Currently, there are several methods of attaching biological ligands to polystyrene particles. These methods include adsorption to plain polystyrene particles, covalent attachment to surface functionalized particles, and attachment of the ligand of interest to particles that are pre-coated with a binding protein such as Streptavidin, Protein A or Protein G. Presented in this Spherotech Technical Note are protocols such as adsorption, covalent coupling, and other methods used to attach ligands to polystyrene particles.

Procedures and Discussion

The following information explains generalized protocols for the attachment of ligands to polystyrene particles. These protocols are easily optimized to meet the requirements of specific applications. The following protocols are developed by Spherotech for the convenience of SPHERO™ microparticle users. They are to be utilized only as initial conditions. Spherotech encourages the optimization of the coating conditions by changing the buffer, pH or reagents concentration.

In general, polyclonal antibodies are coated to polystyrene particles by adsorption without using any coupling agents. The binding of polyclonal antibodies to polystyrene particles is strong. However, care should be taken not to overload the antibodies to the particles. If overloading occurs, leaching of the coated antibody will happen during storage. This is due to the weak interaction between antibody molecules compared to the interaction of antibody molecules on the surface of polystyrene particles.

SpheroTechnical Note #1 will provide protocols for the following coating protocols:

• Particle coating using passive adsorption
• Amino particles with ligands or proteins using EDC and covalent coupling
• Carboxyl particles with avidin or other proteins using EDC covalent coupling
• Covalent coupling using a two-step EDC coupling protocol
• Avidin particles with biotinylated proteins using affinity coupling
• Covalent coupling of protein to hydroxyl particles using cyanogen bromide (CNBr)
• Dimethylamino particles with DNA using ionic interaction coupling
• Ligands to modified amino proteins covalent coupling using SPDP
• Periodate oxidation of polysaccharides and coupling to amino particles
• Carboxyl polystyrene particles with amino-modified oligonucleotides

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Protocol

The amount of the antibody binding to the particles after coating can be determined by using the subtraction method as follows:

1. Measure the absorbance of the antibody solution to be used for coating at 280 nm (OD280). The IgG has OD280 of ~1.4/mg.
2. Pellet the particles to be coated by centrifugation or magnetic separation.

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Materials:

1. Avidin magnetic particles, 1% w/v, Cat. # VM-40-10, Lot No. J01, 4.35 μm
2. Biotin-FA conjugate, Lot No. 021688, 533 nM in 1% diluent. (IBS containing 1% normal goat serum and 1% fetal bovine serum )

Procedures

1. Add 50, 100,150, 200, 300 & 400 μl of Avidin magnetic particles to six 12x75 test tubes.
2. Add 1 mL of Biotin-FA conjugate to each tube.
3. Vortex and incubate at ambient temperature for at least 30 minutes with occasional shaking.
4. Adjust the fluorimeter for excitation and emission at 490 and 520 nm respectively

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Materials:

1. Gt. anti-Ms IgG magnetic particles, 1% w/v, Cat. # MM-40-10, Lot No. J01, 4.35 μm
2. Ms-IgG FITC, Lot No. 10952, 5μg/mL in 1% diluent ( IBS containing 1% normal goat serum and 1% fetal bovine serum)

Procedures

1. Adjust fluorimeter for excitation and emission at 490 and 520 nm respectively.
2. Set 100% emission with the Ms IgG-FITC solution.
3. Vortex the Goat anti-Ms IgG(H&L) magnetic particles and add 50, 100, 150, 200,300, and 400μL into six 12x75 mm tubes. Pellet the particles and aspirate the supernatant.
4. Add 1 ml of Ms IgG-FITC solution to every tube containing the pelleted particles. Vortex and incubate for 30 minutes.
5. Separate the Goat anti-Ms IgG(H&L) magnetic particles from the conjugate and read the fluorescence of the supernatant.

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Materials:

1. Streptavidin magnetic particles, 1% w/v, Cat. # SVM-40-5 Lot No. Y01, 4.35 µm
2. Biotin-FA solution, Spherotech, Lot No. 021688, 533 nM in 1% diluent (IBS containing 1% normal goat serum and 1% fetal bovine serum)

Procedures

1. Adjust the fluorimeter for excitation and emission at 490 and 520 nm respectively.
2. Set 100% emission with the Biotin-FA solution.
3. Add 50, 100, 150, 200, 300, and 400 µL of Streptavidin magnetic particles to six 1.5 ml microfuge tubes.

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Materials:

1. Biotin magnetic particles, 1% w/v, Cat. # TM-40-10 Lot No. J01, 4.32 µm
2. Avidin-FITC solution, Jackson ImmunoResearch, #003-090-083, Lot No. 21452, 5 µg/mL in 1% diluent.(IBS containing 1% normal goat serum and 1% fetal bovine serum)

Procedures

1. Adjust the fluorimeter for excitation and emission at 490 and 520 nm, respectively.
2. Set 100% emission with the Avidin-FITC solution.
3. Add 25, 50, 75, 100, and 200 µL of Biotin magnetic particles to five 1.5 mL microfuge tubes.
4. Separate the particles magnetically and remove the supernatant.
5. Add 1 mL of Avidin-FITC solution to each tube, vortex and rotate at room temperature for one hour.

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Materials:

1. Gt anti-Ms IgG Magnetic Particles, 1% w/v, Cat. # MM-40-10 or MMXA-40-10, ~2e8 particles/mL
2. Appropriate monoclonal antibody (anti-CD3, CD4 or CD8, etc.)
3. Appropriate FITC conjugates
4. Dulbecco PBS
5. Fetal bovine serum
6. Histopaque
7. Paraformaldehyde fixative
8. FlexiMag Separator, Jr., Cat. # FMJ-1000

Procedures

Collect peripheral blood by venipuncture of the antecubital vein. Draw blood into heparin Vacutainer tubes, transfer to 50 mL polypropylene centrifuge tubes, and dilute with an equal volume of calcium and magnesium-free Dulbecco’s phosphate buffered saline.

Layer 10 to 20 mL aliquots of diluted blood onto an equal volume of Histopaque in 50 mL tubes. Centrifuge for 30 minutes at ambient temperature using a centrifugation force of 400g at the blood/Histopaque interface. Aspirate the lymphocyte band into a 15 mL centrifuge tube and bring the volume to 14 mL with Dulbecco PBS containing 2% v/v heat-inactivated fetal bovine serum or 5% plasma protein fraction. Pellet the cells by centrifugation at 4°C for 7 minutes at 450g, wash with 14 mL of PBS, and recentrifuge. Resuspend the final cell pellet in 1-2 mL buffer, and a viable cell count is performed. Only cell preparations with a viability of >95% should be used.

SpheroTechnical Note #7 will provide protocols for the following:

• Separation of Mononuclear Cells From Peripheral Blood
• Coating of Anti-mouse Igg Magnetic Particles With Monoclonal Antibody
• Incubation of Magnetic Particles and Cells
• Detection of Cell Depletion by Immunofluorescence

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The SPHERO™ Calibration Particles are versatile, stable, economical and convenient to use. These particles contain a mixture of fluorochromes which are spectrally similar to many of the fluorochromes used in flow cytometry. As a result, they are used for routine alignment, day-to-day performance verification, and long-term performance tracking of several channels of flow cytometers in one run. These particles are very stable since the fluorochromes are entrapped inside the particles instead of being located on the surface. They are packaged in a convenient dropper bottle to facilitate dispensing and storage. The diluted particles can be stored for later use if desired to reduce costs. These products and their uses are described briefly as follows:

Rainbow Calibration Particles (RCPs):
The RCPs are designed for the routine calibration of most available channels in any flow cytometer. For example, these particles are used to verify the instrument setup and to check the linearity and sensitivity of the instrument. If factory-recommended procedures are used for instrument setup, we recommend that the RCPs be included in QC programs to track the long-term and day-to-day performance.

SpheroTechnical Note #8 will provide the following information:

• Rainbow Calibration Particles (RCPs) for creating calibration curves and determining sensitivity
• Ultra Rainbow Calibration Particles (URCPs) for creating calibration curves and determining sensitivity
• Rainbow Fluorescent Particles (RFPs) for checking system setting and alignment
• Ultra Rainbow Fluorescent Particles (URFPs) for checking system setting and alignment
• Yellow Calibration Particles (YCPs) for the calibration of the FL1 channel
• Allophycocyanin Calibration Particles (ACPs) for calibrating the Allophycocyanin channel
• Blank Calibration Particles (BCPs) for setting the fluorescence threshold
• Recommended Flow Cytometry QA Procedures including the following: Daily Alignment , Setting the Threshold,
  Verification of Setting and Validation of Logarithmic Amplifier Linearity and Sensitivity, Compensation & Creating Levey-Jennings Charts

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The Molecules of Equivalent Fluorochrome (MEF) value is useful for flow cytometer users. However, the Quantum Yield of most fluorochromes usually changes upon binding to the cells. As a result, the actual number of fluorochrome molecules binding to the cells may be different than the expected MEF value. In addition, the MEF value will vary depending on the measurement method.

There are several methods for measuring the MEF value. Most commonly, a standard curve is generated with a fluorimeter using solutions of fluorochrome in various concentrations. The fluorescence of these solutions is then compared to the intensity of labeled cells or particles less the fluorescence of a blank particle or non-labeled cell. This provides the equivalent concentration of fluorochrome. The MEF value is then calculated by dividing the equivalent fluorochrome concentration by the number of cells or particles used.

SpheroTechnical Note #9 will provide information for the following:

• Methods for measuring the MEF value
• Measuring relative MEF values using the Ultra Rainbow Calibration Particles (URCPs) and Rainbow Calibration Particles (RCPs)
• Normalizing different instruments using URCPs or RCPs
• Quantitative flow cytometry using URCPs or RCPs

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Magnetic Particle Enzyme Immunoassay (MPEIA) is an immunoassay method for the isolation of antibody/antigen complexes on a solid phase surface of magnetic microparticles. MPEIA has been used to automate the measurement of large molecules such as markers associated with cardiac, fertility, cancer, metabolic, hepatitis, and thyroid testing.

Procedures

1. In the wells of a microEIA plate, make serial 2-fold dilutions of rabbit, human, or mouse IgG in 1% bovine serum albumin (BSA), adding 100 µL /well. The control wells should receive 100 µL of 1% BSA without IgG.
2. Add 50 µL of a 0.25% (w/v) suspension of Spherotech Cat. No. CM-40-10 magnetic particles coated with antibodies to rabbit, human or mouse IgG. Dilute the particles in a buffer consisting of 1% casein hydrolysate and 0.05% Tween 20 in phosphate buffer saline, pH 7.2.

SpheroTechnical Note #10 will provide the following:

• A protocol for the development of a Magnetic Particle Enzyme Immunoassay (MPEIA)
• The advantages of MPEIA over coated well EIA
• Selected references showing use of magnetic particles in immunoassays

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Preparation of IgG F(ab’)2 Fraction Using Pepsin Coated Magnetic Particles (Spherotech Cat. # PEPM-40-2)
The Spherotech Cat. No. PEPM-40 consists of 4µm magnetic particles covalently linked to pepsin from porcine stomach mucosa. These particles efficiently cleave IgG from various species producing F(ab’)2 and other lower molecular weight products within a few hours. After digestion, Pepsin-Magnetic particles can be easily removed magnetically from the reaction vessel, leaving no pepsin in the solution. As a result, the cleavage of IgG can be effectively controlled. The supernatant containing products from the cleavage and any residual IgG can be further purified chromatographically or by using Protein A magnetic particles (Spherotech Cat. # PAMX-10).

Preparation of IgG Fab Fraction Using Papain Coated Magnetic Particles (Spherotech Cat. # PAPM-40-2)
The Spherotech PAPM-40 consists of magnetic particles (4µm) covalently linked to Papain from Papaya latex. These particles efficiently cleave IgG into Fab and Fc fragments within a few hours. After digestion, Papain-Magnetic particles can be easily removed from the reaction vessel magnetically, leaving no Papain in the solution. As a result, effectively stops further cleavage of IgG. The supernatant containing products of cleavage and any undigested IgG can be further purified chromatographically.

This procedure allows the controlled preparation of essentially pure Fab in simple two steps. In addition, papain magnetic particles can be reused repeatedly without a significant loss in activity.

SpheroTechnical Note #11 will provide the following:

• A procedure for the controlled preparation of essentially pure F(ab’)2 using Pepsin Magnetic Particles
• A procedure for the controlled preparation of essentially pure Fab using Papain Coated Magnetic Particles
• The advantages of using enzymes covalently linked to magnetic particles over enzymes attached to nonmagnetic gel, latex, or soluble enzymes
• Selected references using Magnetic Particles Coated with Pepsin and Papain

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Protein A is a 42kD polypeptide isolated from Staphylococcus aureus which has a specific binding affinity for the Fc region of IgG from several species. Each Protein A molecule has four IgG binding sites. In addition, protein A binds to IgG without interfering with the antigen-binding site of the immunoglobulin. The Protein A-coated magnetic particles provide a quick, easy, and economical way for the capture of antigen-specific antibodies used in the purification of recombinant antigens.

In the past, protein A linked gel matrix has been routinely used for isolating IgG from human, mouse, and rabbit serum. However, protein A covalently bound to magnetic particles increases the reaction kinetics while reducing the capture time of antigen-specific antibodies. As a result, Protein A-coated magnetic particles are uniquely suited for isolating IgG from limited volume samples without dilution or loss. They can also be used to capture and concentrate low-level IgG in large-volume samples. In addition, Protein A-coated magnetic particles can be repeatedly used without a significant loss in their ability to bind IgG.

SpheroTechnical Note #12 will provide the following:

• The affinity of Protein A coated beads to IgG from different species
• Product details for the various Protein A linked magnetic particles offered from Spherotech
• A protocol for the purification of IgG from hybridoma tissue culture for clone selection
• A protocol for the isolation of specific cells from blood (B, T and HLA) using Protein A Magnetic Particles
• A protocol for an All-Purpose “Fish-Hook” for isolating specific antigen from a mixture
• A protocol for the reuse of Protein A Linked Magnetic Particles

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The fluorescence linearity of flow cytometers is affected by optical alignment, laser power, electronic offsets, and amplifier calibration1. In addition, it is important to monitor and validate flow cytometers’ performance due to the nature of the information obtained during diagnostic testing. As a result, it is recommended that the linearity of the flow cytometer is determined on a monthly basis after instrument repair and after instrument relocation(2).

The SPHERO™ Calibration Particles and SPHERO™ PMT Quality Control Excel Template (PMT QC Template) are designed for linearity calibration and long-term performance tracking of flow cytometers. They will help flow cytometer users verify the operation of their instruments. The PMT QC Template is a valuable tool for determining the linearity of log amplifies. The information acquired from this template should be implemented into flow cytometer calibration documentation. The user can determine a schedule for routine maintenance procedures and tolerance limits of linearity based on instrument trends or malfunctions using this template.

The SPHEROTM Rainbow Calibration Particles (RCPs) and Ultra Rainbow Calibration Particles (URCPs) contain a mixture of similar size particles with different fluorescence intensities.

SpheroTechnical Note #14 will provide the following information:

• The use of the Rainbow Calibration Particles (RCPs) for the determination of the logarithmic amplifier linearity
• The use of the PMT QC Template to determine the number of related fluorophores for an unknown sample or other particles
• References using the RCPs for the standardization of flow cytometers

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Assays for cell counting using flow cytometry and calibrated fluorescent particles are rapid and accurate. The single platform method that enumerates T-cells by counting the identifier cells in either a precise known cell volume or an internal ‘spike’ of a known number of calibrated fluorescent particles by flow cytometry is simple and efficient (1). These assays allow the counting of T-cells during anti-T-cell globulin treatment of cardiac, lung, and renal transplant patients (2). In addition, laboratories can determine the absolute count of CD4 and CD8 T-cells to estimate HIV disease progression with the single platform method (3). Calibrated fluorescent particles and flow cytometry are also used to count platelets in a wide range of murine models of platelet disorders (4). It is also possible to count other various cell types with flow cytometry and calibrated fluorescent particles.

SpheroTechnical Note #15 will provide the following information:

• A description of using Spherotech AccuCount beads and flow cytometry for the ratio-metric method of absolute counting
• A protocol for using the Spherotech AccuCount beads for cell enumeration
• Calculation necessary for using absolution counting beads for cell enumeration
• References using the AccuCount beads

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Covalent coupling of lysozyme to amino-polystyrene particles utilizing a heterobifunctional coupling agent, SPDP, and to aldehyde particles was studied using Particle Concentration Fluorescence Immunoassay (PCFIA). The results showed that these methods can be used to couple lysozyme covalently to amino-polystyrene and aldehyde particles.

The modification of amino-polystyrene particles with SPDP offers the advantage that the resulting pyridyl disulfide particles can be used to react specifically with the sulfhydryl group of proteins, or they can be reduced further with DTT to form sulfhydryl particles. The aldehyde particles can be used to couple covalently to proteins without any coupling agent.

The activity of the lysozyme-coated particles depends upon the orientation of lysozyme on the particles. Higher activity was obtained when the amino groups of the lysozyme were utilized to couple to the particles covalently.

SpheroTechnical Note #16 will provide the following information:

• A protocol for covalently coupling lysozymes to amino-polystyrene particles through either EDC coupling on SPDP modification
• A protocol for the covalent coupling of lysozymes to aldehyde particles without the use of a coupling agent
• How the activity of coated particles varies with the coupling methods used and depends on the orientation of lysozyme on the surface of the microparticles
• Various coupling strategies, including passive adsorption on particles with different surface charges and covalent coupling using different functionalized particles and coupling agents, can be used to optimize the activity of immobilized protein on microparticles

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In order to obtain accurate and scientifically sound flow cytometric data it is critical to use standardization techniques and a robust and reliable instrument. The standardization protocol should address specific operational parameters to determine the performance of the instrument at any point in time. The flow cytometer's optic, fluidic, and electronics design should be simplistic while still being sensitive and effective.

Experimental Procedure
One major parameter to be included in the standardization protocol is a test for sensitivity. Sensitivity is an important parameter since it defines the ability to detect particles above the background. However, sensitivity determination should also include resolution, the ability to resolve dim particles. This can be measured using Spherotech Rainbow Calibration Particles with eight intensities, Cat. No. RCP-30-5A (Rainbow Calibration Particles, 8 peaks, 1E7/mL, 3.0-3.4 um, 5 mL). Since each intensity of the RCP-30-5A has been calibrated to the molecules of equivalent fluorophores (MEF), it can be used to quantify sensitivity. Figure 1 is the histogram of the RCP-30-5A on Stratedigm's S1000 cutting-edge flow cytometer in the PE channel. See http://statedigm.com/instrumentation for more information on Stratedigm flow cytometers.

A flow cytometer's sensitivity can be described as the detection efficiency (Q) and the background light level (B). The detection efficiency is how well light is collected in the cytometer, while the background light level shows how much noise is created by the instrument in the background. In order to determine the efficiency (Q) and the background light level (B), the MEF values of blank beads and another dim population must be known. These values can be obtained using the Spherotech PMT QC Template.

SpheroTechnical Note #17 will provide the following information:

• How the Spherotech 8 peak Rainbow Calibration Particles (Cat. No. RCP-30-5A) provides robust and reliable data when a standardization protocol is successfully adopted and implemented
• An introduction to using the Rainbow Calibration Beadsfor the calculation of a flow cytometer's sensitivity by measuring the Detection Efficiency (Q) and the Background Light Level (B)
• Why a flow cytometer standardization protocol should include the statistical analyses of sensitivity measurements to promote innovative, scientifically sound experimental results

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When setting up multicolor flow cytometry experiments proper compensation is extremely important since these experiments provide complex data. Compensation helps correct spectral overlap to match the various fluorophores used during cell staining, after which the data becomes easier to interpret. Compensation using cells for single-color staining provides autofluorescence levels that are the same as those obtained during multicolor staining and are independent of the antibody host or isotype. However, valuable cellular material and antibodies targeting dimly expressed antigens or rare cellular populations create difficulties when using this approach. Furthermore, native cells are difficult to standardize and introduce additional variability. Compensation procedures using antibody-capturing beads overcome some of these limitations. However, many bead kits are host-specific and do not cover the full range of isotypes. In addition, high backgrounds upon violet laser or red laser excitation are observed for the vast majority of capture bead kits. As a result, Spherotech offers the COMPtrol line of antibody capture beads.

The COMptrol beads offer:

• Low autofluorescence regardless of excitation wavelength or detection bandpass
• Enormous breadth of compatible hosts and isotypes makes COMPtrol capture beads a truly universal compensation tool

SpheroTechnical Note #18 will provide the following information:

• Why compensation must be optimized to obtain consistent and allow for proper data interpretation for multicolor applications
• An introduction to Spherotech COMPtrol beads which are designed to capture antibodies with conjugated fluorophores to provide detectable signals
• How the COMPtrol antibody capture beads provide proper compensation values during multiple fluorophores flow cytometer experiments when combined with acquisition and analysis software

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SPHERO T25 DNA Coated Nano Particles and Microspheres:

• Used for the detection and identification of oligonucleotides
• Allow for simple, rapid and reliable binding of Biotinylated PCR hybridized with a poly(dA)-tailed oligo

SpheroTechnical Note #19 will provide the following information:

• A covalent coupling protocol for amino terminated oligonucleotides to carboxyl functionalized superparamagnetic nanoparticles using T25 DNA as an example
• A hybridization procedure using T25 DNA Coated beads as an example
• A protocol for measuring the concentration of hybridized oligonucleotide using T25 DNA and A25-FAM DNA as an example

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Hydroxylic particles are used to facilitate the development of immunoassays, as well as, quantitate, isolate purify and characterize proteins, cells, or various target analytes. In order to provide the benefits of hydroxyl groups, Spherotech offers SPHERO Hydroxyethyl (HEMA)/Polystyrene Copolymeric Magnetic Particles. These copolymeric particles provide a hydrophilic surface which reduces denaturation of immobilized protein. In addition, the hydroxyethyl groups hydrogen bonds with a layer of water in an aqueous solution preventing aggregation of the particles. These two characteristics translate into longer ligand stability, lower nonspecific binding potential, and a more stable particle solution compared to particles of more hydrophobic surfaces.

Although hydroxyl groups do not spontaneously react toward functional groups on biomolecules, they can be activated for covalent coupling by a number of known reaction mechanisms. The reactions result in covalent attachment of ligands and can be performed under aqueous or non-aqueous conditions. By utilizing non-aqueous conditions, the hydrolysis of activating agents and the intermediate groups is prevented.

The SPHEROTM HEMA/Polystyrene Copolymeric Magnetic Particles:

• Minimize non-specificity problems and provide stable particle solutions
• Can be activated using a variety of methods for the coupling of ligands in either aqueous or non-aqueous solutions
• Yield intermediate after activation that will then bind to a thiolated ligand or the amino groups in the ligand being conjugated
• Can utilize cross-linkers that serve as spacers between the bound ligand and the particle
• Serve to expand the repertoire of molecules capable of being conjugated to magnetic beads

SpheroTechnical Note #20 will provide the following information:

• An introduction to the hydroxylic beads and their benefits
• A protocol for the utilization of a maleimide-and-isocyanate crosslinker for attaching hydroxyl magnetic particles to compounds with sulfhydryl groups
• A protocol for the activation of hydroxyl magnetic particles using imidazole carbamestes for the spontaneous binding to proteins
• A protocol for the activation of hydroxyl magnetic particles using disuccinimidyl carbonate to create an NHS-carbonate particle

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Streptavidin is a protein (MW of approx. 66,000) made up of four identical subunits, each containing a high-affinity binding site for biotin (KD = 10-15 M). It has the same biotin-binding properties as avidin, but less non-specific binding is observed. It has been used both in immune assays and genomic assays for target detection.

Spherotech Streptavidin bead surfaces are designed as a matrix for simple and efficient methods such as:

• Protein-coated beads for the isolation of biotinylated compounds such as proteins, immunoglobulins, sugars, lectins or DNA/RNA and microRNA
• Magnetic streptavidin-coated beads can be used as substrates for both immune assays and genomic assays
• Small fluorescently labeled streptavidin particles can be used as probes for detection

SpheroTechnical Note #21 will provide the following information:

• Instructions for the preparations of SPHERO Streptavidin Particles before use
• Optimized biotinylation protocols for various ligands in order to facilitate optimal binding to An introduction to SPHERO Streptavidin Coated Beads capture antibodies with conjugated fluorophores to provide detectable signals
• Protocols for the immobilization of biotinylated nucleic acids, antibodies, and PEG-biotins
• Methods to quantitative the amount of biotinylated DNA after binding to SPHERO Streptavidin Coated Beads

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Since the 1950s, latex-based immunoassays have been used in clinical laboratories. The first described latex agglutination assay was developed by Plotz and Singer as a Rheumatoid Factor test in 1956. Currently, in the life sciences immunoassays are used for detecting different proteins, hormones, and antibodies.

Spherotech Goat anti-Mouse bead particles are designed for:

• The improvement of Sandwich Assay development by optimizing the orientation of monoclonal antibodies for site-specific immobilization of antigens
• Reducing the complexity of Magnetic Immunoprecipitation using Goat anti-Mouse IgG

SpheroTechnical Note #22 will provide the following information:

• Information on how to use Spherotech antibody coated beads in sandwich immunoassays
• Protocols for developing a bead-based assay
• Protocols for washing Spherotech antibody-coated beads and coating with monoclonal antibodies
• Protocols for the Magnetic Immunoprecipitation using Goat anti-Mouse IgG (Fc) Magnetic Bead
• Protocols for regeneration and reuse of Spherotech antibody-coated beads

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Spheroteh Goat anti-Mouse Magnetic beads coated with a primary mouse IgG antibodies are ideal for isolation of cells from different species (e.g. human, rat) depending on the specificity of the primary antibody. Cells can be directly isolated from any sample, such as whole blood, bone marrow, MNC suspensions, or tissue digests.

SpheroTechnical Note #23 will provide the following information:

• Information on how to use Spherotech antibody-coated magnetic beads for cell isolation using positive and negative selection techniques
• Details on the handling, washing, and storing magnetic beads used in cellular isolations
• Protocols for the direct and indirect isolation techniques
• Recommendations to improve the success of cell isolation

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