| Q: What is toxicity of beads in relation to cells and cell culture? |
| A: The concern about toxicity here is that the cells can take the particles in via either phagocytosis or endocytosis (two separate cellular mechanisms for taking in particles of various sorts). Once taken in things go into endosomes and these initial membrane bounded vesicles fuse with a lysosome forming a phagolysosome. In these vesicles things get digested by various enzymes. My guess is that particles would pretty much persist because the polymer bonds are not attacked by the sort of hydrolytic enzymes found in mammalian cells. Whether this would be toxic is hard to predict. There was a study about 12 years back by a group at IU Med center on toxicity of cross linked PS particles in rabbits. This work was about drug delivery studies. There was no toxicity demonstrated. |
 |
| Q: Assay design for a quantitative turbidimetric small hapten (700 MW)? |
| A: Coupling of a peptide via the SH of cysteine directly to the particle surface would require an amino particle. We have in the past converted CML to AML by hexanediamine (HDA) via EDAC. This can give a fully converted amino particle. then one would use SMCC (Pierce Cat. no. 22322) to link peptide sulfhydryl to particle amino.
The alternative route is to prepare an albumin conjugate of the peptide using SMCC and then couple the resulting protein conjugate to a regular CML with direct EDAC protocol. For a detailed conjugation procedure see Hermanson, G.T., Bioconjugate Techniques; Academic Press1996, pp. 439 to 446. this gives detailed procedure including monitoring of the amount of incorporation of the peptide. The particle coupling may be done exactly from the Manual with direct EDAC protocol.
The protein conjugate route is highly preferred. Protein conjugates of haptens are invariably more sensitive than directly coupled haptens. Controlling the colloidal stability of sensitized particles is far easier with albumin conjugates than with direct hapten. Albumin both stabilizes the particle against autoagglutination and presents the hapten in highly reactive form. Albumin is certainly acting as a large scale spacer.
Optimization of the assay should include preparing a conjugates over a wide range of peptide: albumin and then coating each of the conjugates at several protein levels. Obviously, the amount of antibody must also be co-varied. Select particles of 0.2 to 0.3 µM diameter with Parking Areas of 20 to 80. There is a lot of titration required but it can be done surprisingly quickly.
These recommendations are based on actual experience with hapten assays |
|
| Q: What particle diameter should I use for my slide agglutination assay? |
| A: For slide agglutination assays, it has long been considered optimal to use particles with diameters of 0.8 to 1.0 uM. The rationale for this belief is that larger diameter particles will form visible agglutination with fewer crosslinking events. Empirical evaluation of diameter effects in slide agglutination reveals that in fact smaller diameter particles give higher sensitivity than larger particles. We have performed very sensitive slide agglutination assays using particles with diameters of 0.15 uM to 0.30 uM. It seems very likley that smaller diameter particles are more reactive due to the intense Brownian motion associated with smaller diameter particles.
Small diameter particles (less than 0.4 uM) if maintained as monodisperse preparations will remain stably suspended. This eliminates the inevitable settling of larger (0.8 uM to 1.0 uM) diameter particles. Note that many slide agglutination assays have clumped particle suspensions. This will not improve sensitivity and will necessitate mixing of the particles before use. Given all these considerations, the best design for slide agglutination assays is to use particle diameters of about 0.3 uM and maintain the final sensitized reagent as a monodisperse suspension. This will give the maximum sensitivity and the best differentiation positive and negative readings. |
|
| Q: What is the best wavelenth to use for a turbidemetric assay? |
| A: Particle agglutination assays for quantitative determinations can be very sensitive and specific. In general, sensitized particles are reacted with a sample to undergo a degree of agglutination that is proportional to the concentration of analyte in the sample. Particle agglutination is accompanied by a change in the amount of light scattering. Increased light scatter may be detected in spectrophotometers as in increase in absorbance. The maximum amount of light scattering and therefore the maximum absorbance change is obtained when the scattering particles are about the same size as the wavelength of light being scattered. If the particles are very much larger or smaller than the wavelength of the scattered light the absorbance signal will decrease.
In order to take advantage of this relationship it is useful to use a wavelength that is two to three times that of the monodisperse particle diameter. So for particles of 200 to 300 nanometers, wavelengths of 500 to 700 nm should be chosen. For particles of 400 to 500 nm wavelengths in the near infrared should be used. The rationale for this relationship is that when particles aggluntinate the effective diameter of the aggregates should be about that of the wavelength chosen. This is achieved by empirically optimizing such factors as particle concentration, load of sensitizing species on the particle, concentration of reaction accelerators and reaction buffer pH. In performing this optimization the assay developer should try several wavelengths in the “2 to 3 times” range.
A final point on choice of wavelengths is that the choice of longer wavelengths avoids problems with spectral interference from hemoglobin, bilirubin and lipemia. At wavelengths of 600 nm and higher these components of serum samples will have little or no influence on test results. |
|
| Q: What antifoam agents can be used in particle reagents? |
| A: Antifoam agents are usually silicone compounds which are highly surface active. There is a great danger that any antifoam added to a particle suspension would immediately coat out on the particles. This would probably lead to autoagglutination. If antifoam were added to a buffer reagent (not containing particles) it should be compatible for the duration of the assay. Antifoam B or Antifoam C available from Sigma are good general purpose antifoam agents. Incidentally, antifoams can readily grow microorganisms and are frequently contaminated so I would check the material used and store concentrates in the refrigerator. |
|
| Q: What is the best to clean and wash small diameter particles? |
| A: The best way to clean and wash small particles is with tangential flow. Our recommended membrane is from Spectrum Laboratories, Inc. in Rancho Dominquez, CA. You can reach them at 310-885-4600 or at the web site, www.spectrapor.com. The membranes are sold under the trade name MiniKros. These are available in a range of sizes appropriate for volumes ranging from milliliters to liters. Pore sizes are readily available at 0.1 µM and these have been widely used by major diagnostics companies.
Particles around 0.1 µM may be pelleted readily in floor model centrifuges at speeds of about 18K for times of 20 to 30 minutes. This is readily done with Sorval or Beckman Centrifuges. Volumes of 2 ml to 20 ml per tube may be readi8ly processed using round bottom centrifuge tubes designed to deal with the relatively high speed. Note that this speed is below that of an ultracentrifuge.
During assay optimization it is preferable to use centrifugation in order to process numerous tubes in parallel. Once scale up has begun tangential flow becomes the preferred method. It is necessary to use ultrasonication to remix pellets after centrifuging. Tangential flow also causes clumping of microparticles due to the high shear forces involved. It is therefore advisable to use sonication during tangential flow processing. |
|
| Q: Which system can be use for conjugation of synthetic peptides, particles or conjugatiuon? |
| A: Use streptavidin particles to bind through the biotin. This will work. Direct coupling in this case would be very inefficient because the peptides probably adsorb inefficiently. Biotin would not only put the peptides on but would do so in an oriented manner. |
|
| Q: I've used your recommended coupling protocol and I'm not getting any binding of my antigen to the particle. |
| A: In questions like this it is critical to differentiate between chemistry and physical phenomena. When a coupling reaction does not produce the desired activity it is usually assumed that no ligand bound; a chemical explanation. Assuming the antigen is a protein it is useful to perform a BCA assay for the amount of bound protein. If the protein is "there" by BCA assay but you are not getting the desired activity then the problem is almost certainly one of colloidal balance; a physical phenomenon. If a particle system is too colloidally stable there will be little or no activity whether the assay is agglutination or membrane based. The most successful optimization strategy is to couple at multiple loads of the ligand, measure the amount bound with the BCA Assay and then optimize the colloidal balance across this initial panel of samples. Colloidal balance may be altered by using acclerators such as high molecular weight dextran, altering the pH of the final reaction and adjusting the salt concentration. All these things are manipulating the physical parameters of the reaction. |
|
| Q: Do I need to use a linker to bind my ligand to the particle to avoid steric hindrance issues? |
| A: Linkers usually imply a low molecular weight compound such as a heterobifunctional linker with molecular weight below 1000. In coupling to noadsorptive solid phases linkers have proven value by increasing the accessibility of reactive sites on the protein. Hovever, linkers are of no value in coupling to polystyrene microparticles. Proteins bind to PS MP by adsorptive interactions . This is true even with covalent coupling protocols. Adsorption happens very rapidy and covalent interactions follow with slower kinetics. Thus, it is the rapid adsorptive interactions which determine the orientation of a bound protein. Note also that the size of typical linkers is very small relative to any protein that is being coupled to a microparticle. So the belief that a linker will act as a so called spacer arm is incorrect. The use of albumin as a carrier for haptens such as drugs of abuse or monitored drugs does have value as a spacer. Here a large molecule, albumin, presents a much smaller molecule, the drug. In this case the spacer effect is real. |
|
| Q: Is it better to adsorb or covalently link proteins to the surface? |
| A: Although adsorbed preparations are capable of excellent stability covalent is very much to be preferred. With adsorbed preparations it is necessary to be very cautious about additives for adjusting the reactivity and preventing autoagglutination. For example, as shown in the Microparticle Optimization Manual, detergents such as Tween 20 can cause considerable desorption from the particle. Even salt can cause desorption during storage. With covalent coupling there is much greater freedom in the formualtion. In one case where I directly compared the sensitivity of an assay for a hapten I was able to get much better sensitivity of detection and better stability with covalently couple reagents than with adsorbed. |
|
| Q: How can you couple both antibodies and oligos to the same particle? |
| A: The easiest approach is to use biotinylated antibodies and bioinylated oligos to accurately bind them to the particles in appropriate proportions. |
|
| Q: What parking area to use when covalent coupling antibody to the particle? |
| A: Clearly in RF the PA made a big difference, but one must remember that it is the Fc-portion not the Fab portion that is critical in the assay. RE uses a PA of 12, most individuals I've talked to want 50 or greater. I don't know if they truly understand PA or if they're following someone's work. |
|
| Q: What are the dark blue things in the blue latex? Are they hurting anything? How do
we get rid of them? |
| A: These questions usually come from in-house. We have improved the leaching of dye bys team stripping the particles after dyeing. I wonder whether an alk/heat treat would improve it more, noting that the dye is pushed into the particle with heat, but is allowed to cool without a wash. |
|
| Q: Questions on co-coupling of antibody and protein usually BSA together in one step. |
| A: I have experience doing this with adsorbed and covalent methods, frankly neither works well. |
|
| Q: Why does the package insert recommend putting line diluent between Cal F and the first control sample? |
| A: The line diluent was put in place, only as a precaution because the low control is immediately following a high calibrator. A sample carryover study was performed per recommendations in the TDx user’s manual. This study did not show any trends in samples carryover. I there concerned about carryover, each lab should individually perform the sample carryover study. |
|
| Q: How much sample evaporation error is created if a carousel of pretreated samples sits in the instrument for 5-15 minutes? |
| A: Evaporations studies indicate up to 10% over recovery of controls when allowed to sit for 5 minutes on the analyzer and up to 20% over recovery of controls when allowed to sit for 15 minutes. |
|
| Q: Why do I keep getting INSUFFICIENT SPL errors? |
| A: It has been observed that with some TDxs, pipetting 300ul into the sample cup will result in this error. It may be necessary to pipette 320ul for the extracted calibrators and controls into the sample cup.
This also happens if the probe positioning is off. In addition to increasing sample volume, it may be necessary to perform the following on the TDx; Z-Boom Calibration (Test 3.5), Dispense Check (Test 6.3) and Boom Cal (Test 3.2).
|
|
| Q: Why do I keep getting LLS failures? |
| A: LLS fail is caused by a droplet of conductive liquid that connects the level sensing electrodes on the probe. There are many causes of this error; poor probe alignment may cause splashing when the probe dispenses liquid. Air in the diluent lines may cause splattering when the probe dispenses. The probe tips are generally coated with Teflon to make them hydrophobic, if they are damaged or dirty, they will lose the ability to repel aqueous solutions. This assay is a bit more sensitive to LLS failures since the sample matrix is not very viscous, it is more likely to suffer from splashing than a serum based assay. |
|
| Q: Is there any recommended validity criteria suggested for agreement between duplicate patient specimen measurements as there is for calibration duplicates? |
| A: Samples with CVs greater than 20% should be re-assayed. |
|
| Q: We notice that the Calibrator values are value assigned and will thus vary from Lot to Lot. What is the impact of customers forgetting to reprogram new Calibrator values into the instrument? Will Calibration typically fail due to poor curve fit and how will Controls be affected? |
| A: Current specs allow for ±15% deviation from nominal for calibrator assigned values. If the incorrect values are programmed into the instrument, the control recovery will likely fail ranges. It is less likely that calibration will fail. The calibrator and reagent package insert emphasize the need to re-program values into the TDx with each new lot of calibrator. There is also a calibrator value card that is supplied with each new kit of calibrator to help remind the lab technician to check the values on the TDx. |
|
| Q: Explain the qualification for the 2 weeks Calibration Curve stability ("depends on customer use"). Is this instrument run frequency or use or a switch to fresh reagent packs? |
| A: These reagents are rather sensitive to light and to a lesser degree, temperature. If a user runs a kit every day, he will be increasing the amount of time the reagent is exposed to light and elevated temperature. This could result in decreased curve stability. If a user batches this test or only runs once or twice a week, there is less time the material is exposed to light and elevated temperature.
Additionally, some users may improperly leave the reagents out of 2-8ºC storage (or uncapped) longer than is necessary and as such will accelerate the calibration curve instability.
|
|
| Q: How are your particles made? |
| A: Seradyn microparticles are made by emulsion polymerization. For plain polystyrene microparticles, the ingredients are water, styrene, buffer, anionic surfactant, and initiator. When mixed together, the surfactant emulsifies the styrene into the water phase. Upon heating, the initiator forms free radicals which cause polymerization of the styrene into polystyrene. The polymer chains are insoluble in water and form spheres, which are stabilized by the negatively-charged intitiator end groups. Carboxyl-modified (CM) microparticles are prepared similarly except for the additional ingredient of an acid-functional monomer. The carboxyl groups, provided by the incorporation of this monomer into the polymer chain, remain close to the surface of the particles. These groups, which can be adjusted within a wide range of carboxyl contents, control the surface reactivity and colloidal stability of the microparticles. |
|
| Q: What conditions will help reduce NSB to MG-CM particles? |
| A: In order to minimize adsorptive binding to Sera-Mag the suspending buffer
should be at elevated pH (for example, greater than or equal to 8)and
combine high salt (NaCl >= 150 mM) and nonionic surfactant (Tween 20 0.1 %).
The composition of the SA MP suspension buffer is 50 mM Tris, pH 8.0; NaCl
150 mM; Tween 20 0.1%. This combination gave quite low adsorptive binding
of serum proteins to Sera-Mag particles. |
|
| Q: What can I do to prevent NSB? |
| A: Nonspecific binding(NSB) in microparticle assays must be managed at three levels: choice of appropriate particle, blocking agents focused on the particle and additives in the reaction mixture. In choosing the particle the carboxylate modified microparticle (CM MP) is by far the most versatile. Choose a particle of appropriate diameter, see the relevant notes for diameter choice, and with different carboxyl densities. At a minimum choose a low and high acid particle. Blocking agents may be employed most easily with covalently bound ligand. Protein blockers may be bovine serum albumin (BSA) or fish skin gelatin. The nonionic detergents Tween 20or Triton X100 may be used with the particle reagent. Finally, additives may be added in a reaction buffer to prevent unwanted interactions with the particle. This is most useful in quantitative assays where there is a particle reagent and a separate reaction buffer. See the note about blocking for more details. |
|
| Q: What should I use to block with? |
| A: The concept of blocking was developed in solid phase assays such as the ELISA format. The assumption is that blocking agents such as as inert protein or detergents will bind to unoccupied areas of the solid phase and prevent unwanted sample interacitons. Proteins used for blocking have included bovine serum albumin (BSA), gelatin, ovalbumin and casein. Surfactants most commonly used are nonionic surfactants such as Tween 20 or Triton X100. In applying blocking to polystyrene microparticle (PS MP) reagents it is important to take into account the colloidal balance of the reagent. Ovalbumin and casein binding to PS MP causes clumping due to loss of colloidal stability. Bovine gamma globulin is widely used in assays for specific antibodies but is not useful in Microparticle assays due to the profound loss of colloidal stability. BSA and fish skin gelatin are the two preferred blocking proteins for use with PS Microparticles. Bothe these proteins have haigh charge density that will help to stabilize PSD MP. Tween 20 or Triton X100 may be used with PS MP reagents provided that the reagent is covalently bound, see the note about Covalent vs. Passive binding of reagents. There is another aspect to blocking that is illustrated by Human Antimouse Antibody (HAMA) interference. HAMA interference is most efficiently eliminated by adding soluble mouse IgG to the reaction mixture. In this case the rationale is to bind a decoy to the interference in order to prevent it binding to the particles. |
|
| Q: What strength magnet should you use for magnetic particle isolation? |
| A: We recommend the use of samarium cobalt or neodymium magnets. Magnets for Sera-Mag particles should be the rare earth type to get maximum response. All the commercially available magnetic racks for use in biotechnology will work well with Sera-Mag. When working in a "miniprep" mode, it is useful to use a microcentrifuge to pellet the particles. Since Sera-Mag has high density, the particles may be pelleted in 1 to 2 minutes. The pellets will remain tight for prolonged times permitting easy washing and processing of reaction in batch mode. When a magnetic rack is used the tubes must be kept in place during processing to prevent disturbing the pellet. |
|
| Q: What is the amount of dye loading for Color-Rich dyed particles? |
| A: Dyed particles have approximately 8 to 10% (w/w) dye loading |
|
| Q: What is the pI of the beads, in otherwords at which pH are the beads neutrally charged
neutrally charged? |
| A: An aliphatic COOH would be expected to have a pK of about 4 but groups on particle surfaces will have a broad range of pK due to degree of hydration. That is why potentiometric titration of CML gives a broad transition rather than a sharp inflection as in the titration of acetic acid with strong base. We have never done potentiometric titration of our magnetic but I bet it will be a broad distribution. Given that I would guess that the pI is somewhat high; say in the 5 to 7 range |
|
| Q: How do I resuspend the particles? |
| A: Microparticles with diameters greater than about 0.4 micron tend to settle upon prolonged storage, because the Brownian motion of the particles in the fluid is not sufficient to overcome the constant pull of gravity. If particles have settled, they may be resuspended by swirling or rolling the container, or by sonication. Agitation strong enough to produce foam should be avoided. Polystyrene microparticles with diameters over 0.6 micron should be resuspended monthly to prevent irreversible aggregation. |
|
| Q: What is the stability? |
| A: Stability of microparticle reagents may have different meanings and must be defined clearly. Colloidal stability refers to the tendency of particles to aggregate spontaneously. Particle suspensions with low colloidal stability tend to spontaneously aggregate and may show nonspecific agglutination during a test. Particle suspensions with high colloidal stability will remain monodisperse during storage but may show low reactivity. Adjusting the colloidal balance between these two extremes is part of the art of developing microparticle based assays. Stability may also refer to maintenance of activity over time. This is seen when a microparticle reagent is functionally tested over time. A critical factor in maintaining the activity of a microparticle suspension is the control of shedding of the bound ligand over time. |
|
| Q: What is the recommended storage temperature? |
| A: The original bottle of microparticle fluid should be stored at +2 to +8 degrees C to prevent microbial contamination. Once opened, additional precautions should be taken to prevent contamination, including the following: pour from the bottle instead of repeatedly pipetting; make a stock solution to work from; prepare working aliquots; keep the bottle closed when not in use; keep the bottle refrigerated. |
|
| Q: What is the surface chemistry of the particle? |
| A: The polystyrene microparticles have a negative surface charge derived from sulfate initiator end groups and adsorbed anionic surfactant. Carboxyl-modified microparticles have additional negative charge from bound carboxyl groups, and thus are much more hydrophilic. When using microparticles, note that colloidal stability will be reduced by any additive that lowers the net charge on the particle surface. |
|
| Q: Which way is the oligo(dT)14 bound to the particle, 5' or 3'? |
| A: Seramag oligo (dt)14 is covalently bound to the particle via the 5' end. |
|
| Q: What is PA? |
| A: Parking area (PA) is the average surface area containing one functional group, calculated from the diameter of the particles (in microns) and the titrated milli-equivalents (of acid, sulfate, hydroxyl, or amino groups) per gram of polymer solids. The higher the PA value, the fewer functional groups will be found on a given area of particle surface. For particles with carboxyl groups, a low PA (below about 30) indicates a hydrophilic surface with many COOH groups available for covalent coupling. A high PA (above about 60) means a more hydrophobic surface with less coupling capability. |
|
| Q: What PA particle should I use for turbidimetric assays? |
| A: Carboxyl Parking Area (PA) should be treated as an explicit experimental varialble in assay optimization. Choose particles with same diameter and two to three different PA. Good choices for initial experimentation are Low (20 to 30), Medium (40 to 60) and High (80 to 120). |
|
| Q: What PA particle should I use for lateral flow assays? |
| A: Carboxyl Parking Area (PA) should be treated as an explicit experimental varialble in assay optimization. Choose particles with same diameter and two to three different PA. Good choices for initial experimentation are Low (20 to 30), Medium (40 to 60) and High (80 to 120). Lateral flow assays are affected by colloidal factors in the same way as turbidimetric assays. That is, Low PA (high acid) particles will tend to be more colloidally stable therefore more difficult to capture. The final colloidal stability of the sensitized microparticle depends on the ligand that is coupled and the impact of the buffer formulation. |
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