IL-1β and IL-18 ELISA

IL18 Elisa kit from Sigma, RAB0810

IL 1β Elisa protocol is attached 

Enzyme-Linked-Immunoabsorbant-Assay (ELISA)                                                                      

The IL1-beta ELISA Ready-Set-Go reacent set (with or without high-affinity binding microwell plates) contains the necessary reagents, buffers and diluents for performing quantitative enzyme linked immunoabsorbent assays (ELISA).  This ELISA reagent set is specifically engineered for accurate and precise measurement of mouse IL-1beta protein levels from samples including serum, protein lysate, and supernatants from cell cultures.  The ELISA set will recognize the cleaved, mature form of uncleaved pro-form of Mouse IL-1 beta.
 

Reagents:  (if purchase kit)

Capture Ab: Purified anti-mouse IL-1beta (B122)   eBiosciences 14-7012-67A

Standard: Recombinant mouse IL-b   eBiosciences 39-8012-60

Detection Ab: Biotin Conjugate anti-mouse IL1-beta (Polyclonal)   eBiosciences 13-7112-67A

Enzyme: Avidin-HRP   eBiosciences 00-4100-93

5x Assay Diluent   eBiosciences 00-4202-43

Substrate: 1x TMB solution   eBiosciences 00-4201-52

10X Elisa coating buffer  eBiosciences 00-0000-50

If Making own solutions:

1X Coating Buffer:

1X DPBS (dilute 10X DPBS: 10mls + 90mls diH2O)

1X Assay Diluent:

1 part FBS in 9 parts 1XDPBS

Wash Buffer:     1x PBS

                          0.05% Tween-20

                          (or eBioscience ELISA Wash Buffer Powder – 00-0400)

Stop Solution:    1M H₃PO₄ (recommended) or 2N H₂SO₄ (1ml 36N H₂SO₄ plus 17 mls H₂O)

Storage Conditions:

Purified anti-mouse IL-1beta (B122):  4 degrees C

Recombinant mouse IL-1b: -80 degrees C

Biotin Conjugate anti-mouse IL1-beta (Polyclonal):  4 degrees C

Avidin-HRP:  4 degrees C

5x and 1X Assay Diluent:  4 degrees C

1x TMB solution:  4 degrees C

10X Elisa coating buffer: 4 degrees C

Stop Solution:  r.t.

Wash Buffer:  r.t.

Solutions (for 96-well plate):

Capture Antibody: Purified anti-mouse IL-1beta (B122)

For 1:250 dilution, add 48ul Ab solution to 12ml coating buffer or appropriate amount to coat 100ul/well X # wells

Standard: Recombinant mouse IL-1b

Aliquot standard once received into 3ul/tube and re-freeze -80C.  At time of assay add 3ul standard  to 3 ml assay diluent to prepare the top standard sol’n (1000pg/ml).  Note: these are single-use tubes.  Use once and discard.  Dilute as suggested above.

Detection Ab: Biotin Conjugate anti-mouse IL1-beta (Polyclonal)

For 1:250 dilution, add 48ul of Ab sol’n to 12ml assay diluent or appropriate amount to coat 100ul/well X # wells

Enzyme: Avidin-HRP

For 1:250 dilution, add 48ul of enzyme sol’n to 12 ml assay diluent or appropriate amount to coat 100ul/well X # wells

Substrate: 1x TMB solution

100ml in one bottle = working concentration
 

Method:

1. Coat Plate: coat each well with 100ul of capture antibody (tube #1) in 1x coating buffer.  Seal plate and incubate overnight at 4 degrees C.
2. Wash: Aspirate wells and wash 5x with 200ul Wash Buffer (PBS w/ Tween sol’n), allowing 1 min soak time per wash. (5min)
3. Block: Dilute 1 part 5x ELISA Diluent with 4x DI water or add 1part FBS to 9parts 1XDPBS.  This is your black/assay buffer for the rest of the procedure. Block wells with 200ul each well. Incubate 1 hr.
4. Wash: 5x, 200ul each well, 1 min soak (5 min)
5. Standard: Standard column dilutions will be as follows: 1000 (no assay buffer), 400, 160, 64, 25.6, 10.2, 4.1, 0 (no sample). Label 8 tubes accordingly.
   - 1 ng/ml or 1000pg/ml “stock” sol’n.
   - For sequential dilution: 
   o place 300ul block/assay buffer in every tube except for 1000.  
   o Place 500ul stock solution in 1000 pg/ml  tube
   o Transfer 200ul from 1000 to 400.  Mix.
   o Transfer 200ul from 400 to 100. Mix. Continue this process until you get to the 4.1 tube.  Stop before placing 200ul from the 4.1 into the 0 tube.
   - Add 100ul of each dilution to appropriate wells in the standard column. (30min)
   - Add 50-100ul assay buffer to each of the remaining wells. Incubate plate at room temp for 2 hrs (or at 4 degrees C overnight), sealed.
6. Wash: 5x, wash buffer, 200ul/well, 1 min soak (5min)
7. Detection: Dilute detection Ab (tube #2) in buffer appropriately.  Add 100ul/well. Seal plate and incubate 1hr.  Remove TMB from fridge at this point so it’s at RT when you need it in step 11.
8. Wash: 5x, 1 min, 200ul/well (5min)
9. Enzyme (HRP): Dilute enzyme (tube #3) appropriately.  Add 100ul/well.  Incubate at RT 15 min.
10. Wash: 5-7x, 2min soak, 200ul buffer/well (10-15 min)
11. TMB: Add 100ul/well. Incubate at RT protected from light 2-5 min. Note: watch plate closely and do not over develop as there will be a black ppt. formed when adding stop reagent.  Final OD for top std concentration should be between 2-3.
12. Stop: 50ul stop sol’n to each well (2N HCl)
13. Read Plate 450 nm/595nm

 Blue = active prep time

Red = incubation time