Focus Simplexa NATtrol

They represent the preferred industry standard for molecular diagnostic testing and can be used as independent (third party) quality control materials. NATtrol products are prepared from purified microorganisms that are grown in cell culture, in microbial culture, or isolated from the plasma of infected individuals.

NATtrol treatment modifies surface proteins and makes organisms non-infectious and stable in the refrigerator while retaining the complete genome. Inactivation of the organisms is verified by the absence of growth invalidated infectivity tests based on tissue cultures or in validated growth protocols (as appropriate)

  • Non-infectious and stable in the refrigerator
  • Purified and intact organisms
  • Comprehensive process controls to monitor extraction and amplification steps
  • Available in different formats (one or more analytes)
  • Can be used on various molecular assay platforms and analyzes
  • Traceable to WHO international standards (if applicable)
  • Shelf life of 12 to 24 months
  • Extensive coverage of patents * and/or pending patents

Frequent use of NATtrol controls can be used to:

  • Train and supervise laboratory staff
  • Assess lot-to-lot consistency of test kits and test reagents
  • Monitor daily variations in reported results
  • Provide an impartial and independent assessment of the competence of the tests
  • Provide consistent, reliable, and accurate quality control solutions
  • Help identify lab trends

3 COMMON PROBLEMS WHEN DOING AN ELISA

The ELISA immunoassays are among the most versatile to detect, identify and / or quantitate a particular antigen present in our sample. It is a highly sensitive, robust and relatively inexpensive test, quick and easy to carry out.

But despite the apparent simplicity of its protocol, as with most biological assays, there are times when the results are not as expected.

In this post we will focus on  3 common problems when doing an ELISA , and we will try to give some guidelines to be able to  solve them successfully .

Some of the common problems when doing an ELISA are obtaining a weak , low intensity or even non-existent signal , generating a high background noise that distorts the results, or the high inconsistency of the results between wells .

Next we leave you a list of Possible Causes (PC) and Solutions (S) for these 3 common problems when doing an ELISA.

1.- THE SIGNAL INTENSITY IS VERY LOW OR NON-EXISTENT:

PC: Problems with the protocol

S: The essay may not have been prepared correctly, and to fix it it is necessary to review several points:

  • Check that each step of the protocol has been applied correctly
  • Check that the wavelength and filters used in the plate reader are appropriate. For this, the test can be repeated using a positive control.

PC: Antibody problems

S: Antibodies are the most critical reagents in ELISA tests, and it is important to pay attention to the following points:

  • Use the dilution recommended by the manufacturer, or failing that, optimize the dilution of the antibody to be used.
  • Make sure you use enough antibodies, testing different concentrations of the antibodies to identify the optimal concentration for your assay.
  • Make sure that the antibodies have been stored correctly and have not undergone excessive freeze / thaw cycles .

PC: Problems with antigen

S: When the plates are covered with the antigen and when adding the primary antibody the signal is weak or null, there are two main aspects to check:

  • Sometimes it is possible that a sufficient amount of antigen has not adhered to the plate, try adding more.
  • In the case of peptide antigens, they must be previously conjugated to a large carrier protein in order to facilitate the recognition of the epitope by the antibody.

PC: Problems with reagents

S: Always make sure that:

  • The reagents are at room temperature before starting the assay
  • Check expiration dates
  • Check that they have been prepared correctly and have been added in the corresponding order.
  • Make sure that they do not interfere with other buffers or compounds present in the sample such as sodium acid, EDTA, etc.
  • Avoid mixing reagents from different kits.

PC: Low incubation times and temperature

S: Be sure to incubate the samples for the time specified by the manufacturer and optimize the incubation temperature for your assay. Remember that the reagents must be at room temperature before starting the assay.

PC: Storage conditions

S: Stores reagents to your specifications, keeping in mind that all reagents may not need the same storage requirements.

2.- THE BACKGROUND NOISE IS VERY HIGH

PC: Antibody problems

S: Regarding antibodies, there can be several cases:

  • That the concentration of antibody used is too high. This will be resolved by reducing its concentration, or by carrying out an optimization test to determine the ideal concentration.
  • There are cross reactivity problems, for which a negative control should be used.
  • Non-specific binding where the antibody is detecting other molecules besides the target antigen. The solution is through the use of affinity purified antibodies and the use of the appropriate blocking buffers .

PC: Problems with reagents

S: Regarding the different reagents, we must make sure that:

  • The stop solution is added to avoid an over development of the reaction.
  • The detection reagent has been properly diluted, and if so, retest with an even higher dilution.
  • The appropriate blocking buffers have been used and the blocking time has been sufficient.

PC: Incubation problems

S: In case of obtaining high background noise, it may be necessary to reduce the incubation temperature. On the other hand, it is necessary to ensure that the incubation of the substrate is carried out in dark conditions.

PC: Insufficient washes

S: Carefully wash the bottom of the plate and repeat the reading.

3.- THE VARIATION OF RESULTS BETWEEN WELLS IS VERY HIGH

PC: Inadequate incubation

S: Regarding the incubation step, it is important:

  • Do not stack the plates during the process to ensure that the temperature is evenly distributed.
  • Make sure there are no bubbles inside the wells before incubation.

PC: improper washing

S: Make sure to thoroughly wash all wells, as inhomogeneous washing could result in a high coefficient of variation.

 

 

How To Improve Protein Quality

When working with protein samples , there are critical factors that must be closely monitored to avoid their degradation or dysfunction and guarantee the reliability of the results.

These factors therefore reflect the quality of the proteins and can be basically summarized as:

  • Integrity
  • Purity
  • Homogeneity
  • Solubility
  • Stability
  • Storage

In this post we bring you some tips to improve the quality of proteins and thus optimize the results of the tests.

1.- OPTIMIZATION OF PROTEIN PURITY AND INTEGRITY

Optimizing the purity and integrity of proteins involves avoiding or minimizing contamination of the sample with impurities, modified proteins or degradation products, among others.

Some of the steps that can be taken to this end are:

  1. Change the purification protocol by modifying for example the washing and elution conditions or adding additional purification steps such as ion exchange chromatography.
  2. Modify the conditions of induction of protein expression
  3. Use a different cloning vector
  4. Use a different expression system

2.- OPTIMIZATION OF PROTEIN HOMOGENEITY AND SOLUBILITY

To prevent or eliminate the formation of aggregates and increase the solubility of the protein s interest, there are several points that can affect, such as:

  1. Always perform size exclusion chromatography as the last step in the protein purification process.
  2. Do not over-concentrate the sample, since the processes applied for this purpose usually induce the aggregation of proteins (Remember this entry about 5 techniques to concentrate proteins ).
  3. Modify the composition of the buffer in which the protein is located until optimizing the pH, salinity, additives, etc. best suited in each case.

3.- OPTIMIZATION OF THE STABILITY AND STORAGE OF PROTEINS

Regarding the stability and medium or long-term storage of proteins, it is common to think that freezing and cryopreservation is the ideal method. And although this is true in a large part of the cases, it is not always the case due to the intrinsic variability of each individual protein.

That is why it is advisable to study the stability of each protein of interest to identify the optimal storage method in each case, whether it be simple refrigeration, freezing, lyophilization, etc.

You can expand the information in this post on How to store proteins to avoid their degradation .