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Why is it important to purify my antibody from free amines such as sodium azide or tris buffers when performing a covalent conjugation?

Covalent conjugation with our carboxyl and NHS nanoparticles uses Sulfo-NHS esters that couple rapidly with amines on target proteins. Having other free amines in the reaction will compete with your target molecule for binding sites on the nanoparticle. We recommend purification using Amicon Ultra centrifugal filters as a quick and easy way to purify and perform buffer exchanges (click here for a link to our antibody purification protocol). 

My antibody is already purified, do I really need to purify it again?

Many antibodies are purified using protein A or Protein G affinity columns.  During this purification process, TRIS is often used to elute the antibody off of the columns, and may be present in varying amounts.  We strongly encourage purification of antibodies to remove any possible sources of primary amines which interfere with covalent conjugation.   

What should I purify my antibody into, and at what concentration?

Typically, 1× PBS or 10 mM potassium phosphate at pH 7.4 are suitable antibody purification buffers.  Always check the certificate of analysis from the vendor, and contact them if the buffer the antibody provided in is different, as there may be a reason the antibody is provided in a unique buffer formulation (e.g. increased stability).  In general, proteins are more stable at higher concentrations, so we recommend storing your antibody at a concentration greater than 1 mg/mL. 

What if my antibody is provided with BSA or glycerol?

It is best to avoid antibodies that come in a storage buffer with BSA or glycerol.  If that is the only option, some vendors are able to provide antibodies without these additives on a custom basis. 

What are the advantages of carboxyl nanoparticles over NHS nanoparticles?

The NHS nanoparticles are a great tool for quickly evaluating antibody pairs – especially for small scale “proof of concept” studies, or in lateral flow where it is critical to pair antibodies on a strip in order to mimic appropriate kinetic conditions.  However, the NHS nanoparticles are limited by scale.  The semi-stable NHS-ester moiety hydrolyses in water. We rely on a quick lyophilization of the particles to “pin” the NHS ester reactivity.  Performing this process with large volumes of material slows down the process, and reduces the amount of active NHS-ester on the surface of the particles.  

What are the advantages of carboxyl nanoparticles over carbonate nanoparticles (covalent conjugation over passive adsorption)?

Covalent conjugates are more stable than conjugates prepared by passive adsorption because the amide bond is permanent, and the antibody won’t dissociate over time.  This is especially useful when working with challenging sample matrices, which require high concentrations of detergents or surfactants to reduce sample variation and increase availability of the target.  Additionally, the covalent coupling procedure is not dependent on the isoelectric point of the antibody, removing the need for extensive pH sweeps saving time and reducing costs.  Furthermore, the amount of antibody required per unit particle for covalent conjugates is often less than that required for passive adsorption. 

Covalent conjugation is more reproducible compared with passive conjugation and it is easier to control the amount of antibody you are attaching to each particle.  Limiting the number of antibodies on the conjugate can help to modulate the dynamic range in a competitive format assay.  Limiting the number of antibodies while still producing a stable conjugate can be incredibly challenging with passive adsorption methods, and is much easier to control and reproduce using covalent chemistry. 

Why is the buffer selection important when performing covalent conjugations?

While the conjugation pH is not dependent on the isoelectric point of the specific antibody, the pH for covalent coupling is still greatly important.  The activation with EDC and Sulfo-NHS is most efficient at pH 4.5-7.2.  Covalent amide bond formation, or reaction of sulfo-NHS-activated molecules (NHS ester is the semi-stable intermediate formed during EDC/NHS coupling) is most efficient at pH 7-8.  NHS esters have a half-life of 4-5 hours at pH 7, 1 hour at pH 8, and only 10 minutes at pH 8.6 

Unconjugated gold nanoparticles are not particularly salt stable, so it’s important to handle them in a low-salt environment until they are protected and stabilized by a protein.  Proteins are made up of amino acids, and each protein is unique in how it folds and unfolds in different buffer and salt environments.  When optimizing conjugations, we recommend empirically testing a few different buffer formulations at pH 7.4 to determine the optimal conditions for amide bond formation for each protein. 

Do you recommend using MES buffer for activation?

MES buffer is often used in activation protocols for Latex and fluorescent beads to adjust the reaction to pH 5.  nanoComposix’s BioReady materials are provided in a mildly buffered aqueous solution that adjusts to pH 5 upon addition of EDC and sulfo-NHS.  It is not necessary to activate BioReady materials in MES buffer. 

How can gold nanoshells increase sensitivity in lateral flow?

Our 150 nm gold nanoshells are 30× visibly brighter per particle than traditional 40 nm gold used in lateral flow.  Because they have been engineered with a silica core, they are half as dense as a solid 150 nm gold particle and flow easily through a nitrocellulose membrane.  It is important to note that for any OD per volume, there are about 30X less nanoshells by particle number, so conjugate volumes will need to be adjusted appropriately) to maximize binding events. As a starting point, increasing OD or conjugate volume per strip two-fold will give you a boost in sensitivity. 

What if I see false positive results?

When a test line is visible in the absence of the desired analyte, the false positive result may be caused by a number of factors such as non-specific binding, cross-reactivity, or heterophilic antibodies.  In order to optimize the assay and eliminate the false positive result, it is important to understand which of these factors or combination of factors is giving rise to a false positive result. Non-specific binding occurs when there is a non-specific interaction between the antibody-nanoparticle conjugate and the antibody at the test line, regardless of the presence or absence of the target analyte in the sample.  Non-specific binding can often be overcome by simply optimizing the conjugation procedure for a particular protein.  Conjugation optimization includes optimizing the antibody/protein loading (too little or too much antibody can lead to non-specific binding), optimizing antibody incubation time, and optimizing the reaction buffer.  Blocking agents such as proteins, surfactants, or polymers  can  be incorporated in a component of the test strip (e.g. conjugate diluent, sample pad pre-treatment, conjugate pad pre-treatment, running buffer, etc.). 

Cross-reactivity is different than non-specific binding and occurs when the antibody has an affinity for an analyte in the sample that is NOT the target analyte. This issue is more difficult to address, and usually will result in the need to change antibody systems that do not have cross-reactivity to unwanted analytes. 

The presence of heterophilic antibodies (endogenous antibodies that bind assay antibodies) in a sample can result in a false positive result. There are multiple types of heterophilic antibodies that can cause a type of cross-linking between the antibody conjugated to the nanoparticle and the antibody at the test line, even in the absence of the target analyte.  To test if your sample contains heterophilic antibodies, perform a serial dilution of the sample.  If the false positive result remains the same signal intensity even after diluting the sample instead of showing a linear decrease in signal intensity, it may be due to heterophilic antibodies. To prevent heterophilic interference, heterophilic blocking reagents are commercially available (http://scantibodies.com/hbr/), or a mouse IgG conjugate can be added to the assay to sequester- human anti-mouse monoclonal antibody (HAMA) if that is determined to be the cause of the non-specific binding.

What do you recommend as a blocking agent to eliminate non-specific binding?

Blocking steps will need to be optimized for each specific assay system.  We often start with a 1-2 hour block in 4 mM sodium tetraborate with 1% BSA at pH 8.2.  There are countless options available both using proteins and synthetic blocking reagents such as casein, fish skin gelatin, sea block from East Coast Bio, and synthetic blocking reagents from Biolipidure. 

Why do I see decrease of test and control line when switching from “clean” (analyte spiked in buffer) system to a real sample (saliva, whole blood, etc.)?

A decrease or loss of a test and control line when switching from a “clean” system to a clinical sample is observed in many cases and may be due to the many additional components that exist in the clinical sample such as proteins, salt, or additional metabolites or molecules. The addition of blocking agents such as proteins, surfactants, or polymers into the conjugate diluent or conjugate pad pre-treatment buffer can help recover the signal intensity. 

What other factors can influence conjugation results?

If running under the correct pH conditions and the antibody incubation time has been optimized, confirm that EDC and Sulfo-NHS has been stored properly and that they are prepared in solution just prior to conjugation. EDC should always be stored at -20°C and Sulfo-NHS between 4-8°C.  It is important to allow reagents to come to room temperature prior to opening the bottles to avoid condensation from the atmosphere as both EDC in particular and Sulfo-NHS are moisture sensitive.  For preparation, we recommend bringing bottles to room temperature for ~45 minutes before opening vials, weighing out a precise mass into a microcentrifuge tube, and then dissolving into a volume of water immediately before adding to the colloid solution. If the cold powders are exposed to moisture in the atmosphere, they may work immediately, but will degrade before you are able to use the reagents in subsequent experiments.  Improperly handled EDC is one of the biggest culprits for an unsuccessful covalent conjugation. 

Are your particles tolerant to detergents such as DMSO or tween?

After covalent conjugates are prepared, they are very stable with almost all detergents and polymers commonly used in bioconjugation applications.  Detergents at concentrations up to 1% are typically fine for covalent conjugates which is higher than what passively conjugated gold will usually tolerate. 

WHy is there PEG in the reaction buffer, is it necessary?

The PEG is inert and helps provide steric protection during the centrifugation steps to help produce a more stable conjugate.  While we do recommend including PEG, it is not critical in preparing a functional conjugate.

What is 1× PBS?

PBS is phosphate buffered saline.  Saline is 0.9% NaCl solution, and formulations of PBS vary.  The PBS we use is a preparation which comes pre-weighed from Sigma Aldrich (1× PBS (Sigma Aldrich) cat #P3813).  Reconstitution of the contents of one pouch, when dissolved in one liter of distilled or deionized water, will yield 0.01 M phosphate buffered saline (NaCl 0.138 M; KCl - 0.0027 M); pH 7.4, at 25°C. 

Dulbecco's phosphate-buffered saline has a lower phosphate concentration than standard PBS. Some formulations do not contain potassium and magnesium, while others contain calcium and/or magnesium.  It’s important to be consistent with your formulation across conjugations as different salt concentrations can result in different outcomes in an assay.   

Can I just add an excess of antibody rather than performing an optimization?

In many cases, too high or too low antibody addition can lead to non-specific binding, and a decrease in sensitivity.  We often sweep the mass recommended in the protocol for a specific particle +/- 5 µg/mL antibody loading and empirically test negative and positive samples.  If one of the three loadings gives better performance, we will perform additional antibody loading experiment to fully optimize the particle/antibody ratio. For example, if a conjugate prepared with 20 µg/mL performs better than 25 µg/mL and 30 µg/mL loading in terms of lower non-specific and higher sensitivity, we might try an optimization closer to that range and examine 18, 20, and 22 µg/mL loadings. 

Are your particles salt-stable?

Unconjugated gold nanoparticles are not salt stable, but after successful conjugation, conjugates are generally stable in 1X PBS, and some customers have observed stability in much higher salt concentrations.  If your application requires a high-salt environment, we recommend preparing your conjugate at low salt concentrations, and then adding additional salt and monitoring the conjugate by UV-VIS.  A broadening in the peak and drop in optical density are indicative of instability. 

Why do you recommend specific tubes for covalent conjugation?

All plastics will contain residual monomer, catalysts, and additives that support the high temperature molding of the polymer, and also lubricants to release the plastic off of the mold.  Although these are present in relatively small amounts, they can and will leach to some degree into the fluids you put inside them.  The amount of leaching is dependent on many factors including time, temperature, the mobility of the additive in the polymer, and the interaction of the container contents with the polymer (solvent effectiveness).  The use of the wrong tubes is one of the most common problems we discover when working with clients doing lateral flow covalent conjugations. 

Some of these residuals interfere with EDC and sulfo-NHS chemistry, and will cause the gold to plate out onto the tube or reduce the sensitivity of the assay.  We recommend the use of tubes manufactured by LABCON, sold by VWR or other vendors. 

Lo-bind tubes are pre-treated specifically to not bind protein.  If you are conjugating a protein you have found to be “sticky” we suggest activating the gold nanoparticles and performing the centrifugation step to remove excess EDC and sulfo-NHS in the recommended tubes, and then transferring the activated nanoparticles to a lo-bind tube prior to adding your protein.  

There is a lot of background color when I run the conjugate in a lateral flow test, are your gold nanoshells too large to flow through the nitrocellulose?

No, nitrocellulose membranes were designed to perform with latex beads which are much larger than our gold nanoshells.  Even slow membranes such as Millipore HF180 have >10 micron pore sizes.  We have tested the gold nanoshells on membranes from Millipore, Sartorius, GE/Whatman, and mdi and haven’t found any membranes that our gold shells are incompatible with or won’t clear, however some do perform better universally.  We have had a lot of success with nitrocellulose membranes from mdi. For a slower/more sensitive membrane, MDI 10µ-CNPF is a good starting point with a read time of ~20 minutes. If you’re looking for a faster option, MDI 70 or 90 CNPH works well.

When encountering problems with high background or a slow clear-rate, we often incorporate detergents such as tween 20 or surfactant 10-G into the running buffer, or a pre-treatment of the sample pad.  Our first-pass running buffer is 1× PBS or 0.9% normal saline with 1% tween 20. 

The image below of nanoshells bound to a nitrocellulose membrane shows the nanoshell size compared to the size of the pores in nitrocellulose.  

Nanoshells on Nitrocellulose for Lateral Flow Assays

Do you perform custom conjugations or assay development (consulting)? 

Yes!  Please contact us regarding our custom capabilities and collaboration efforts.