Cat. # MK138. For Research Use. Rat IgG EIA Kit. Product Manual. v1012

Transcription

1 Cat. # MK138 For Research Use Product Manual

2 Table of Contents I. Description... 3 II. Principle... 3 III. Kit Components... 4 IV. Materials Required but not Provided... 4 V. Storage... 4 VI. Intended Use... 4 VII. Protocol... 4 VIII. Performance... 6 IX. Experimental Example...11 X. Precautions for Use URL:

3 I. Description Immunoglobulins (Ig) are antibody proteins that play a key role in antibody-mediated immunityone of the host defense mechanisms. There are five classes of immunoglobulins (IgG, IgA, IgM, IgE and IgD) present in animals, with IgG accounting for 80% of the immunoglobulins in the blood. IgG levels vary greatly depending on the immune response; however, IgG is generally present in the blood at 3-10 mg/ml. This is a high enough concentn that IgG can be detected without using a high sensitivity ELISA assay. Monoclonal antibodies are uniform immunoglobulins produced by hybridoma cells. These cells are a fusion between antibody-forming cells and tumor cells grown in culture. Monoclonal antibody production technology is well established, and it is now common to establish cell lines capable of long-term production of an antibody against a mouse antigen by constructing heterohybridomas through fusion of rat lymphoma cells with mouse myeloma cells. Assaying rat IgG production has become indispensable in the maintenance of rat-monoclonal antibody-producing heterohybridomas. Unlike IgG levels in blood, IgG is present in heterohybridoma supernatants at low levels and a high sensitivity assay is required for detection. With a rat IgG-specific polyclonal antibody, this assay kit is applicable not only for measuring IgG in all rat blood samples regardless of strain, but also for monitoring IgG production in rat-monoclonal antibody-producing hybridomas with high sensitivity. This kit comes with a ready-to-use plate coated with solid-phase capture antibody. Its usage and dosage are designed to allow simple detection of rat IgG with good sensitivity and reproducibility within a very short time - only 60 minutes for the entire assay. II. Principle Substrate Rat IgG Labeled antibody POD POD Color development Antibody plate Sandwich complex URL: 3

4 III. Kit Components (for 96 reactions x 2) (1)Antibody Coated Microtiter plate Anti-rat IgG polyclonal antibody (96 wells: 8 wells 12 strips) 2 plates (2)Antibody - POD Conjugate (lyophilized) Peroxidase-labeled anti-rat IgG polyclonal antibody for 11 ml x 2 (3)Standard (lyophilized) Purified rat immunoglobulin 640 ng for 1 ml x 2 (4)Sample Diluent 25% Block Ace -containing PBS (with preservative) 60 ml (5)Substrate Solution(TMBZ) 3,3,5,5 -Tetramethylbenzidine solution 12 ml x 2 IV. Materials Required but not Provided 1. Reagents 2. Materials V. Storage 4 - Washing Buffer: Phosphate-buffered Saline (PBS) containing 0.1% Tween 20 (Dissolve 8.0 grams of NaCl, 0.2 grams of KCl, 2.9 grams of Na2HPO412H2O and 0.2 grams of KH2PO4 in 1000 ml of distilled water, and add 1 ml Tween 20.) Alternatively, a PBS tablet (Cat. #T900) is useful to prepare PBS solution. - Stop Solution: 1 N H2SO4 - Precision pipettes with disposable tips: 20 and 100 µl micropipettes, µl adjustable multiwell pipetter or 20 µl and 100 µl multiwell pipetters - Beakers, flasks, cylinders necessary for prepan of reagents - Disposable pipettes and test tubes - Microtiter plate reader (can assay absorptions up to 3.5 at 450 nm) VI. Intended Use To assay rat immunoglobulin in serum. Measure culture supernatants of hybridomas for rat monoclonal antibody concentn. VII. Protocol 1. Sample Suitable samples include rat serum and plasma, or rat hybridoma culture supernatant and ascites, etc. Samples may be stored up to 12 hours at 4. If the assay will be performed longer than 12 hours after sample prepan, then store samples frozen at -20. Use (4) Sample Diluent for dilution if necessary. The recommended dilution for normal rat serum is 1,000- to 10,000-fold. The recommended dilution for rat hybridoma culture supernatant is 10- to 500-fold. (With the antibody productivity varies depending on the cell strain, a preliminary assay is required.) 4 URL:

5 2. Prepan of Solutions (1) Antibody Coated Microtiter Plate Allow the unopened plate to reach room temperature in its package before use. POD-labeled Antibody Solution Reconstitute (2) Antibody - POD Conjugate with 11 ml of distilled water. Once reconstituted, it is stable for up to 1 week at 4. For longer storage, freeze at -20, at which it is stable for up to 1 month. However, no more than 1 cycle of freeze-thaw is allowed. Rat IgG Standard Solution Add 1 ml of distilled water to reconstitute (3) Standard, yielding a Rat IgG Standard Solution (640 ng/ml). Dilute with (4) Sample Diluent before use to prepare fresh serial dilutions (Standard Solutions at concentns of 640, 320, 160, 80, 40, 20 and 10 ng/ml). Use (4) Sample Diluent as the 0-concentn standard. The Rat IgG Standard Solution (640 ng/ml) is stable for up to 1 week after prepan when stored at 4, or for up to 1 month at -20. Note: the detection titer varies depending on the IgG subclass. For accurate assays, please perform assays using an independently established IgG standard. Substrate solution Return the (5) Substrate Solution to room temperature before use. It is supplied ready to use in reactions. Check before use that it has not developed a dark blue color. A reaction with metal ions will result in colon; make sure it is not contaminated with any tap water. 3. Procedure If the Substrate Solution will be used for several reactions, divide it into aliquots of the required volume in advance. Standard Protocol: Total time = 1 hour Assay samples in duplicate. Return each reagent in the kit and samples to room temperature and make sure solutions are mixed uniformly without creating bubbles before use. Note: Prepare reagents and samples in a separate 96-well plate in advance so that they can be added to the antibody-plate quickly (within 5 minutes) using an 8-channel pipette or similar apparatus. 1. Add prepared samples (100 µl/well) to the Antibody Coated Plate. In order to provide highly reliable results, it is recommended to place serial dilutions of the Standard Solution (100 µl/well) in the 1st and 12th rows. Perform this reaction at room temperature (20-30 ) for 25 minutes; incubation at 37 may compromise antigenicity. (First reaction) 2. Remove reaction mixtures from wells, discarding the liquid. Wash wells 3 times with Washing Buffer (100 µl/well). Remove excess Washing Buffer and then add 100 µl of the Labeled Antibody Solution per well using an 8-channel pipette and allow to react for 25 minutes at room temperature. (Second reaction) 3. Remove reaction mixtures from wells, discarding the liquid. Wash wells 4 times with Washing Buffer (100 µl/well). Remove excess Washing Buffer and then add 100 µl of (5) Substrate Solution (TMBZ) per well using an 8-channel pipette and allow to react for 10 minutes at room temperature (20-30 ). (Third reaction) 4. Add 100 µl of the Stop Solution (1N H2SO4) to each well in the same order as for (5) Substrate Solution (TMBZ) to stop the reaction. Then mix well. URL: 5

6 VIII. Performance 5. To make a zero adjustment, use distilled water as a blank and measure absorbance at 450 nm. The color is stable for up to 1 hour after reaction termination. 6. Plot a standard curve based on the results obtained from the standard solutions (with concentn as x-axis and absorbance as y-axis) and use it to determine the corresponding concentns of mouse IgG based on the absorbance of samples. Note: Cover the plate with film or the like to prevent evapon of solutions during reactions at room temperature or in an incubator. It is recommended that the Washing Buffer be completely discarded to get rid of the residual fluid. <High-sensitivity protocol> You may raise the detection sensitivity by prolonging the reaction times. This will increase the measuring range of the calibn curve, giving a detection limit of 2.5 ng/ml. <Standard protocol> <High-sensitivity protocol> Standard ng ng First reaction 25 min 2 h Second reaction 25 min 1 h Third reaction 10 min 15 min 1. Standard curve () The following is a typical standard curve. The standard curve for each lot is provided in the attached Certificate of Analysis. In order to obtain accurate results, it is preferable that a standard curve be plotted for each assay. Limit of detection: 10 ng/ml Standard Curve Mean Value Conc (ng/ml) CurveFit : 4-Parameter : y (A D)/(1 (x/c) B ) + D : A B C D R Rat IgG (ng/ml) Color development: 15 min 6 URL:

7 2. Reproducibility <Intra-assay precision test (n = 16)> A reproducibility test was performed using 3 different concentns of control prepared from dilutions of rat hybridoma culture supernatant. Sample (n = 16) Mean (ng/ml) SD (ng/ml) CV (%) Control A Control B Control C <Inter-assay precision test (n = 3)> A reproducibility test was performed by assaying 3 different concentns of control over 3 days. 3. Recovery test Sample (n = 3) Mean (ng/ml) SD (ng/ml) CV (%) Control A Control B Control C Equal volume of samples in different concentns were combined and assayed. The assay results were compared with the theoretical values to determine recovery rates. Sample A Sample B A+B (Assay Result) A+B (Theoretical Value) Recovery Rate (%) Strain comparison Unit: ng/ml Blood IgG concentns were investigated between rat strains and between individual animals. Methods: 6-week-old male LE, F344 and Donryu rats, and 7-week-old and retired female SD rats. Serum samples were collected from the 7-week-old SD rats prior to primary immunization and following the second and third immunizations (antigen boosts). IgG concentns were measured using10,000-fold dilutions of serum. URL: 7

8 Animal No. IgG in blood (mg/ml) LE, no LE, no Donryu, no Donryu, no F344, no F344, no SD retired, no SD retired, no SD 7W (pre-immune) SD 10W (post-second-boost) SD 11W (post-third-boost) Results: There is a strain-specific difference in steady state blood IgG concentn among the 6- to 7-week-old rats. Steady state IgG concentn in SD rats increased with age (including acquiring natural immunity and other extrinsic factors). As the number of antigen immunizations (boosts) increased, the serum IgG level climbed dramatically. The high level of IgG in retired animals was presumably attributable to the immunity acquired over their lifetime. 5. Monitoring blood IgG levels after transnasal immunization with ovalbumin The change in blood IgG concentn after transnasal immunization with ovalbumin was tested between rat stains and between individual animals. IgG concentns were measured using10,000-fold dilutions of serum samples. Immune-induced increase in blood IgG level by rat strain and by individual animal mg/ml Days post-immunization Result: There was great variability in blood IgG levels between strains and individuals. 8 URL:

9 6. Rat IgG subclass reactivity Rat polyclonal antibodies [1 mg/ml] were serially diluted to test for reactivity with each rat IgG subclass and IgM. Standard Rat IgG-subclass ( ) IgG 1 IgG 2a IgG 2b IgG 2c IgM , , , , , , blank Results: No cross-reactivity with IgM was detected, but reactivities with all IgG subclasses were confirmed. Reactivity to IgG 2a was particularly strong. When using this kit for antibody assays, it is recommended to use a subclass standard corresponding to the antibody being evaluated. 7. Improvement of sensitivity by prolonging reaction time The high-sensitivity protocol improves the measuring range. Standard protocol First reaction 25 min Second reaction 25 min Third reaction 10 min Standard curve: 10 to 640 ng/ml Rat IgG (ng/ml) URL: 9

10 High-sensitivity protocol (Normal conjugate) First reaction 2 h Second reaction 1 h Third reaction 15 min Standard curve: 2.5 to 160 ng/ml Rat IgG (ng/ml) Result: Prolonging reaction times improved the detection limit to 2.5 ng/ml. Note: The high-sensitivity protocol may be useful when assaying a large number of samples or when your schedule does not allow completion of the assay in one hour. 8 Effects of hemolysis Method: Each blood sample from the orbital vein was divided into two, with one half subjected to centrifugation (3,000 rpm 20 min) to isolate serum (untreated) and the other half subjected to pre-centrifugation hemolysis by passing it through an injection needle 3 to 4 times. Hemolysis results in lysis of red blood cells and release of their contents into the serum sample. Rat IgG (ng/ml) Untreated (1) Untreated (2) Untreated (3) Untreated (4) Hemolyzed (1) Hemolyzed (2) Hemolyzed (3) Hemolyzed (4) Result: Hemolytic components did not excessively interfere with IgG detection using this kit. 10 URL:

11 9. Rat IgE cross-reactivity To check for cross-reactivity to rat IgE, serial dilutions of a commercially available rat IgE (10 µg/ml) were assayed. Rat IgE (ng/ml) Measurement (ng/ml) Result: The rat IgG polyclonal antibody shows cross-reactivity against rat IgE. IX. Experimental Example 1. Measurement of IgG purified from a rat hybridoma culture supernatant The protein-free culture supernatant of a rat hybridoma was concentrated by ammonium sulfate precipitation, buffer-exchanged by dialysis, and subjected to cation exchange chromatography to purify antibody protein. Rat antibody levels in the gradient elution fractions were assayed using this kit. Cation chromatography carrier: HiTrap SP cartridge column Eluate condition: A) 50 mm MES Buffer (ph6.0) B) 50 mm MES Buffer (ph6.0) + 1M NaCl Absorbance A280 Flow through (Fr. No.) URL: 11

12 Sample (before chromatography) Flow-through Fr.3 Fr.4 Fr.5 Fr Fr.7 Fr.8 Fr.9 Blank Result: The majority of rat antibody protein is concentrated in Fr. 4. X. Precautions for Use 1. Do not mix-use kits or reagents from different lots. 2. Do not expose reagents to strong light during storage or reactions. 3. Use pipettes free of metal when handling (5) Substrate Solution (TMBZ) and the stop solution (1N H 2 SO 4 ). 4. Exercise care to prevent (5) Substrate Solution (TMBZ) or the stop solution (1N H 2 SO 4 ) from coming into contact with hands or mucous membranes. 5. Do not use a (5) Substrate Solution (TMBZ) that has developed color. 6. Each reaction varies subject to length of time and temperature. Therefore, a new standard curve must be generated for each assay. 7. Handle blood samples with great care. NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic procedures for humans or animals. Also, do not use this product as food, cosmetic, or household item, etc. Takara products may not be resold or transferred, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please contact us by phone at or from our website at 12 URL: