Quantitative Enzyme-linked Immunosorbent Assay (ELISA) to Measure Serum Levels of Total Murine Immunoglobulin   

Abstract

Materials and Reagents

1. Coating antibody: goat anti-mouse IgX* unlabeled (Southern Biotech)
   Note: IgX*: the isotype of the Ig you wish to detect.
   
2. Murine immunoglobulin of the desired isotype (Sigma- Aldrich, catalog number: M5284 )
   
3. Horseradish peroxidase (HRP) conjugated goat anti-mouse isotype specific antibodies (Southern Biotech)
   
4. 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) Peroxidase Substrate Solution A (Kirkegaard & Perry Laboratories, catalog number: 50-64-00 )
   
5. ABTS Peroxidase Substrate Solution B (Kirkegaard & Perry Laboratories, catalog number: 50-65-00 )
   
6. ABTS Peroxidase Stop Solution (Kirkegaard & Perry Laboratories, catalog number: 50-85-01 )
   
7. 10x PBS-Tween 20 (see Recipes)
   
8. Blocking solution (see Recipes)
   

Equipment

1. Clear 96-well Microtest polystyrene assay plate (BD Biosciences)

Procedure

1. Add 100 μl/well of coating antibody (1 μg/ml in PBS) to a 96-well Microtest assay plate.
   
2. Wrap the plate with plastic wrap and incubate at 4 °C for overnight.
   
3. Discard the coating antibody solution and wash the plate with 1x PBS-Tween 6 times.
   
4. Dry the plate and add 100 μg of blocking solution per well to the plate.
   
5. Incubate the plate at room temperature (RT) for 1.5 h.
   
6. Discard the blocking solution and wash the plate with 1x PBS-Tween 5 times.
   
7. Dilute the mouse serum in 1% BSA in PBS.
   
8. Add 100 μl/well of diluted serum in duplicates or triplicate to the plate. Serum should be diluted in 1% BSA in PBS and titration is required to achieve optimal detection.
   
9. Add 100 μl/well of serial dilutions of standard murine Ig (starting at 100 ng/ml) to the plate.
   
10. Incubate the plate at 37 °C for 1 h.
    
11. Discard the diluted serum and wash the plate with 1x PBS-Tween 10 times.
    
12. Add 100 μl/well of HRP conjugated goat anti-mouse isotype specific antibodies (1/4,000 in PBS/1% BSA) to the plate and incubate at 37 °C for 1 h.
    
13. Discard the diluted serum and wash the plate 10 times.
    
14. Add 100 μl/well of 1:1 mix of ABTS Peroxidase Substrate Solution A and B to the plate.
    
15. Develop the plate at RT in dark. Incubation times will vary depending on your assay.
    
16. Stop the reaction by adding 100 μl /well of ABTS Peroxidase Stop Solution.
    
17. Read the plate using an ELISA reader with a wavelength of 410 nm.
    
18. Calculate the concentration of the serum samples using the standard curve established with the serial dilutions of standard murine Ig.
    

Recipes

1. 10x PBS-Tween 20 [0.1 M PBS, 0.5% Tween 20 (pH 7.4)]

   Na2HPO4 (anhydrous)	10.9 g
   NaH2PO4 (anhydrous)	3.2 g
   NaCl	90 g
   Distilled water	1,000 ml

   Mix to dissolve and adjust pH to 7.4 and then add 5 ml of Tween 20, store this solution at RT.
   Dilute 1:10 with distilled water before use and adjust pH if necessary.
   
2. Blocking solution
   5% FBS and 3% BSA in PBS
   

Acknowledgments

This protocol was developed or modified in Dr. Anne Davidson’s lab at Feinstein Institute for Medical Research, NY, USA. This work was supported by grants from the NY SLE Foundation (RB), Rheuminations, NIH AI082037 and AR 049938-01, NIH (PO1 AI51392 and the Flow Cytometry and Protein Expression and Tetramer Cores of PO1 AI51392).

References

1. Mihara, M., Tan, I., Chuzhin, Y., Reddy, B., Budhai, L., Holzer, A., Gu, Y. and Davidson, A. (2000). CTLA4Ig inhibits T cell-dependent B-cell maturation in murine systemic lupus erythematosus. J Clin Invest 106(1): 91-101.