Enzyme Linked Immunosorbent Assay

Categorized: Immunology Based Methods

ELISA Buffers:

Coating Buffer:

1 litre 500 mls 250 mls
Na2CO3 1.59 gm 0.80 gm 0.40 gm
NaHCO3 2.93 gm 1.47 gm 0.73 gm
NaN3 0.20 gm 0.10 gm 0.05 gm
MgCl2 (5 mM) 1.02 gm 0.51 gm 0.25 gm

pH 9.6 (Don’t usually need to adjust). Best pH during first 2 weeks.


Washing Buffer (1 liter):

10 X PBS 100 mls
MgCl2 1.0 gm

Note: adding Tween 20 (0.05% vol/vol = 5 ml/1 litre buffer) is optional for some ELISAS but do not use if testing membrane proteins. If using Tween, wash 3X, then wash 1X with no Tween.

Blocking/Conjugate Buffer (1 liter):

10 X PBS 100 mls
MgCl2 1.0 gm
BSA/FCS 10g/10ml

Substrate Buffer:

Diethanolamine 100 mls

Conc. HCl (approx. vol.) 10 mls

pH 9.8. Store in the dark, in a dark bottle. The older the buffer, the longer the substrate will take to develop. Make fresh at least every 3 months; more often if necessary.

10 X PBS
NaCl 80 gm
KH2PO4 2 gm
Na2HPO4-12 H2O 29 gm
NaN3 2 gm
KCl 2 gm
pH 7.4. Can autoclave instead / as well as adding NaN3.

Store all ELISA buffers at 4oC.

Remember COATING and SUBSTRATE BUFFERS work best fresh.

ELISA Assay:

Use ELISA 96 well plates. (Currently using Dynatech flexible PVC or Falcon Pro-Bind) Use lids or wrap plates during incubations to avoid evaporation.

Leave 1st vertical row as antigen blank (easiest to blank machine when reading plates). If possible avoid using all “outer wells” of plate.

  1. Coat wells with 50 – 100 ul of antigen diluted to 20 – 50 ug/ml in COATING BUFFER.
  2. Incubate 1 hr at 37oC (or overnight at 4oC).
  3. Discard liquid. Wash plates 3X with WASHING BUFFER. Discard all liquid.
  4. Add 100 ul of BLOCKING/CONJUGATE BUFFER. Incubate 30 min. at 37oC.
  5. Add 50 ul undiluted culture supernatant or antiserum diluted in BLOCKING/CONJUGATE BUFFER. Incubate 1 hr. at 37oC.
  6. Wash as in Step 2.
  7. Add 100 ul of 1/1000 dilution of conjugate in CONJUGATE BUFFER. (Conjugate = Alkaline phosphatase alpha mouse IgG). Incubate 1 hr at 37oC in dark (alk. phos. = light sensitive)
  8. Wash as in Step 2.
  9. Add substrate ( 1 Sigma tablet paranitrophenyl phosphate to 5 mls SUBSTRATE BUFFER. Don’t make ahead!). Add 150 ul and incubate at 37oC in dark.
  10. Turn on ELISA plate reader at least 10 minutes before estimated reading time. Read plates after 45 minutes. Time may vary. Read before substrate is intense yellow color (over “1.00” on reader).
  11. Read first clones and other weak reactions overnight.

Note:– incubations can be performed 1 hr at 37oC, 2 or > hr at RT or overnight at 4oC.