Materials

MATERIALS

Solid Phase

  • To adsorb the antigen or antibody onto the solid phase, the 96 well microtitre plate manufactured from polyvinyl chloride or polystyrene is commonly used.
  • The flat bottomed wells are recommended in which spectrophotometric reading is easily employed to assess the colour development.

Coating

  • Coating is attaching antigens or antibodies on surface easily by passive adsorption.
  • The protein adsorb to plastic surface probably as a result of hydrophobic interactions between nonpolar protein substructures and plastic matrix.
  • The interactions are independent of the net charge of protein.
  • The rate and extent of the coating depends on
    • Diffusion coefficient of the attaching molecule
    • Ratio of the surface area coated to the volume of the coating solution.
    • Concentration of the substance being adsorbed.
    • Temperature
    • Time of adsorption
  • A protein concentration range of 1-10 µg/ml of protein in a volume of 50 µl needed to saturate available sites on the plastic microtitre plate.
    • Carbonate bicarbonate buffer (pH 9.6)
      • Sodium carbonate - 1.59 g
      • Sodium bicarbonate - 2.93 g
      • Distilled water to make - 1000 ml

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Washing

  • The purpose of washing is to separate bound and unbound (free) reagents. It is performed at least three times for every well.
  • The phosphate buffered saline (0.01M) with Tween 20 (0.05%) is commonly used. Mechanical action of flooding wells with solutions and shaking (3-5 minutes) is enough to wash the wells.
    • Stock Phosphate buffered saline (PBS) pH 7.4
      • Na2HPO4 - 1.21g
      • KH2PO4 - 0.20g
      • NaCl - 8.00g
      • KCl - 0.20g
      • Distilled water- 1000ml
    • Working PBS  solution
      • PBS – 10x - 100 ml
      • Tween 20 - 0.5 ml
      • Distilled water to make up 1 Litre
    • Washing solution
      • PBS 10x - 100 ml
      • Tween 20 - 50ul
      • Distilled water to makeup 1 Litre

Incubation

  • The reaction between antigens and antibodies affected by their respective concentrations, distribution, time and temperature of incubation. 
    • The temperature of incubation are most commonly at 37◦ C, or room temperature for 1-3 hours.
    •  The other one is longer incubation (over night) at 4◦ C.

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Blocking

  • Subsequent to coating blocking of unsaturated sites on the wells is essential to prevent non specific adsorption of immunologic reagents.
  • Substances added should not react with the solid phase antigen nor the conjugate used.
  • The concentration used often depends upon dilution of the test samples.
  • Some of the commonly used blocking agents are 
    • Skimmed milk powder
      • It is used at 5 percent solution prepared in PBS.
      • Inexpensive, suitable for different enzyme system but can not be used in case of direct ELISA or when biotin avidin system are used.
    • Bovine serum albumin(BSA)
      • It is used as 2 to 3 percent solution prepared in PBS. It is relatively more expensive.

Conjugation

  • Addition of Anti species specific immunoglobulin to an enzyme commonly termed as conjugate.
  • The analytical sensitivity of the ELISA depend on the ability of the antibody to bind and the specific enzyme activity of the labeled immunoreactant and the conjugate.
  • Conjugates may be obtained commercially or made in individual laboratory.
  • The features of conjugates are 
    • The species in which the anti serum is produced. Thus rabbit antichicken  Ig G denotes that a rabbit have been used to prepare antiserum against chicken Ig G.
    • The donating serum and immunogen are probably subjected to   immunochemical treatments. Thus, the donating serum should be crudely fractioned before conjugation, so that the conjugate is 100% reactive against the immunogen.
    • Conjugates must be titrated to optimum condition by chessboard or checkboard titration.
  • Some of the commonly used enzymes conjugates are
    • Horse radish peroxidase (HRP)
    • Alkaline phosphatase (AP)
    • Beta galactosidase
    • Urease

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Substrate

  • The substrate is usually chosen to yield a coloured product.
  • The rate of colour development will be proportional to the amount of enzyme conjugate present.
  • The coloured product must be stable with in a definite time.
  • The Product that are unstable in bright light or temperature at which assay is performed should be avoided.
  • The physiochemical parameters that affect the development of colour includes
    •  Buffer composition and pH
    •  Reaction temperature
    •  Substrate concentration , stability and enzyme stability
  • Some of commonly used substrates are

Enzyme conjugate

Substrate

Chromogen

 Buffer

Horse Radishperoxidase (HRP)

Hydrogen peroxide(H2O2) (0.004%)

Orthophenylene diamine dihydrochloride (OPD)

Phosphate/Citrate.pH 5.0

HRP

Hydrogen peroxide (H2O2) (0.02%)

Diaminobenzidine (DAB)

Tris or PBS,pH 7.4

Alkaline Phosphatase (AP)

Paranitrophenyl phosphate(PNP)

Paranitrophenyl phosphate(PNP)

Diethanolamine,pH 9.8

B-Galactosidase

Ortho-nitrophenyl beta-D- galactosidase (ONPG)

Ortho-nitrophenyl beta-D- galactosidase (ONPG)

Magnesium chloride and 2-mercapto-ethanol,pH7.5

Urease

Urea

Bromocresol

pH 4.8

Stopping reaction

  • Reagents are added to prevent further enzymatic reaction in ELISA at a time as determined for the specific assay.
  • The process is called stopping and reagent used is the stopping reagent.
  • Molar concentration of strong acids or strong bases stop enzymatic activity by denaturing enzymes.
  • The addition of stopping reagents should not varies the total volume of reactants, as it affects the photometric reading.
  • Table shows the stopping reagents ,color produced and wavelength for appropriate substrates for reading.

Enzyme

Substrate

Color(Non stopped)

Color(stopped)

Stopping reagent

Wavelength (nm)

HRP

OPD

Green/orange

Orange/Brown

1M H2So4

492

 

DAB

Brown

Brown

No stop

N/A

ALP

PNP

Yellow/green

Yellow/green

3M NaOH

405

B-Galactosidase

ONPG

Yellow

Yellow

1M Na2Co3

410

Urease

Urea bromocresol

Purple

Purple

Merthiolate (1%)

588

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Reading Results

  • The product of substrate catalysis is colored. It can be read in two ways.
    • By eye inspection
    • Using a Spectrophotometer

Reading by Eye

  • A varies range of colour product will be formed, from partial colour to no colour.
  • The known strong positive samples will give strong colour.
  • Weak positives will give partial colour and negative will give no colour.
  • Some difficulties arise in differentiating weak positives from negative by eye.
  • The interpretation of reading by eye can vary from operator to operator and hence results are more subjective than by using a spectrophotometer.

Spectrophotometer reading

  • The product of the substrate catalysis is measured by transmitting light of a specific wavelength through the product and measuring the amount of adsorption of that light.
  • Special machines are available for the reading of colored products in micro plates.
  • Either one well can be read at a time (manual reader) or more suitably a column of eight wells can be read simultaneously (semi automatic or automatic multi channel reader).
  • The results from such machines are expressed as absorbance units and are recorded on paper rolls or connected to computers.
  • A range of software is available to manipulate and store data.

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Last modified: Tuesday, 23 August 2011, 10:55 AM