免疫比浊法技术
TEL：
免疫比浊法技术
Blocking agents are coated onto microspheres by adsorption and just after the coupling step.
Blocking agents are used to reduce nonspecific interactions between the coated microspheres and non-target molecules in sample (e.g. hydrophobic interaction between proteins and polymer surface).
The blocking agent should be selected carefully as certain blockers may interfere with the assay, and contribute to NSB.
Blockers are often added to the storage buffer in varying amounts from 0.05% to 0.1% (w/v). Blocker
concentration should be determined for efficient blocking without loss of activity.
In order to saturate any exposed surfaces of the microspheres, a separate incubation in a higher
concentration of blocker (up to 1%) is also recommended before storage,
Some recommended blockers :
• BSA (bovine serum albumin): Often used alone but may be combined with other blockers, most
commonly surfactants.
• PEG (polyethylene glycol): A very versatile blocker, available in a number of sizes and MW, length and
charges.
• Irrelevant’ IgG: Often used when conjugating a specific IgG to microspheres. For example, if coupling mouse
IgG, rabbit (or any non-cross-reacting) IgG may be adsorbed as a blocker.
• Casein: A milk-based protein, containing indigenous biotin, which should be avoided when working
with systems involving biotin to prevent interference.
• Pepticase (casein enzymatic hydrolysate): An enzymatic derivative of casein, should also be avoided when
working with systems involving biotin.
• Non-ionic surfactants: Tween 20 and Triton X-100 are typical. When used in combination with another
blocker, a ratio of 1% blocker: 0.05% surfactant is common.
• FSG (fish skin gelatin): Pure gelatin or gelatin hydrolysate may also be used.
To avoid aggregation during scale up phase , we suggest:
1) Continous mixing is required during incubation with Ag/Ab , (US bath or probe depending on ligand)
Continous mixin
2) Choice of ultrasonics bath or probe depends on:
• test
• size of the lot (usually, bath is preferred up to 50ml lot)
Sonication time depends on:
• size of the lot (usually 10’’ to 5’)
3) Blocking with Glycine buffer pH6.5 then pH2.5
Blocking agents are used to reduce nonspecific interactions between the coated microspheres and non-target molecules in sample (e.g. hydrophobic interaction between proteins and polymer surface).
The blocking agent should be selected carefully as certain blockers may interfere with the assay, and contribute to NSB.
Blockers are often added to the storage buffer in varying amounts from 0.05% to 0.1% (w/v). Blocker
concentration should be determined for efficient blocking without loss of activity.
In order to saturate any exposed surfaces of the microspheres, a separate incubation in a higher
concentration of blocker (up to 1%) is also recommended before storage,
Some recommended blockers :
• BSA (bovine serum albumin): Often used alone but may be combined with other blockers, most
commonly surfactants.
• PEG (polyethylene glycol): A very versatile blocker, available in a number of sizes and MW, length and
charges.
• Irrelevant’ IgG: Often used when conjugating a specific IgG to microspheres. For example, if coupling mouse
IgG, rabbit (or any non-cross-reacting) IgG may be adsorbed as a blocker.
• Casein: A milk-based protein, containing indigenous biotin, which should be avoided when working
with systems involving biotin to prevent interference.
• Pepticase (casein enzymatic hydrolysate): An enzymatic derivative of casein, should also be avoided when
working with systems involving biotin.
• Non-ionic surfactants: Tween 20 and Triton X-100 are typical. When used in combination with another
blocker, a ratio of 1% blocker: 0.05% surfactant is common.
• FSG (fish skin gelatin): Pure gelatin or gelatin hydrolysate may also be used.
To avoid aggregation during scale up phase , we suggest:
1) Continous mixing is required during incubation with Ag/Ab , (US bath or probe depending on ligand)
Continous mixin
2) Choice of ultrasonics bath or probe depends on:
• test
• size of the lot (usually, bath is preferred up to 50ml lot)
Sonication time depends on:
• size of the lot (usually 10’’ to 5’)
3) Blocking with Glycine buffer pH6.5 then pH2.5
