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Moby 06-22-2011 11:49 AM

Freezing serum in assay diluent?
Does anyone know whether serum samples diluted in assay diluent can be frozen and used for ELISA afterwards?

luisillo 06-22-2011 05:26 PM

Re: Freezing serum in assay diluent?
I've never done it before, but I think it's possible. However, I think it would be wise to freeze the diluted samples at -70C in order to prevent protein degradation.

Elisadeveloper 06-23-2011 12:15 AM

Re: Freezing serum in assay diluent?
In regulated studies, stabilities are established in any matrix type used.

Typically assay diluents contain stabilizers and blockers of non-specific binding which will prevent adherence or degredation of analytes. However, this is dependent on the diluent and the analyte.

One way to be sure, would be to prepare stability controls by spiking in analyte to serum, serum/assay diluent and assay diluent (ideally at concentrations equivalent to the low mid and high mid curve region) and store these in parallel to your serum samples. Assess these immediately after preparation to get a day zero value, and then after storage for your stability assessment.

Appropriate recoveries in the spiked samples would give you confidence in your serum/diluent samples.

danfive 06-23-2011 05:37 PM

Re: Freezing serum in assay diluent?
I wouldn't do it. First if its propietary buffer (from a kit) then you can't really be sure how it reacts over many hours. Secondary, best practice for proteins is not to store the diluted solution but the more concentrated sample. Dilute protein solutions tend to produce protein unfolding, the proteins with exposed hydrophobic regions then pair up in unnatural pairs and can even precipitate out of solution.

If you know your analyte and know that nothing (within normal working buffer) makes it precipitate or unfold--then you can certainly use that as justification for saving the diluted samples.

Also be certain to inactivate proteases, (PI cocktail).

danfive 06-23-2011 05:51 PM

Re: Freezing serum in assay diluent?
What's up with only 2 minutes for editing---that sucks.

Couldn't edit my previous post to include the following.

Regarding the dilute solution producing protein unfolding, hydrophobic regions aggregating and some of these interactions precipitating. Add Freeze thawing to the dilution, and you'll see the typical rec. protein tend to lose 10% protein to this type of precipitation (that is 10% loss at each freeze-thaw cycle). The mM conc. is reported to be the primary factory affecting this, very concentrated solution loses less, more dilute loses more.

Consider that since serum dilutions can be 1:60,000 to 1:100,000 (that I know of).

I hope this helps and gives you context for some basic rules of protein manipulation in the lab.

1. Concentrated protein solutions are better for storage.
2. Aliquot concentrated protein samples to avoid multiple freeze-thawing (Notice no reliance on super duper dilution buffers).

Elisadeveloper 06-24-2011 12:17 AM

Re: Freezing serum in assay diluent?
I concur dan...however, if the samples are precious and you need data, then simple stability assessments will answer your question for you. Serum, even at 100 fold dilution is still in the 1mg/mL total protein concentration range, however, higher dilutions that this may be problematic if the diluent itself doesn't contain protein stabilizer.
Proteases within the serum will be diluted by the same factor as the analyte, so the dilution per se will not require more protease inhibitors than the serum itself if the molecule is subject to proteolysis.

Zagami 07-18-2011 01:17 PM

Re: Freezing serum in assay diluent?
Proteins comprise an extremely heterogeneous class of biological macromolecules, what are often extremely unstable when not placed in native environments. To make more stable, the organism submits proteins to some chemical process (e.g., sialylating) by means this become over stable and resistant to stress conditions (variation pH, strength ionic, temperature and others) or enzymatic degradation, what cannot only be verified under normal conditions, what from intra- to extracellular compartments proteins passage, but also and particular way in pathological conditions which stress situations that’s the rule. When protein biological fluids (serum/plasma, urines, CSF, etc..) extracted by it environment, that it participates to keep stable to guarantee the structure-function, it is able not to correctly work, until loss activity, if is placed in an environment with non optimal pH or as result of the enzymatic action, or to lose solubility for molecular aggregation. Biological proteins during their purification are deprived by natural environment be it stabilizing lipids, lipoproteins and/or proteins. Further, as the protein is purified, its concentration usually far exceeds the in vivo concentrations or contrary in dilution process. Other factors such as sheer stress during concentration or dilution are co-solvents, pH, etc. may also influence the stability of the protein. On the inside protein structure, different stability is observed, in which primary and secondary showed bounds more stable that tertiary structure, which can be easily disgregated (denaturation) while handling "in vitro", that entail loss of the biological function. When protein is researched with immunometric method, using antibodies against tertiary structure (conformational epitopes), sample denaturation can determine underestimation for epitope target loss. Insofar, is to need guaranteed protein stability in it’s native complex structure, when must have determined it’s biological activity or to quantize it by means corformational epitopes detection. Probability to denaturation protein is proportionally increased at handling steps necessary for to complete determination process. At this handling preparation of the sample containing protein, it’s purification, and subsequent storage must be included. Besides preventing contamination from other laboratory reagents, the use of gloves prevents contamination from proteins commonly found on the skin, particularly proteases which may degrade the protein sample and destroy activity. Store of the protein purified or crude (multiproteins mixture of the biological fluids), it has to guarantee the maintenance of the original structural integrity and/or activity. The extent of storage “shelf life” can vary from a few days to more than a year and is dependent on the nature of the protein or of the protein mixture, and the storage conditions used. This conditions for storage become is optimize and are distinctive to each protein, also if is possible to suggest some general guidelines for protein storage and stability. For example, proteins can be stored in solution at 4C it requires a condition of sterility obtainable with physical (instrumental) or chemical (antimicrobial) methods, well as the addition of components that prevents the proteolytic degradation. This protein solutions may not be stable for more than a few days or weeks. By contrast, lyophilization allows for long-term storage of protein with very little threat of degradation, but may be damaged during the lyophilization process, with loss of the initial nominal value. Analyzing the various parameters of storage, storage temperature and protein concentration of the solution, result to be primary. The temperature, for which the best range is of 2-8 C, while the room temperature, conducts more often to the degradation and/or inactivity of the protein whose factor is almost always connected to the microbial growth. Temperatures of -20 or -80 C, is the more common form of cold protein storage, that needs a freeze-thaw cycle, process which it has its drawbacks. During the freezing process, intra-molecular protein crystals water is formed that can determine several protein damages, while during the thaw phase a series of structural modifications can be determined, also determine from the possible heating of the solution, that can compromise the molecular identity. Opposed to the temperature and the behavior of the concentration of the protein in the solution that shows an easier inactivation and loss when the protein concentration of the diluted solution results < 1 mg/ml, that is a value to the limit (1.4 mg/ml) for dilutions 1:50 of the whole serum (average total serum proteins 70 mg/ml) done in ELISA procedure. The high solution concentrations protein has the tendency to stabilize it in native structure, preventing protein to stick at inactive glass or plastic surface tube, what in such a way won't be brought away by the solution. From such considerations deduces that it is not possible to dilute a protein or proteins mixture, below certain concentrations, in water or in buffer (phosphate or Tris) and store in freezing state without causing structural-functional damages. To avoid such drawbacks is necessary to use as diluent a buffer solution with optimized pH and salt concentration, content : a) cryoprotectant : such as glycerol or ethylene glycol at concentration between 25-50%; b) anti-protease : that inhibits the cleavage action of the proteolitic enzymes that can to destroy the protein; c) antibacterial : such as NaN3, at final concentration among 0.02 and 0.05% (w/v), or thimerosal at 0.01 % (w/v) final concentration, or one of the Proclin series or Kathon; d) chelating agents of metals as EDTA at final concentration between 1 and 5 mM to avoid SH groups oxidation metals induced; e) reducing agent helping to maintain protein in reduced state (inactive) such as dithiothreitol (DTT) or 2-mercaptoethanol (2-ME) at final concentration among 1 mM that prevent the cysteine oxidation. This cocktail variedly combined and optimized is commercially available (e.g. from Pierce) that allow to extend the middle life of the diluted proteins, stored between +4 and -20 C. Nevertheless all try must have conducted, starting from this knowledges and that sample diluent furnished in the diagnostic kits doesn't possess the chemical characteristics that allows a suitable stability of the proteins content of the sample diluted in freezing condition.

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