Coupling by Adsorption
ProceduresPassive Adsorption
Reagents
1°) Polymeric microspheres (often supplied at 10% solids)
2°) Adsorption Buffer (low ionic strength buffer of pH at or near pI of protein)
3°) Purified ligand
4°) Storage buffer (adsorption buffer with 0.01-0.1% blocking molecule added)
Procedure
1°) Dilute the microspheres to 1% solids (10 mg/ml) with adsorption buffer.
Note: Although surfactants or detergents, in which microspheres are normally shipped, may sometimes interfere with binding, it is often not necessary to clean the microspheres prior to use.
If cleaning is desired, this can be done by techniques like centrifugation, dialysis, or ion exchange as described in our
2°) Dissolve appropriate amount of purified ligand in adsorption buffer.
3°) Add the microsphere suspension to the appropriate volume of dissolved protein, and mix gently for 1-2 hours.
Note: By adding microspheres to protein, rather than protein to microspheres, efficiency is maximized and even distribution of adsorption is more likely.
4°) Incubate suspension overnight at 4°C, with constant mixing.
Note: Although the vast majority of ligand adsorption occurs very rapidly, the extended incubation seems to aid in achieving correct orientation by allowing a equilibrium to be reached.
Other options are to incubate at room temperature for 1-2 hours, or at 37°C for 15-30 minutes (in cases where the ligand will not be adversely affected by elevated temperatures).
5°) Centrifuge, remove supernatant*, and resuspend microspheres pellet in storage buffer to desired storage concentration (often 10 mg/ml). A separate blocking step may be added here if necessary.
* Supernatant can be saved to determine the amount of free protein, from which the amount of adsorbed protein can be indirectly quantified.
A common assay to determine the amount of free protein in solution is the BCA assay (Pierce Chemical Company).
A more crude measurement can be made by measuring the A 280 of the supernatant on a spectrophotometer.
Absorbance at 280 nm: A280
Equipment
1°) Spectrophotometer (equipped for UV reading)
2°) Matched Quartz Cuvettes
3°) Pasteur pipettes and pipette bulbs for solution transfer
Reagents
1°) Adsorption Buffer (for blank)
2°) Supernatant (from Procedure a)
Procedure
Single Beam Spectrophotometer
1°) With no cuvette present in instrument, set A 280 to zero.
2°) With adsorption buffer in cuvette, read A 280, then reset to zero (this step determines whether
the adsorption buffer has a significant absorbance.)
3°) Remove buffer and add supernatant to cuvette, then record absorbance.
Dual Beam Spectrophotometer
1°) With matched, empty cuvettes in machine, set instrument zero.
2°) Add adsorption buffer to sample cuvette, leave reference cuvette empty. Record absorbance (this step determines whether the buffer has a significant absorbance.)
3°) Remove buffer from sample cuvette. Add supernatant and adsorption buffer to sample and reference cuvettes, respectively, then record absorbance.
Comments
1°) It is a common laboratory shortcut (although a very imprecise one) to assume that an absorbance of 1.0 in a 1 cm cuvette roughly approximates 1 mg/ml of protein.
For comparison, measured A 280 values of a sampling of proteins at 1 mg/ml follow: Bovine Serum Albumin 0.70 ; Ovalbumin 0.79 ; gamma-Globulin 1.38 ; Trypsin 1.60 ; Chymotrypsin 2.02 ; a-Amylase 2.42
2°) If absorbance is off scale, the sample can be diluted with buffer and the assay repeated. Alternatively, a cuvette with a shorter path length may be used.
3°) Glass or plastic cuvettes absorb light in the UV range and should not be used for this assay.
Covalent Coupling to Non-functionalized Polymeric Microspheres
Although adsorption of hydrophobic ligands to polymeric microspheres is advantageous in many situations, there are times when the hydrophobic attractive forces may not be strong enough to resist the incubation and wash steps included in many assay procedures.
In other cases, the antibody in question might not be able to be adsorbed and still retain its immunoreactivity. One answer to such situations is to modify the surface of the microspheres so that covalent coupling becomes an option. Following are approaches that can be taken for such modification.
1°) A practical and reproducible coating method for plain polystyrene involves the adsorption of dial and coupling of the required ligand.
The poly phe-lys fulfills two important requisites.
First, the strong hydrophobic interactions between the phenylalanine residues and the polystyrene surface create an essentially irreversible adsorption.
Secondly, the introduction of ‘reactive’ amino groups offers a means of co-valent attachment via
standardized chemistries.
2°) It is possible to directly derivatize, and covalently couple ligands to, non-functionalized polystyrene microspheres.
Many other surface functionalized microspheres are available for easy covalent coupling.
Buffers
Following are some basic recipes for buffers commonly used in adsorption protocols. Generally, maximal adsorption occurs at or near the pI of the protein, and so the choice of buffer should be made accordingly.
Additionally, many researchers have reported that the addition of NaCl to the coupling buffer, in physiological concentrations of about 0.15 M, increases adsorption efficiency.
This information is intended only as a general guideline. Feel free to substitute buffers and/or adjust concentrations as your application demands.
Phosphate Buffered Saline (PBS); pH 7.4
(a) Potassium Phosphate dibasic: 1.82 g/l (MW 174.2)
(b) Sodium Phosphate monobasic: 0.22 g/l (MW 120.0)
(c) Sodium Chloride: 8.76 g/l (MW 58.4)
Bring to final volume of 1 L using DI water. Adjust pH to 7.4 using either 1N HCl or 1N NaOH.
Borate Buffer, pH 8.5
(a) Boric Acid, H3BO3 : 12.4 g/l(MW 61.8)
(b) Sodium Tetraborate: 19.1 g/l(MW 381.4)
Add 50 ml of (a) to 14.5 ml of (b).
Bring to final volume of 200 ml using deionised water. Adjust pH to 8.5 with NaOH (3M).
Acetate Buffer; pH range 3.6 to 5.6
(a) 0.1 M Acetic acid (5.8 ml made to 1000 ml)
(b) 0.1 M Sodium acetate; 8.2 g/l (anhydrous, MW 82.0)
Mix acetic acid and sodium acetate solutions in the proportions indicated and adjust the final volume to 100 ml with deionised water. Adjust the final pH using HCl (1 N) or NaOH (.1 N).
Citrate-Phosphate Buffer; pH range 2.6 to 7.0
(a) 0.1 M Citric acid; 19.2 g/l (MW 192.1)
(b) 0.2 M Dibasic sodium phosphate; 35.6 g/l (dihydrate; MW 178.0)
Mix citric acid and sodium phosphate solutions in the proportions indicated and adjust the final volume to 100 ml with deionized water. Adjust the final pH using 1 N HCl or 1 N NaOH.
Carbonate-Bicarbonate Buffer; pH range 9.2 to 10.4
(a) 0.1 M Sodium carbonate (anhydrous), 10.6 g/l (MW 106.0)
(b) 0.1 M Sodium bicarbonate, 8.4 g/l (MW 84.0)
Mix sodium carbonate and sodium bicarbonate solutions in the proportions indicated and adjust the final volume to 200 ml with DI water. Adjust the final pH using 1 N HCl or 1 N NaOH.
Note: Small concentrations of anti-microbial agents (0.05-0.1% w/v) such as sodium azide or merthiolate are often added to the storage buffer, especially for long-term storage.
Blocking agents
Blockers can be added to the storage buffer in varying amounts, a standard concentration being 0.05 %(w/v).
Using a substance dissolved in the storage buffer that will block the exposed hydrophobic surfaces of the polymeric microspheres will reduce non-specific binding and self-aggregaton of the microspheres.
A separate incubation in a higher concentration of blocker (up to 0.1%) is also recommended before storage, in order to saturate the exposed hydrophobic surfaces of the microspheres.
Some commonly used blockers are as follows:
1°) BSA (Bovine Serum Albumin): Often used alone, but can be combined with other blockers, most commonly surfactants.
2°) Casein: A milk-based protein, containing indigenous biotin, which should be avoided when working with systems involving biotin to prevent interference.
3°) Pepticase(Casein Enzymatic Hydrolysate): an enzymatic derivative of casein, should also be avoided when working with systems involving biotin.
4°) Non-Ionic Surfactants: Tween 20 and Triton X-100 are typical. When used in combination with another blocker, a common ratio is 1% Blocker: 0.05% Surfactant.
5°) "Irrelevant" IgG: Often used when conjugating a specific IgG to microspheres. For example, if coupling mouse IgG, rabbit (or any non-cross reacting IgG)can be adsorbed as a blocker.
6°) FSG (Fish Skin Gelatin): Pure gelatin or gelatin hydrolysate can also be used.
7°) Polyethylene Glycol: A very versatile blocker, available in a number of sizes, configurations, and charges.
8°) Sera: Non-cross-reacting serums, such as horse or fish se-rum, are very inert in terms of cross- reactivity with various types of antibodies.
9°) Commercial Blockers: Many companies offer preparations which are a composite of 2 or more single blocking substances of various molecular weights, and which can be used effec-tively over a wide range of conditions.
These go under various trade names, and most chemical vendors will offer a variety of these.
References
1°) Hechemy, K. and E. Michaelson, "Latex Particle Assays in Laboratory Medicine. Part I and Part II.", Laboratory Management, 27, #40, 26-34 (1984).
2°) Cantarero, L.A., J. E. Butler and J. W. Osborne, "The Adsorptive Characteristics of Proteins for Polystyrene and Their Sig-nificance in Solid-Phase Immunoassays", Analytical Biochem-istry, 105, 375-382, (1980).
3°) Bale, M.D., S. J. Danielson, J. L. Daiss, K. E. Goppert and R. C. Sutton, "Influence of Copolymer Composition on Protein Adsorption and Structural Rearrangements at the Polymer
Surface", Journal of Colloid and Iterface Science , 132, 176-1874 (1989) .
4°) Bollag, D.M., M.D. Rozycki and S.J. Edelstein. 1996. Protein Methods, New York: Wiley-Liss.
5°) Wood, W.G. and A. Gadow, "Immobilisation of Antibodies and Antigens on Macro Solid Phases-A Comparison Between Adsorptive and Covalent Binding", J.Clin. Chem. Clin.
Biochem., 21, 789-797, (1983).
6°) Tenoso, H.J. and D.B. Smith. 1972. "Covalent Bonding of Antibodies to Polystyrene Latex Beads: A Concept." NASA Tech. Briefs
7°) Cheung, S.W. 1993. "Methods of making fluorescent microspheres." US Patent # 5,194,300.
Coat on the right surface
How many times have you observed differences in reactivity of your coated reagents when assessing lot-to-lot reproducibility of the raw microspheres ? Same conditions of coating, same antibody at the same concentration, same volume of particles, same...And what about the surface?
One critical concern is to consider the number of molecules immobilised per unit of surface, rather than per number or per volume of particles. Diameter and percentage of solid (this information is indicated precisely on the Certificate of Analysis) vary from one particle to the other, providing differences in the surface available for coupling.
You only need to know the surface area of a spherical particles :
S = pD2 and the number of particles per ml :
E is the percentage of solid
d the density of the particle (1.059 g/cm3 for polystyrene)
D the diameter in µm
100 µl of a 10% solid suspension of K080 particles (diameter 0.800 µm) yield a total surface of : S x N = 708.2 cm2
Adding 0.16 mg of a monoclonal antibody (IgG, M.W. = 150 000 g/mol) to this suspension gives a theoritical surface coverage of : 1.5 pmol/cm2
As a comparison, adding the same amount of antibody to the same volume of particles (100 µl) at 10% solid and of diameter 0.75 µm gives, in the same experimental conditions, a surface coverage of : 1.4 pmol/cm2
The variation (6.6%) between the two reagents is likely to generate unvaluable effects.
Which coverage?
For Latex Agglutination Tests involving an antibody as a coated protein, most optimisations lead to an antibody coverage within the range [0.5 - 5 pmol/cm2].
Coat at the right pH
Passive adsorption is said non pH dependent. This is particularly true for polyclonal antibodies.However, numerous results indicate that pH should be considered during passive adsorption.
For example, it has been shown that passive adsorption can be affected by a variation of pH. The maximum of adsorbed IgG onto poly(vinyltoluene) particles is 2.6 mg/m2 at pH = 4 and pH = 10, whereas it is 7.5 mg/m2 at pH = 7.8.
When changing the pH from 7.8 to 4 (or 7.8 to 10), a partial desorption of IgG was observed (values changed from 7.5 to 3.2 mg/m2).
Literature
Bagghi P. and al. J.Colloid Interface Sci. 1981, 83 : 460-478.
Coupling on COOH beads
Covalent coupling procedure : N-hydroxysuccinimide ester activationMaterials
- Particles EM1-100 / 40- 10 % solid 250 µl
- Water soluble carbodiimide
- [1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide] 30 mg
- N-hydroxysuccinimide 18 mg
- Buffer A : NaH2PO4 – 10 mM – pH 6.0 10 ml
- Buffer B : HCl – 2 mM 10 ml
- Buffer C : NaH2PO4 /Na2HPO4-20 mM – pH 7.5 10 ml
Method
1°) Wash particles twice 1 ml of buffer A.
2°) Dissolve 30 mg of carbodiimide and 18 mg of NHS in 3 ml of buffer A, in a glass tube. Add
2.5 ml of this solution to the pellet of magnetic particles.
3°) Place the tube under agitation for 15 minutes at room temperature. Wash the particles with
1 ml of buffer B.
4°) Resuspend the pellet of particles with 1.480 ml of buffer C. Add immediately the proteins (*1)
and mix. Place the tube under agitation 2 hours at room temperature.
5°) Wash the coated particles three times with buffer C and resuspend in the appropriate buffer (*2).
Note:
*1. Most proteins bind to particles within the range [5-60] 10-4 nmole/ cm2. For IgG, 15 104 nmole / cm2 can be tried as a basic example. Concentration of IgG : 5 mg / ml. Volume to be added : 71µl. The protein suspension must be free of primary amines.
*2. Example of appropriate buffer :
- Na2HPO4-20 mM
- Glycin – 100 mM
- Adjust to pH 7.5 with NaH2PO4
- Bovine albumin - 1 to 10 g / l
- Preservative ( avoid sodium azide for peroxidase based immunoassay )
Coupling on NH2 or COOH beads
Covalent coupling procedure : use of soluble carbodiimide (CDI)Materials
- Particles at 10% solid
- Water soluble carbodiimide 250 µl
- [ 1-ethyl-3-(3-Dimethylaminopropyl) carbodiimide] 40 mg
- Buffer A : NaH2PO4 – 10 mM pH 6.0
Method
1°) Dialyse the protein 2 x 1h in buffer A.
2°) In a 5 ml clean glass tube, mix 1000 µl of Buffer A with 250 µl of particles at 10 %.
3°) Dissolve water soluble carbodiimide at a final concentration of 20 mg / ml in Buffer A.
4°) Add 125 µl of solubilised water soluble carbodiimide ( 3 ) to the diluted particle suspension ( 2 ) and mix.
5°) Add protein at the desired final coverage (see *1).
6°) Incubate for 2 h at room temperature or overnight at +4°C, under weak agitation.
7°) Wash the particles twice with Buffer A and resuspend in the appropriate buffer (see *2) at the desired concentration.
Note:
*1. Most proteins bind to particles within the range [5-60] 10-4 nmole/ cm2. For IgG, 15 10-4 nmole / cm2 can be tried as a basic example. Note that only the total surface of particle must be considered (do not consider the number of particles).
The protein buffer must be free of primary amines.
*2.Example of appropriate buffer :
- Na2HPO4-20 mM
- Glycin – 100 mM
- Adjust to pH 7.5 with NaH2PO4
- Bovine albumin – 1 to 10 g / l
- Preservative (avoid sodium azide for peroxidase based immunoassay)
Coupling on Estapor® NH 2 - Modified Microspheres with Glutaraldehyde
Introduction of glutaraldehyde:The first cross-linking agents used for modification and conjugation of proteins were bireactive compounds containing the same functional group at both ends.
Glutaraldehyde is the most popular homobifunctional reagents used for protein conjugation, especially for creating antibody-enzyme conjugates.
Amino groups on proteins may react with the bis-aldehyde compound glutaraldehyde to form derivatives able to cross-link with amino-groups on the particle.
When using these homobifunctional reagents for coupling proteins to microspheres, one main disadvantage is the potential for creating cross-links between two proteins leading to undefined complexes and precipitated.
To overcome these problems, two-step reaction was developed, removing the excess of cross-linkers before adding the proteins.
The reaction mechanism for this modification can proceed by different ways. The more simple of these is the formation of a Schiff base linkage between one of the aldehyde ends and amines on proteins (e-amine from lysine or a-amine from most amino acid or primary amine from the particle) to leave the other aldehyde terminal free to conjugate with another molecule.
Schiff base interactions between aldehydes and amines typically are not stable enough to form irreversible linkages, and have to be reduced with suitable reductants.
Principle: a two-step procedure
Glutaraldehyde, used in large excess when compared to the number of amino groups of the particle, is first allowed to react with the NH2 groups at the surface of the bead. (a molar ratio would lead to cross linking of the particles).
The excess of Glutaraldehyde is removed.
The second aldehyde group reacts in a second step with the amino groups of the protein to be bound to the microspheres.
Particle Characteristics
- Reference : Estapor K2 Seri
- Polymer : Polystyrene
- Mean sizes : 0.20 – 0.25 – 0.50 or 0.75 µm
- Number of NH2 : from 10 to 50 µeq/g
Protocol
In a Eppendorf tube, add 100 µl of Latex amino-modified microspheres (10% solid)
1°) Add 700 µl of a NaH2PO4 (10 mM) pH 6.8 Add glutaraldehyde to a final concentration of 1.25%
React 4 hrs at room temperature
2°) Wash the activated particles to remove the excess of glutaraldehyde in the same buffer.
Dissolve the protein to be conjugated at a concentration of 1 to 10 mg/ml in a sodium carbonate buffer 0.1 M pH 9.5
1°) Mix the particle suspension with the protein at the desired ratio and react overnight at +4°C
2°) Wash the particles twice in the same buffer
3°) To reduce the Schiff bases and any excess aldehyde, add sodium borohydride to a final concentration of 10 mg/ml
4°) Reduce for 1 hr at +4°C
5°) Wash the particles twice and resuspend in the desired buffer.
Literature
Avrameas, S. (1969) coupling of enzyme to proteins with glutaraledhyde. Immunochemistry 6, 43-52
Avrameas, S. and Ternynck, T. (1969) The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunosorbents. Immunochemistry 6, 53-66
Hermanson, G.T., Mallia, A.K., and Smith, P.K. (1992) in "Immobilised Affinity Ligand Techniques." Academic Press, San Diego
Hermanson, G.T., (1996) in "Bioconjugates Techniques." Academic Press, San Diego
Coupling on thiol-modified microspheres with heterobifunctional agents
Thiol-functionalised microspheres allow covalent coupling of numerous molecules that can be derivatized into an iodoacetyl or maleimide compounds. Commercially available heterobifunctional cross-linkers are routinely used for chemical modifications of proteins at one end, and covalent attachment to one microspheres at the other end.Thiol-Reactive Chemical Reaction
Reactive groups able to couple with thiol contained in molecules (or coated onto the surface of the microspheres) are frequently used on cross-linking, especially in the design of heterobifunctional cross-linkers where sulfhydryl-reactive groups are present on one of two ends. The other end of such cross-linkers is often an amine-reactive functional group.
The primary coupling chemical reactions for modification of Thiol-microspheres proceed by alkylation or disulfide interchange. Many of the reactive groups involved in these reactions are stable in aqueous environment and allow a two-step conjugation strategy.
Moreover, these reactions present the advantage to be rapid and to give stable thioether or disulfide bonds. Chemical reactives able to effect a coupling with a thiol functional group are numerous: Haloacetyl and Alkyl halide derivatives; Maleimides; Aziridines; Acryloyl derivatives; Arylating Agents; Thiol-Disulfide Exchange reagents.
Main Chemical reactions
Main functional groups involved in reactions with Thiol to create sulfhydryl-reactive compounds are Haloacetyls and Maleimides.
Haloacetyl and alkyl Halide Derivatives
Three forms of activated halogen derivatives can be used to create sulfhydryl-reactive compounds: haloacetyl and benzyl halides that react through a resonance activation process with the neighboring benzene ring; alkyl halides that possess the halogen β to a nitrogen or sulfur atom. In each of these compounds the halogen is easily displaced by an attacking nucleophilic substance to form an alkylated derivative with loss of HX. The iodoacetyl derivative has the highest reactivity toward sulfhydryl and may be directed specifically for SH modification. The specificity of this modification has been used in the design of heterobifunctionnal cross-linking reagents, where one end of the cross-linker is an iodoacetamide derivative and the other end contains a different group directed at another chemical target.
Maleimides
The double bond of maleimide may undergo an alkylation reaction with sulfhydrylgroups to form stable thioether bonds. Maleimide reactions are specific for sulfhydryl groups in the pH range 6.5-7.5. At pH 7, the reaction of the maleimide with sulfhydryl proceeds at a rate 1000 times greater than its reaction with amines. At higher pH values, some cross-reactivity with amino groups takes place.
Some examples of heterobifunctionnal cross-linkers with an sulfhydryl reactive group
- N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB) with amine-reactive and sulfhydryl-reactive ends.The NHS ester of SIAB can couple to primary amine-containing molecules, forming stable amide linkages. The iodoacetyl group creates stable thioether bonds. SIAB is water-insoluble; it must be first dissolved in organic solvent (DMSO and DMF) prior to addition to an aqueous reaction medium.
- Succinimidyl-4-(N-maleimidomethyl) cyclohexana-1-carboxylate (SMCC) with NHS ester andmaleimide ends. The SMCC is a heterobifunctional cross-linker with significant utility in cross-linking proteins (antibody, enzyme) and hapten-carrier conjugates. The maleimide end of SMCC is specific when the reaction pH is in the range of 6.5-7.5. At pH 7 the reaction of the maleimides with sulfhydryls proceeds at a rate 1000 times greater than its reaction with amines. SMCC is a water-insoluble cross-linker; it must be dissolved first in organic solvent (DMF) before it is added to a protein to be modified.
- m-Maleimidobenzoyl-N-hydroxysuccimide ester (MBS) with NHS ester and maleimide ends. The NHS ester can react with primary amines in proteins and other molecules to form stable amide bonds, while the maleimide end nearly exclusively reacts with sulfhydryl groups to create stable thioether linkages. These characteristics allow highly controlled conjugation to be done with MBS using two- or three-step processes. In this sense, the NHS ester end of the reagent typically is reacted with the first protein to be cross-linked, forming an maleimide-activated intermediate. The maleimide group is more stable to breakdown by hydrolysis than the NHS ester, so the activated intermediaries can be quickly purified from excess cross-linker and reaction by-products before it is added to the sulfhydryl present onto the surface of microspheres. MBS is a water-insoluble reagent so it must be first dissolved in organic solvent (DMF or DMSO).
- N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) may be the most popular heterobifunctional cross-linking agent available. The activated NHS ester end reacts with amine groups in proteins and other molecules to form an amide linkage. The 2- pyridylthiol group at the other end reacts with sulfhydryl residues to form a disulfide linkage with sulfhydryl present onto the surface of microspheres.
Coupling IgG on SH-Microspheres with SMCC
Particle Characteristics
- Reference: Estapor R00-07
- Polymer : Polystyrene
- Mean size : 1.46 µm
- Number of SH : 18 µeq/g (Ellman method) or 290 µeq/g (Sulphate assay)
Protocol
1°) Dialyse the protein against 50 volumes of PBS.
2°) Incubate SMCC (1 mg/ml in DMF/PBS) and protein (1-10 mg/ml in PBS) for 75 min at +4°C.
3°) Eliminate free SMCC and unmodified proteins on a Sephadex G15 column Elute with HEPES 0.1 M pH 7.5. The elution of the protein-SMCC can be monitored at 206 nm with a spectrophotometer.
4°) Prepare 100 µl of Estapor® microspheres at 10% solid, previously washed twice with PBS.
5°) Incubate the Estapor® microspheres with 1 ml of DTT (dithiothreitol 3 mg/ml) for 15 min at room temperature, and wash the Estapor® microspheres 3 times with PBS.
6°) Mix 100 µl of the reduced Estapor® microspheres with the protein-SMCC conjugate, and incubate for 12hrs at +4°C.
7°) Wash the Estapor® microspheres and resuspended in the desired buffer.
Literature
Hashida, S., and Ishikawa, E. (1985) Use of normal IgG and its fragments to lower the non-specific binding of Fab’-enzyme conjugates in sandwich enzyme immunoassay. Anal.Lett.18 (B9), 1143-1155
Dewey, R.E. et al. (1987) A mitochondrial protein associated with cytoplasmic male sterility in the T cytoplasm of maize. Proc.Natl.Acad.Sci.USA 84, 5374-5378
Smyth, D.G. et al (1964) Reaction of N-ethylmaleimide. J.Am.Chem.Soc.82, 4600
Hermanson, G.T., (1996) in "Bioconjugates Techniques. Academic Press, San Diego
Coupling on Estapor® OH-Modified Microspheres
1°) Washing twice MS at 10 % with wash or coupling buffer: pH 8-9 ( 1 ml in 10 ml). Buffer : avoid amine-containing buffers such as Tris or Glycine, which will compete with the ligand for coupling sites.2°) Resuspend in 9.5 ml of activating buffer. Buffer : Sodium Carbonate 2M (vortex, ultrasound or rolling).
3°) Dissolve ( in a fume hood) 1.0 g of CNBr (for 100 mg of MS) in 0.5 ml acetonotrile.
4°) Add CNBr solution to the stiring MS suspension allowing the activation reaction for precisely 2 minutes at room temperature ; Concentration of MS suspension is now 10 mg/ml.
5°) Wash quickly the activated MS in a large volume of ice-cold water, then with cold coupling buffer. Resuspend MS in 5 ml of coupling buffer (4°C). Dissolve the ligand to be coupled in 5 ml of coupling buffer at a concentration corresponding to a 1-10 X excess of calculated monolayer. Combine MS suspension and protein solution.
6°) Keep suspension at 4°C for 24 hours with constant mixing.
7°) Wash, resuspend in 10 ml of quenching solution, and mix gently for 30 minutes. Quenching solution with primary amine source: 30-40 mM (hydroxylamine, ethanolamine or glycine, etc) with 0.05-1% (w/v) blocking molecule. Wash, and resuspend in storage buffer to desired concentration : often 10 mg/ml. Storage buffer: pH 7-7.5 with 0.01-0.1% (w/v) blocking molecule and NaN3 (0.09%).
8°) Store at 4°c until used.
We supply OH-modified microspheres packaged at 10% or 1% solids in water.
| Product Reference | Type of Microspheres | Size (µm) | Main Application |
| K4 030 | White | 0.3 | Agglutination |
| K4 080 | White | 0.8 | Agglutination |
| EM4 100/40 | SuperParaMagnetic | 1.6 | ImmunoAssay |
| F4-U400 | Fluorescent | 4.0 | Calibration |
Should you need additional information regarding our OH-modified Microspheres, please contact us.
Coupling on Chloromethyl (CH2Cl)-Modified Microspheres
As the CH2Cl groups onto the MS will directly react with available NH2 groups from the proteins, our modified Microspheres are classified as pre-activated product. No pretreatment steps required.Due to the high reactivity of these CH2Cl groups, they will dehydrohalogenate in an aqueous suspension over time, and therefore have a limited shelf life after synthesis. Once coupled, their stability matches that of any other ligand coated Microspheres.
1°) Washing twice MS at 10 % (100mg/ml) with washing/coupling buffer (1 ml in 10 ml). Buffer : Phosphate Buffered Saline (PBS) pH 7.5.
2°) Resuspend in 5 ml of wash/coupling buffer, (vortex, ultrasound or rolling).
3°) Dissolve protein (1-10 fold excess of calculated monolayer) in 5 ml wash/coupling buffer. Combine Microspheres suspension and protein solution. Concentration of MS suspension is now 10 mg/ml.
4°) React at room temperature for 3-4 hours with constant mixing.
5°) Wash, resuspend in 10 ml of quenching solution, and mix gently for 30 minutes (at room temperature). Quenching solution with primary amine source: 30-40 mM (hydroxylamine, ethanolamine or glycine, etc) with 0.05-1% (w/v) blocking molecule (BSA, Casein, Pepticase, Tween 20, Triton X-100, PEG, Sera, or IgG ).
6°) Wash, and resuspend in storage buffer to desired storage concentration : often 10 mg/ml. Storage buffer: pH 7-7.5 with 0.01-0.1% (w/v) blocking molecule and NaN3 (0.09%).
7°) Store at 4°c until used.
We supply CH2Cl-modified microspheres packaged at 10% in water.
| Product Reference | Type of Microspheres | Size (µm) | Main Application |
| R02-25 | White | 0.094 | ImmunoTurbidimetry |
| R02-25 | White | 0.104 | ImmunoTurbidimetry |
| K9-020 | White | 0.220 | ImmunoTurbidimetry |
| K9-020 | White | 0.840 | Agglutination |
Biocatalysis - The Use of Estapor® Magnetic Microspheres
Bio-catalysis has been used for several decades in the food, agricultural and also paper industry. Using enzymes rather than conventional chemical transformations offer many advantages in terms of specificity and selectivity. Moreover, this method requires fewer steps and allow higher yields.The use of enzymes for industrial processes has greatly increased, therefore different methods have been proposed to enhance both enzyme stability and enzyme Bio-activity.
It is well known that the use of immobilised systems is really efficient to optimise stability and Bio-activity of enzymes. Binding of enzymes to membrane could be used, but the surface area available for an efficient Bio-transformation is not very large. Binding of proteins to "classical polystyrene microspheres" is quite easy and largely used for diagnostic, biotech and life science applications. Proteins could be bound to plain microspheres using the passive adsorption method (see our technotes 3, 4, 5, 6), or to functionalised microspheres (i.e. with COOH or NH2 groups) using the covalent coupling method.
With small microspheres (<1µm), the surface area is very large, but in fact, if the microspheres are too small, their recovery is very difficult and they can not be easily re-used. With large microspheres (>2µm), their recovery is easier but surface area is insufficient.
An other interesting option is the possible use of super para-magnetic microspheres for bio-catalysis. For many years, magnetic microspheres have been widely used in diagnostic, biotech and life science fields. Binding of proteins, especially antibody or streptavidin, to magnetic microspheres is now very common and offer several advantages :
- Magnetic microspheres could be very easily retained in the reaction system by a magnetic field.
- Recovery is much easier than conventional methods.
- Magnetic microspheres can be re-used and offer an economical tool in Bio-processing.
Estapor® microspheres offer the widest range of super para-magnetic microspheres in terms of size (from 0,3µm to 3µm) and surface properties (COOH, NH2, OH). If it is necessary, our encapsulated magnetic microspheres enable to protect bio-molecules, such as enzymes, from toxic exposure to iron oxide.The following Estapor® super para-magnetic microspheres could be used for Bio-catalysis.
| Product Reference | Size (µm) | Ferrite content (%) | Functional group (µEq/g) |
| M1-030/40 | 0.31 to 0.35 | 40 to 45 | COOH (80 to 120) |
| M1-070/60 | 0.7 to 1.3 | 55 to 65 | COOH (100 to 160) |
| M1-180/20 | 0.8 to 1.2 | 16 to 25 | COOH (30 to 70) |
| EM1-100/40 | 0.9 to 1.8 | 36 to 45 | COOH (15 to 120) |
| EM2-100/40 | 1.3 to 1.7 | 36 to 45 | NH2 (15 to 100) |
| EM4-100/40 | 0.8 to 1.6 | 45 to 50 | OH |
| M1-200/20 | 1.7 to 2.5 | 15 to 25 | COOH (30 to 40) |
Presentation
- Particle content: 10% (100 mg/ml).
Particles are suspended in water - preservatives NaN3 is added on request. - Pack sizes: From 5ml, 10ml and 50ml. Larger volume on request.
Storage
Product must be stored at 4-6°C and under constant agitation (use a roller at 1-2 rpm). Do not freeze.
Streptavidin Coated Magnetic Microspheres
The Estapor® super paramagnetic microspheres are worldwide known as means of magnetic separation and the streptavidin -biotin interaction is very strong (Kd = 10-15M). Then, once coupled with streptavidin, our bioactivated beads provide you with one of the best tools for biomagnetic separation and purification.Our streptavidin coated magnetic microspheres provide an efficient solid-phase for separation or purification of all kind of biotinylated biomolecules, including antigens, antibodies or nucleic acids from different sources such blood, sera, tissues and food.
An increasing number of industrial or research applications presently include:
- Immuno-assays, Chemiluminescent-assays or Radioimmuno-assays
- Molecular Biology, Cell Biology
- Microbiology and Virology
- High Throughput Screening (HTS)
Magnetic bioseparation or biopurification of antigens, antibodies or nucleic acids with Estapor® coated microspheres are both a simple and reproducible technique which provide an easier, faster and cheaper alternative to column purification, centrifugation or extraction with organic solvents.
| Characteristics | Advantages |
| Streptavidin coverage is optimized | Binding capacity is improved |
| Streptavidin is covalently coupled | Reagent is more stable |
| Estapor® microspheres are perfectly uniform | Non specific binding is really reduced |
| Estapor® microspheres are perfectly spherical | Aggregation problems are lower |
| Estapor® microspheres are encapsulated | Biomolecules are protected from iron |
| Estapor® microspheres have a 1µm size | Surface area and reactivity are higher |
| Estapor® microspheres are super paramagnetic | After attraction beads re-suspension is easier |
| Estapor® microspheres contain 40% of ferrite | Magnetic attraction is very fast |
Protocol for the immobilization of biotinylated nucleic acids to Estapor® streptavidin coated microspheres
1°) Estapor® streptavidin microspheres should be washed twice with buffer before use. An
appropriate buffer to the desired application should be used. Nuclease-free material must be
used.
2°) Resuspend Estapor® streptavidin coated beads thoroughly (vortex) before use.
3°) Prewash the streptavidin coated beads with 10 x SSC-buffer (1.5 M NaCl, 0.15 M tri-sodium citrate at pH 7.0). DNase and RNase inhibitors : add 15 to 20 Units.
4°) Mix the biotinylated DNA/RNA/Oligonucleotides with the streptavidin coated beads,and then incubate the solution at room temperature for 15 min (oligonucleotides) or 30 min (longer sequences) respectively. During this step, maintain the streptavidin-coated beads in suspension, even their small size ensures that they remain in solution.
5°) Place the tube for 2 to 3 min in the magnetic rack. Remove the supernatant.
6°) Wash the streptavidin coated beads with bound nucleic acids, twice with 10 x SSC.
7°) Resuspend the streptavidin coated beads in an appropriate buffer according to your application.
Binding capacity
1 mg of Estapor® streptavidin coated beads will bind at least 400 pmole of biotinylated oligonucleotides and more than 5 µg of biotinylated proteins.
Due to high COOH group density on their surface, the protein streptavidin is covalently attached to the Estapor® beads. For this reason, Estapor® streptavidin coated beads can be regenerated and reused for the same application with no significant loss of yield. Once used, Streptavidin coated beads should be stored at 4-6° C in the storage buffer - antiprotease (Trasylol®) could be added.
According to the evidence that microsphere requirements for capturing microbial cells (such as E.coli or Salmonella) are different from those for isolating nucleic acids (DNA, RNA or PCR products), we have launched a new program for developing smaller and larger super paramagnetic microspheres
Estapor's new proprietary polymerisation technology (patent pending) has recently created a new line of COOH microspheres from 0.3µm to 3.0 µm. Then, we have selected five different sizes of microspheres in order to better cover all the applications using streptavidin coated beads.
High surface area, high content of ferrite, high level of COOH groups and low non-specific-binding are among the main characteristics of this selection.
Now, Estapor® offers a complete range of streptavidin coated beads well suited for industrial or research applications involving biotinylated ligands.
| Size (µm) | 0.31 | 0.86 | 1.00 | 1.91 | 2.60 |
| Ferrite (%) | 40 | 55 | 40 | 13 | 30 |
| Particle number (n/ml) | 4.2 1011 | 1.6 1010 | 1.2 1010 | 2.4 109 | 0.8 109 |
| Binding capacity of a biotinylated IgG (µg/mg) | 7.7 | 7.5 | 6.6 | 4.4 | 3.3 |
| Product number | BE-M08/03 | BE-M08/08 | BE-M08/10 | BE-M08/19 | BE-M08/26 |
| Main advantages | |||||
| Surface area | +++ | ++ | ++ | + | + |
| Binding capacity | +++ | +++ | ++ | + | + |
| Magnetic separation | ++ | +++ | +++ | ++ | ++++ |
Presentation
- Particle content : 1% (10 mg/ml).
- Pack sizes : 2ml, 10ml and 50 ml. Larger volume on request.
- Product is suspended in Phosphate Buffer pH 7.4 containing bovine albumin and sodium azide
- 0.09%.
Stability and Storage
Product must be stored at 4-6°C. Do not freeze. Product is stable for 24 months.
Estapor® Microspheres for Lateral Flow Assays : the "Rainbow Effect"
Over the past decade, membrane based-lateral flow assays have become an important tool not only in medical diagnostics but also in veterinary diagnostics and in environment and agricultural fields. This test format is well known for its simplicity, its utility and its production cost. Together, researchers and physicians have demonstrated the clinical interest of more than 150 different analytes. Many of these have been introduced as products and are designed for the use in hospital, doctor’s offices or pharmacies.Now, most assay developers choose between gold particles (from 20 to 45 nm) and dyed polystyrene microspheres (from 150 to 400 nm). Several rapid-tests, using our Estapor® dyed microspheres, are already marketed.
Today, Estapor® dyed microspheres offer several advantages over the gold particles such as good lot to lot reproducibility, batch size of 1kg and more, uniform size distribution, easy to use for passive adsorption or covalent coupling, and above all, more than 100 different colours.
The last advantage is significant, in particular when several tests are run together, we can easily used different colours of dyed microspheres. In fact the reading is faster, easier and of course more secure. Using the gold particles, the colour lines are always the same and this could induce mistake or, at least, false interpretation. With the dyed microspheres, the different analytes are perfectly and easily identified.
Finally, dyed microspheres offer several advantages over the classical gold particles which should be of interest, not only for R&D people, but also for Marketing and Sales Managers. The competition is always stronger; it is one way to be different!
By Estapor, we recently developed new Estapor® dyed microspheres in the range of 200 nm and highly carboxylated (500 µEq/g)! These new products, very well suited for lateral flow assays, are available in red and blue and, very soon, will be available in green and yellow. Ask for our complete inventory.
Due to their specific light emission properties, our Estapor® dyed microspheres in the range of 150 to 400 nm could be of special interest for developing new lateral flow assays or improving existing ones with a better sensitivity.
Our simple recommendations for using Estapor® dyed microspheres in lateral flow assays are available on simple request.
It is sometime difficult to coat directly antibodies or proteins onto the microspheres. This is the reason why, we developed our own bio-activated dyed microspheres called BioEstapor®. Used as capture-intermediates, BioEstapor® dyed microspheres are coated and bio-activated with streptavidin or secondary antibody (anti-IgG or IgM) and offer many advantages for immobilising easily antibodies or antigens onto their surface.
Until now, lateral flow assays are widely used for the qualitative detection of several analytes : pregnancy test (b-hCG); fertility and ovulation tests (LH and FSH); Infectious diseases (HIV, Strep A, Helicobacter Pylori, HbsAg, Chlamydia, Infectious mononucleosis); cancer detection (PSA, aFP, human haemoglobin faecal); cardiac markers (troponin I); drug abuse (amphetamine, cocaine, BZO).
The quantitative determination of individual human proteins in biological fluids such blood, plasma, serum, urine or cerebrospinal fluid serves as an important tool not only in diagnosing diseases but also in monitoring the course of a disease and, of course the effect of the treatment. During the last ten years, visual techniques are increasingly being replaced by fast and automated optical detection systems.
It is now evident that optimal programming of the instrument is a very important factor for obtaining reliable and reproducible results.
Several analysers are designed to read strip-tests, based on colloidal gold or polystyrene microspheres labelled with different analytes, by remission or retransmission photometry.
More recently, new analysers can detect the amount of magnetic material that passes through a detector. This instrument enables a very good quantification but also, increases the sensitivity by thousand folds in comparison to existing methods of reading.
By Estapor, we recently developed small Estapor® superparamagnetic microspheres of 300nm and containing 30 to 40% of ferrite (M1-030/40) . These new and unique magnetic beads are well suited for developing new lateral flow assays. Then, it is now quite simple to use Estapor® superparamagnetic microspheres as labels in lateral flow assays.
Development of lateral flow assays using these different new solid supports, dyed, fluorescent, bio-activated or superparamagnetic microspheres are really promising.
Estapor® Biomagnetic Technology
Estapor® Biomagnetic Technology is rapid, simple, reproducible and cost effective for :- Immunoassays
- Nucleic Acid Purification
- Cell Separation
- Bacteria Detection
Estapor® Super Paramagnetic Microspheres is your choice material :
- Either for automated or manual detection system.
- Either for automated or manual quantification system.
Along with its Classical "M" and Encapsulated "EM" microspheres, Estapor® has recently introduced Small and Large Super-paramagnetic Microspheres (patent pending) that perfectly complement its product range.
Today, Estapor® Microspheres offer the widest range commercially available of Super-paramagnetic Microspheres in terms of Size, Ferrite content, Surface properties and Density.
| Classical "M" | Encapsulated "EM" | Small M1 030/40 | Large M1 200/20 | |
| Size (µm) | 0.70 - 1.30 | 0.90 - 1.80 | 0.30 - 0.50 | 1.7 - 2.8 |
| Ferrite (%) | 10 - 65 | 15 - 45 | 35 - 45 | 15 - 25 |
| Surface | COOH, NH2 | COOH, NH2, OH | COOH | COOH |
| Density (g/ml) | 1.15 - 2.20 | 1.20 - 1.65 | 1.45 - 1.65 | 1.20 - 1.30 |
This large choice is constantly being adapted and expanded to meet the specific needs of the users
| Classical "M" | Encapsulated "EM" | Small M1 030/40 | Large M1 200/20 | |
| Main Characteristics | High ferrite content | Core shell structure Iron oxide encapsulated |
Large surface area High number of beads | Extremely uniformed size |
| Main Advantages | Magnetic response | Non specific binding Protected from iron exposure | Binding capacity Sensitivity | Dynamic range Linearity Precision |
| Main Applications | Immunoassays | Immunoassays Cell separation Nucleic acid purification Bacteria detection |
Immunoassays Cell separation Flow cytometry Bacteria detection Lateral flow assay |
Immunoassays Cell separation |
With Estapor ®, it is now easy to find the right Magnetic Microsphere which meets your exact and precise specifications.
- Packages: from 5 ml to 5000 ml.
- Presentation: Solid content : 10% (100 mg/ml).
- Storage: Product must be stored under gentle agitation at 4-6°C. Do not freeze.