Polymeric Microspheres and Magnetic Beads

Procedures
Passive 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 use 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

1. Potassium Phosphate dibasic: 1.82 g/l (MW 174.2)
2. Sodium Phosphate monobasic: 0.22 g/l (MW 120.0)
3. 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

1. Boric Acid, H3BO3: 12.4 g/l(MW 61.8)
2. Sodium Tetraborate: 19.1 g/l(MW 381.4)
   
   Add 50 ml of (1) to 14.5 ml of (2).
   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

1. 0.1 M Acetic acid (5.8 ml made to 1000 ml)
2. 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

1. 0.1 M Citric acid; 19.2 g/l (MW 192.1)
2. 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

1. 0.1 M Sodium carbonate (anhydrous), 10.6 g/l (MW 106.0)
2. 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.

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].

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.

Covalent coupling procedure: N-hydroxysuccinimide ester activation

• 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

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 )

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® 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, 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.

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, 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.

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.

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.

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 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.
this table

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 (cocaine or 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.

This large choice is constantly being adapted and expanded to meet the specific needs of the users

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.