTEM I M S AB Dag Hammarskj lds vag 26 S 751 83 Uppsala Sweden Phone 46 18 53 89 09 Fax 46 18 53 89 97 www immunsystem com
4. Product no 03 96 Protein A ELISA Instruction Manual for samples with or without IgG 0e 4 vo IMMUNSYSTEM I M S AB www immunsystem com Edition KI 090921M All rights reserved Copyright IMMUNSYSTEM I M S AB Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 Table of content Wis Background sasse daner ren Sr aren er see render rek redde 2 2s ASSAY principle ss verss sst sssseveasss asbensssds neder de RP E ser s d ns 4 Bi AA KKIUCOIUCIHUS oe apere rea u aes es poasaatuwtan BIE Eass 5 4 Materials not provided sssssssssrrrssssssrrrrrrrrrrrrnnrrrrrrrrrrrrnr rr ren 6 3 PRECAUTIONS 0542 2560 000 ar sa E AS 6 6 Reagent preparation sw ssccsssssessssererrrererrsrrrrrnrrrrrrrrnrrrr rr nn 7 7 Sample and standard preparatiOn sssssseerersrssrrsorrrrrn 8 S VESt Proce mune s vosrrshol aoc E a oarsunceus E RR 10 9 PAMALYSIS OF TESUNS x coxyasiabeasiedteregseantenateetenmesninbvuegnteszyeedes 12 10 Performante sssrin iiri iaaii 13 tI Tipsand hits s s 0 004s500500 200000 Eros patos doresnabeeeeataes 16 12 Related products inne nentrn nanana dar nde liane stor ra vs 16 3 References os ss vocss des etesse eniten bad beses aS bean de NNE SNS 17 14 Quick start guide samples without IgG sssesss 18 15 Quick start guide samples with IgG ssssssessoeorrressnn 19 16 Contact and order informatiOn sssssssssrrerssrrssrrrrrrrrnn 20
5. ation the samples and standard references are prepared before the assay is performed The steps for sample preparation and test procedure are also summarised in the Quick Start Guides on pages 18 and 19 7 1 Preparing samples samples with and without IgG Neutral samples SpA easily binds to glass and plastic material However in the presence of 0 05 Tween 20 this binding is inhibited In order to avoid this add Tween solution 1 400 to the sample tubes before adding sample Next add sample and mix well Example For 2 mL sample volume add 5 uL Tween solution Acidic samples Samples should be neutralised to improve the specific immunological detection of SpA Determine the elution volume of your acid eluted samples Add 1 10 volume of Neutralisation buffer to each sample dilution factor 0 9 Example Add 100 uL Neutralisation buffer to 0 9 mL sample volume Measure the pH and add Neutralisation buffer until neutral pH is reached Note the volume of buffer added in order to calculate dilution factor 7 2 Preparing samples only for samples with IgG 7 2 1 Determine the IgG concentration in your samples by measuring the optical density at 280 nm OD2g0 Divide the ODzgo value with 1 36 to get the concentration 7 2 2 Dilute the samples with Dilution buffer so that all samples contain the same IgG concentration note the volume of buffer added in order to calculate dilution factor 7 2 3 Transfer 500 uL of each sample with adj
6. er words the assay is standardised with the same IgG that is present in the samples The possible error from the blocking of antigenic epitopes is therefore eliminated 2 Assay principle This ELISA kit is designed to detect SpA in IgG containing solutions e g monoclonal antibody preparations in acid eluates from SpA columns or in other liquid preparations It is a sandwich ELISA based on microtitre strips precoated with the primary antibody affinity purified chicken anti SpA SpA from the sample is bound to the microwell and detected by biotinylated chicken anti SpA antibody Next a streptavidin horseradish peroxidase conjugate is bound to the biotin conjugate Finally the substrate is added and reacts with the horseradish peroxidase to produce a colour change Colour development indicates a positive result and the colour can be read visually or by a microplate spectroohotometer at 450 nm Samples to be tested with this assay are often of acidic pH In order for the test to work properly such samples should be neutralised If a sample contains mammalian IgG SpA may interact with the IgG and be partially blocked falsely lowering the signal To avoid this problem samples containing IgG are treated to denature the IgG and expose SpA epitopes A schematic outline of the assay is shown Page 4 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 D SpA from the sample is bound to wells precoated with the p
7. erformed after boiling of samples can be omitted This may be desired if a ten fold dilution of samples makes them too diluted In this case Standard Ill in the table section 7 4 should be further diluted 1 10 in solution G giving Standard IV at 20 ng mL Do not mix these two procedures It is important to keep the same exact procedure for samples and standards 12 Related products MouSelect Product number 09 04 The MouSelect kit contains the basic components needed to set up a sandwich ELISA for the assessment of Protein A binding The components are designed to aid in an early selection of polyclonal or monoclonal antibodies to a preferential purification on Protein A from Staphylococcus aureus The kit can also be used to optimise the Protein A binding of antibodies anti Protein A Product number 01 008 IgY fraction After purification the antibodies can be used in various immunological applications to detect Protein A from Staphylococcus aureus Page 16 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 13 References Forsgren A et al SpA and its exploitation In Staphylococci and Staphylococcal infections 2 Eds C S F Easmon and C Adlam pp 429 480 Academic Press Inc London 1983 Lindmark R et al Binding of immunoglobulins to SpA and immunoglobulin levels in mammalian sera J Immunol Methods 62 1 13 1983 Langone J J et al Radioimmunoassays for SpA of S
8. h IgG 160 140 120 100 80 60 40 20 0 Recovery SpA rSpA 1 rSpA 2 rSpA 3 Page 14 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 10 5 Precision Coefficients of variation CV for duplicate measurements are typically lt 10 The average between day CV was lt 15 when tested on 6 different occasions for three samples The samples were tested in a concentration range 0 5 150 ng mL ppm 10 6 Recovery Samples at three different concentrations were spiked with three different amounts The recovery was within 15 for all samples and amounts spiked 10 7 Matrix effect The binding affinity of SpA differs between IgG from different species but also between isotypes of IgG from the same species Thus the accuracy of the results will depend on the IgG that is used for the standards Even with the same IgG the graph shows the importance of normalizing the IgG concentration in samples and standards Standardsin 1mg mL IgG 120 100 a0 60 Recovery 40 20 0 1 2 4 8 IgG in sample mg mL 10 8 Antigen excess No antigen excess effect was seen for standards tested up 1600 ppm Page 15 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 11 Tips and hints Dilution of standards and samples for samples with IgG The 1 10 dilution of standards and samples that is p
9. his dilution of samples and standards after boiling can be omitted For more information see section 11 Tips and Hints Page 9 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 8 8 1 8 1 1 8 1 2 Test procedure Performing the assay Add 100uL of PBS Tween for samples WITHOUT IgG or Dilution Buffer for samples WITH IgG into row B to H for wells allocated for the standard references Add 200 uL of Standard IV to wells in row A It is recommended to test standard references in duplicate Dilute the standard in the ELISA plate according to figure 1 Figure 1 1 2 dilutions of Standard IV in the ELISA plate 8 1 4 8 1 5 8 1 6 Std IV Add 200uL of Std IV into row A 20ng mL Std V Transfer 100uL of Std IV into row B Mix well 10ng mL Std VI Transfer 100uL of Std V into row C Mix well 5ng mL Std VII Transfer 100uL of Std VI into row D Mix well 2 5ng mL Std VIII Transfer 100uL of Std VII into row E Mix well 1 25ng mL Std IX Transfer 100uL of Std VIII into row F Mix well 0 625ng mL Std X Transfer 100uL of Std IX into row G Mix well 0 313ng mL Discard 100uL of Std X from row G Blank 100uL PBS Tween or Dilution Buffer to row H Add 100 uL of samples to appropriate wells It is recommended to test samples in duplicate Cover with plastic wrap or tape and incubate the plate for 1 hour at room temperature Wash the strips by emptying the wells and then ri
10. lisation and Dilution buffer needed depends on the pH and concentration of the samples and therefore varies with each test e Microtitre wells Substrate solution and Stop solution are ready for use e Protein A reference Should be diluted according to step 7 4 e Tween solution Prepare Tween solution by diluting the concentrate 1 5 in ultra pure water Example Mix 1 mL Tween concentrate with 4 ml water Vortex e Neutralisation buffer Prepare by diluting Tween solution 1 5 in Tris Buffer Example Mix 1 mL Tween solution with 4 mL Tris buffer Vortex e Dilution buffer Prepare by diluting Neutralisation buffer 1 10 in your acid elution buffer i e sample matrix buffer Example Mix 1 mL Neutralisation buffer with 9 mL elution buffer Vortex e PBS Tween PBS T buffer Dissolve 1 tablet in 500 mL ultra pure water Store prepared PBS T buffer at 4 C but not for more than 3 days e Biotinylated anti SpA IgY Concentrate Dilute 1 100 in PBS T buffer The solution is not stable for more than 24 hours Example Mix 120 uL Biotinylated anti SpA IgY Concentrate with 12 mL PBS T buffer Vortex e HRP conjugate Concentrate Dilute 1 100 in PBS T buffer The solution is not stable for more than 24 hours Example Mix 120 uL HRP conjugate Concentrate with 12 mL PBS T buffer Vortex Page 7 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 7 Sample and standard preparation After reagent prepar
11. nsing 4 times with PBS T buffer filling all wells to the top for each rinse Empty the wells by tapping the plate upside down onto a paper towel to remove the last traces of fluid Add 100 uL Biotinylated anti SpA IgY diluted to each well Cover with plastic wrap or tape and incubate the plate for 1 hour at room temperature Page 10 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 8 1 7 Wash the strips see step 8 1 5 Add 100 uL HRP conjugate diluted to each well Cover with plastic wrap or tape and incubate the plate for 30 minutes at room temperature 8 1 8 Wash the strips see step 8 1 5 Add 100 uL Substrate solution to each well and incubate the plate 10 minutes at room temperature in the dark Begin timing after the first well is filled 8 1 9 Stop the reaction by adding 100 uL Stop solution to each well Add the Stop solution in the same order as the Substrate solution was added 8 1 10Measure the absorbance of the samples and standard references at 450 nm within 30 minutes after adding the Stop solution Page 11 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 9 Analysis of results To determine the exact SpA concentrations in your samples use data reduction software to automatically generate an appropriate standard curve such as a four parameter curve fit Most ELISA reader software programs can correct for background automatically so consult the u
12. primarily IgG och IgG gt 10 1 Validity of assay For samples without IgG Standard IX 0 625 ng mL should have a corrected absorbance value greater than 0 1 For samples with IgG Standard VIII prepared as 1 25 ppm should have a corrected absorbance value greater than 0 1 e Std with IgG Standards w o IgG Note These plots should not be used for quantification of samples 0 5 10 15 20 25 Concentration ng mL OD value at 450 nm 10 2 Sensitivity The functional sensitivity is 0 15 ng mL 0 15 ppm This is set as the concentration where the intra assay CV lt 10 and the OD signal is higher than ODegrank 3 SDaiank The unit parts per million ppm is the concentration of Protein A relative to the concentration of IgG e g 1 ng mL Protein A in 1mg mL of IgG equals 1 ppm Page 13 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 10 3 Linearity The linear range is 0 15 33 ng mL 0 15 33 ppm This is set as the concentration where recovery of standards is 80 120 and a plot of Quantified vs Theoretical concentration gives R gt 0 990 Samples with IgG Theoretical conc 5 0 5 10 15 20 25 30 35 Quantified conc 10 4 Specificity Recovery has been tested with different SpA sources both native and recombinant Recoveries were in the range 75 150 E Samples withoutlgG M Samples wit
13. r Additional materials needed to assay samples with IgG e SpA free IgG solution e Water bath 5 Precautions e Carefully read and follow all instructions e Store the kit and all reagents at 2 to 8 C 35 to 45 F e All reagents should equilibrate to room temperature 18 to 25 C 64 to 77 F before use e Unused microtitre wells should be stored sealed at 2 to 8 C e Do not mix components or instruction booklets from kits with different expiration dates different batches e Be careful to prevent contamination of kit components e Do not use the kit beyond expiration date e Do not eat drink or smoke where specimens or kit reagents are handled e Centrifuge 2000g e Vial rack or floating aid e Use a separate pipette tip for each sample e Do not pipette by mouth e Standard references must be used for each new test series e Use only ultra pure water for preparation of reagents e The Substrate solution and 2 M Tris buffer are irritating to eyes respiratory system and skin Avoid contact with skin and eyes e The Stop solution contains HCl This is a strong acid that can cause burns Handle with care e This kit is for research use only Page 6 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 6 Reagent preparation The examples below calculate the approximate amount needed to assay 96 microtitre wells i e 12 strips The amount of Neutra
14. rimary antibody chicken anti SpA IgY Biotinylated chicken anti SpA IgY is added as secondary antibody Strepavidin HRP is added and binds to the biotin conjugate Substrate Coloured product 4 Substrate is added and and reacts with HRP to produce a coloured product Finally stop solution is added to stop the chromogenic reaction 3 Kit contents Incubate 1 hr at room temperature Incubate 1 hr at room temperature Incubate 30 min at room temperature Incubate 10 min at room temp dark e Microtitre strips 8x12 precoated e Biotinylated anti SpA IgY 200 uL with chicken anti SpA antibody 100x solution Contains preservative e PBS Tween tablets 3 tablets e HRP Conjugate 200 uL Streptavidin Sufficient for 3x0 5 L Phosphate horseradish peroxidase conjugate Buffered Saline with 0 1 Tween 100x solution Contains preservative e Protein A reference 200 uL 0 5 e Substrate solution 25 mL mg mL SpA solution recombinant TMB H202 solution origin Contains preservative e Tween 20 concentrate 2 mL e 2M Tris Buffer 25 mL e Stop solution 20 mL 1 M HCI Caution Strong Acid Page 5 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 4 Materials not provided e Pipettes 10 to 1 000 uL e Microplate spectrophotometer e Disposable pipette tips e Eppendorf tubes e 10 mL tubes e Ultra pure wate
15. ser s manual for your specific software 9 1 Manually estimating SpA sample concentration Standard curve Calculate the mean for the standard reference duplicates and correct for background by subtracting the mean absorbance value for the blank Plot Corrected OD45 vs Concentration on a semi log paper ODaso Standard IV to X OD4s9 PBS Tween or Dilution Buffer Corrected OD s9 Samples Calculate the mean absorbance value for the sample duplicates and correct for background by subtracting the mean absorbance value for the blank From the corrected OD values approximate the sample concentration Cs from the standard curve To calculate the final SpA concentration CF in each of the samples divide Cs by the final dilution factor DF for each sample When a sample has been diluted several times i e with neutralisation buffer and then dilution buffer multiply the dilution factors to get the final dilution factor Final SpA concentration ng mL Ce Cs DF Page 12 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 10 Performance The kit has been validated according to general guidelines An excerpt of results is shown below The performance information should only be taken as guidance For quality control purposes the assay should be validated in house with respect to common parameters In the shown results we have used recombinant SpA from GE Healthcare and human IgG mix of subclasses
16. taphylococcus aureus J Immunol Methods 63 145 157 1983 Berglund A et al GO1N 33 563 GO1N 33 569 Ways to determine Fc binding bacterial polypeptides The Swedish Patent Administration Edition number 448 190 1987 Larsson A et al A microELISA useful for determination of SpA binding monoclonal antibodies Hybridoma 9 289 294 1990 Page 17 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 14 Quick start guide samples without IgG Add Tween or neutralise samples if necessary Step 7 1 page 8 Prepare standards in PBS Tween Step 7 4 page 9 Add standards and samples to wells 20 to 0 313 ng mL Step 8 1 1 8 1 4 page 10 100 uL well Incubate 60 min in RT Wash x4 and add 1 100 diluted 100 uL well Biotinylated anti SpA IgY Incubate 60 min in RT Step 8 1 5 8 1 6 page 10 Wash x4 and add 1 100 diluted HRP 100 uL well conjugate Incubate 30 min in RT Step 8 1 7 page 11 Wash x4 and add substrate solution 100 uL well Step 8 1 8 page 11 Incubate 10 min dark Add stop solution and read plate at 100 pL well 450 nm Step 8 1 9 8 1 10 page 11 Page 18 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 15 Quick start guide samples with IgG Add Tween or neutralise samples if necessary Step 7 1 page 8 Determine and normalise IgG concentration of IgG OD2s80 1 36 samples Step 7 2 page 8 Prepare standards
17. usted IgG concentration to a labelled Eppendorf tube not provided Page 8 of 20 Instruction manual for Protein A ELISA kit IMMUNSYSTEM Product No 03 96 7 3 Preparing Solution G only for samples with IgG 7 3 1 Determine the concentration of a SpA free IgG solution see 7 2 1 7 3 2 Add the IgG solution to Dilution buffer in a 10 mL tube not provided Example Add the IgG solution to 5 mL Dilution buffer approximately 4 mL is needed to prepare standards II and Ill Adjust the IgG concentration in Solution G to the same as in the samples see 7 2 2 7 4 Preparing standard references 7 4 1 Label four Eppendorf tubes from to IV and prepare the standards as indicated below Samples w o Samples with IgG IgG PBS Tween Solution G SpA conc Source solution ng mL 40 uL Protein A ref 760 uL 760 uL 25000 40 uL Std 960 uL 960 uL 1000 200 uL Std Il 800 uL 800 uL 200 100uL Std III 900 uL See step 7 5 20 7 5 Removal of IgG only for samples with IgG 7 5 1 Make a hole in the cap of the Eppendorf tubes Place your samples and Standard III 200 ng mL in a rack or floating aid and boil in a water bath for 4 minutes Remove from the water bath and let cool for 5 10 minutes at room temperature 7 5 2 Centrifuge the boiled tubes at 2000g for 60 seconds 7 5 3 Add 100uL of the supernatant of your boiled samples and Standard III into 900uL of Dilution Buffer This gives Standard IV at 20 ng mL Note T
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