ELISA/assay

Rat SRBC IgM ELISA Kit

DEIASL073

96T

Rat SRBC IgM ELISA Kit

PRKCDBP

Rat SRBC IgM ELISA Kit

sELISA

PRKCDBP

Rat

Quantitative

serum, plasma

96T

The kit should be stored at 4°C and the microtiter plate should be kept in a sealed bag with desiccant to minimize exposure to damp air. Test kits will remain stable for six months from the date of purchase provided that the components are stored as described above.

PRKCDBP

Rat

PRKCDBP, protein kinase C, delta binding protein, cavin 3, CAVIN3, HSRBC, MGC20400, sdr related gene product that binds to c kinase, SRBC

This test kit allows rapid and quantitative measurement of rat anti-SRBC IgM levels in serum or plasma samples.

Recent studies have demonstrated that suppression of anti-SRBC (sheep red blood cell) IgM levels by therapeutic agents serves as a useful indicator of immunosupression. 1,2 This test kit allows rapid and quantitative measurement of rat anti-SRBC IgM levels in serum or plasma samples.

The rat anti-SRBC IgM test kit is based on a solid phase enzymelinked immunosorbent assay (ELISA). The assay uses detergent solubilized SRBC ghosts 2 for solid phase (microtiter wells) immobilization and horseradish peroxidase (HRP) conjugated antirat IgM antibodies for detection. Test serum or plasma samples are diluted and incubated in the microtiter wells for 45 minutes. The microtiter wells are subsequently washed. HRP conjugate is added and incubated for 45 minutes. Anti-SRBC IgM molecules are thus sandwiched between immobilized SRBC antigens and the detection antibody conjugate. The wells are then washed to remove unbound HRP-labeled antibodies. TMB Reagent is added and incubated for 20 minutes at room temperature. This results in the development of a blue color. Color development is stopped by the addition of Stop Solution, changing the color to yellow. Optical density is measured spectrophotometrically at 450 nm. The concentration of anti-SRBC IgM is proportional to the optical density of the test sample.

1. SRBC Coated 96-well Plate (provided as 12 strips of 8 wells) 2. Enzyme Conjugate Reagent, 11 ml 3. Reference Standard Stock (lyophilized) 4. 20× Wash Solution: 50 ml 5. Diluent: 30 ml 6. TMB Reagent (One-Step): 11 ml 7. Stop Solution (1N HCl): 11 ml

1. Precision pipettes and tips 2. Distilled or deionized water 3. Polypropylene or glass tubes 4. Vortex mixer 5. Absorbent paper or paper towels 6. Micro-plate incubator/shaker with mixing speed of ~150 rpm 7. Plate washer 8. Plate reader with an optical density range of 0-4 at 450nm 9. Graph paper (PC graphing software is optional)

General Note: Studies at Life Diagnostics, Inc. indicate that anti-SRBC IgM is present in serum or plasma from SRBC immunized rats at concentrations in excess of 4000 u/ml. To obtain values within the range of the standard curve, we suggest that samples initially be diluted 200-fold using the following procedure: 1. For each test sample dispense 298.5 μl of diluent into separate tubes. 2. Pipette and mix 1.5 μl of the serum/plasma sample into a tube containing 298.5 μl of diluent. This provides a 200-fold diluted sample. 3. Repeat this procedure for each sample to be tested. Important: Do note use dilutions lower than 25-fold.

GENERAL INSTRUCTIONS 1. Please read and understand the instructions thoroughly before using the kit. 2. All reagents should be allowed to reach room temperature (25°C) before use. 3. The assay was designed for use with serum or plasma obtained from rats five days after immunization with SRBC, at which point the immune response originates almost exclusively from IgM. 4. Serum or plasma samples must be diluted at least 25-fold in diluent. 5. The optimal sample dilution should be determined empirically. However, studies performed at CD suggest an initial sample dilution of 200-fold. 6. Optimal results are achieved if, at each step, reagents are pipetted into the wells of the microtiter plate within 5 minutes. WASH SOLUTION PREPARATION The wash solution is provided as a 20× stock. Prior to use, dilute the contents of the bottle (50 ml) with 950 ml of distilled or deionized water. STANDARD PREPARATION 1. Reconstitute the vial of the lyophilized rat anti-SRBC IgM standard stock with distilled or deionized water as described on the standard vial label (the reconstituted standard should be aliquoted and frozen at -20°C after reconstitution if additional use is intended). 2. Label 6 polypropylene or glass tubes as 100, 50, 25, 12.5, 6.25, and 3.125 u/ml. 3. In the tube labeled 100 u/ml, prepare a 100 u/ml stock by mixing the volume of reconstituted standard stock with the volume of diluent detailed on the reference standard stock vial label. 4. Dispense 250 μl of diluent into the tubes labeled 50, 25, 12.5, 6.25, and 3.125 u/ml. 5. Prepare a 50 u/ml standard by diluting and mixing 250 μl of the 100 u/ml standard with 250 μl of diluent in the tube labeled 50 u/ml. 6. Similarly prepare the 25, 12.5, 6.25, and 3.125 u/ml standards by serial dilution.

1. Secure the desired number of coated wells in the holder. 2. Dispense 100 μl of standards and diluted samples into the wells (we recommend that samples be tested in duplicate). 3. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (25°C) for 45 minutes. 4. Aspirate the contents of the microtiter wells and wash the wells 5 times with 1× wash solution using a plate washer (400 μl/well). The entire wash procedure should be performed as quickly as possible. 5. Strike the wells sharply onto absorbent paper or paper towels to remove all residual wash buffer. 6. Add 100 μl of enzyme conjugate reagent into each well. 7. Incubate on an orbital micro-plate shaker at 100-150 rpm at room temperature (25°C) for 45 minutes. 8. Wash as detailed in 4 to 5 above. 9. Dispense 100 μl of TMB Reagent into each well. 10. Gently mix on an orbital micro-plate shaker at 100-150 rpm at room temperature (25°C) for 20 minutes. 11. Stop the reaction by adding 100 μl of Stop Solution to each well. 12. Gently mix. It is important to make sure that all the blue color changes to yellow. 13. Read the optical density at 450 nm with a microtiter plate reader within 5 minutes.

1. Calculate the average absorbance values (A450) for each set of reference standards and samples. 2. Construct a standard curve by plotting the mean absorbance obtained from each reference standard against its concentration in u/ml on linear graph paper, with absorbance values on the vertical or Y-axis and concentrations on the horizontal or X-axis. 3. Using the mean absorbance value for each sample, determine the corresponding concentration of anti-SRBC IgM in u/ml from the standard curve. 4. Multiply the derived concentration by the dilution factor to determine the actual concentration of anti-SRBC IgM in the serum/plasma sample. 5. PC graphing software may be used for the above steps. 6. If the OD450 values of samples fall outside the standard curve when tested at a dilution of 200, samples should be diluted appropriately and re-tested (do not use dilutions lower than 25-fold).

A typical standard curve with optical density readings at 450nm on the Y-axis against anti-SRBC IgM concentrations on the X-axis is shown below. This curve is for the purpose of illustration only and should not be used to calculate unknowns. Each user should obtain his or her data and standard curve in each experiment.

1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of and in accordance with the instructions detailed above. 2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

1. GS Ladics. Use of SRBC antibody responses for immunotoxicity testing. Methods 41:9-19 (2007) 2. L Temple, TT Kawabata, AE Munson, and KL White. Comparison of ELISA and Plaque-Forming Cell Assays for Measuring the Humoral Immune Response to SRBC in Rats and Mice Treated with Benzo[a]pyrene or Cyclophosphamide Fundamental and Applied Toxicology 21(4):412-419 (1993)

This test kit allows rapid and quantitative measurement of rat anti-SRBC IgM levels in serum or plasma samples.

1. SRBC Coated 96-well Plate (provided as 12 strips of 8 wells) 2. Enzyme Conjugate Reagent, 11 ml 3. Reference Standard Stock (lyophilized) 4. 20× Wash Solution: 50 ml 5. Diluent: 30 ml 6. TMB Reagent (One-Step): 11 ml 7. Stop Solution (1N HCl): 11 ml

A typical standard curve with optical density readings at 450nm on the Y-axis against anti-SRBC IgM concentrations on the X-axis is shown below. This curve is for the purpose of illustration only and should not be used to calculate unknowns. Each user should obtain his or her data and standard curve in each experiment.

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet