COATING

Immunodetection

Coating

Immunodetection
Coated microplates
Immunoglobulin binding proteins
Saturating agents

Immunodetection

The term Immunoassay (ELISA, Blotting, IHC, etc.) describes a wide range of assays used to detect and quantitate antigens and antibodies. Antibodies are host proteins produced in response to the presence of a foreign molecule (antigen). The antigen antibody interaction is specific, which makes antibodies an important reagent for immunological research.

An example of a typical assay is shown in figure 1. It involves the immobilization of an antigen onto a solid support (polystyrene plate, bead, membrane, etc), a primary antibody (anti primary Ab) specifically raised against the antigen, a secondary antibody which is labeled with an enzyme (ELISA and blotting) or a fluorophore (flow cytometry and microscopy), and the addition of a substrate (in case of an enzyme labeled antibody), which gives a coloured end-product. The intensity of the colour when the substrate is added should correlate directly to the concentration of the primary antibody and the respective antigen.

figure 1

Coated microplates

ELISA immunodetections usually require first to coat microplates with either an antigen or a secondary antibody.

One useful way consists to use functionalized plates that will capture Ag or Ab.

Uptima ready-to-use coated microplates offer several advantages over home made coatings, including gain of time, lower background, higher signal, and plate to plate consistancy

Streptavidin

coated microplates

UPL76161

5 plates

Supplied pre-blocked with Seablock agent (UP40301) for both colorimetric and chemiluminescence detections

Easy and consistant coating of biotinylated ligands, notably very usefull for peptides
Lowest background
Ready-to-use (pre-satured)
More reproducible

Technical Sheet

Anti Mouse IgG (Gt)

coated microplates

UP47246A

5 plates

Anti Rabbit IgG (Gt)

coated microplates, 5 units

UP42458A

5 plates

Supplied pre-blocked with Seablock agent (UP40301) for both colorimetric and chemiluminescence detections

Oriented coating for an optimal binding capacity (superior to passive direct coating)
Species specific coating
Ready-to-use (pre-satured)
More reproducible

Technical Sheet

Supplied pre-blocked with Seablock agent (UP40301) for both colorimetric and chemiluminescence detections

Oriented coating for an optimal binding capacity (superior to passive direct coating)
Species specific coating
Ready-to-use (pre-satured)
More reproducible

Technical Sheet

Ask Uptima custom services for other coatings and saturations !

Immunoglobulin binding proteins

Protein A, Protein G and Protein L are bacterial receptors eliciting very high affinity and specificity for immunoglobulins. They can be used for :
Coupling to a wide variety of reporter molecules including fluorescent dyes, enzyme markers, biotin, colloidal gold and radioactive iodine without affecting the antibody binding site Coating to polystyrene microplates allows easy to perform and very quick and efficient indirect coating of IgGs Coupling to matrices for purification of polyclonal or monoclonal immunoglobulins Ligands for coating
Other ligands for coatings
Streptavidin	UP51558	--	-
For the immobilisation of biotinylated ligands
Neutralized avidin	UP25527	-	-
Even lower background than streptavidin

Protein A	UP40290A	5mg	-
Approx. M.W. 42 000 Da
Protein A binds the Fc portion of human IgG subclasses, IgM (medium), IgA and IgE; and mouse IgG1 (weakly), IgG2a and IgG2b. Protein A also binds IgGs from various other laboratory and domestic animals.
rProtein G	UP75194A	10 mg	-
M.W. 22 600 Da
Protein G binds the Fc portion of IgGs from many species including all human and mouse subclasses, and Rabbit, Sheep, Bovine, Goat, Monkey IgGs, Rat IgGs but IgG2b), and Horse IgGT. It does not recognize Hu and Mo IgM, IgA, IgE or serum albumin.
rProtein L	UP56874A	1 mg	-
M.W. 35824 Da	UP56874B	5 mg	-
Protein L is a protein receptor from Peptostreptococcus magnus that binds to kappa light chains without interfering with antigen binding. An important feature is the binding to Fab and ScFv fragments, rendering it the most effective ligand used to detect and purify these fragments. One proteinL molecule contains 4 binding sites. Optimal binding occurs at pH7.5. Technical Sheet

Saturating Agents

SeaBlock

UP40301A

500ml

saturating agent

Fish serum based saturating agent formulated for immuno-enzymatic detection techniques, especially ELISA and Blotting. This new reagent suits chromogenic and chemiluminescent sytems, as well as immunochemistry, improving results in most applications where antibodies generate a background with other common saturating reagents.

Do not interact with mammalian antibodies
Works with biotin, HRP (both chromogenic and chemiluminescent substrates), AP and fluorescence labels
Lower background than BSA, Gelatin, Milk... immunoglobulins
Better than Milk for biotinylated antibodies/ (strept)avidin conjugates
Improves results for Blotting and ELISA, as well IHC

Technical Sheet

Albumins		-	-
Bovine serum albumin is widely used in immunologicals dilution buffers, and for saturation of coatings (ELISA) and nitrocellulose membranes (blotting).
MaxiBind BSA	UP84225		-
For special coatings / conjugations
Ovalbumin	UPR5851		-
May replace advantagely BSA
Normal sera and IgG		-	-

Choosing a saturation agent is crucial for background and eventual unspecific detection. Examples amongst classic saturants and some drawbacks :

milk based saturants are not recommanded for biotin / streptavidin detection systems
casein based saturants may also generates high background for cell detections
gelatin masks certain antigens.
cross reactivity should be avoided with a carrier that was used for immunization (i.e.BSA-hapten conjugate).

There is no all-purpose and ideal saturant, so different agents should be assayed for optimal results

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