Diluting samples for proteomics – biomarker profiling case studies (I)

To dilute or not to dilute biological samples? And if yes, how much? That’s the question!

Following our post on whether samples should be pooled or not when performing studies for Biomarker discovery, it’s now time to discuss sample dilution (yes or no) and dilution factor (how much) when performing proteomic analysis of Biomarkers. [Read more…]

From RUO to IVD – the acronym guide to reagents’ intended use

Most of us use (and abuse) acronyms in chats and texts. Even in posts. Today, we will have a look at some acronyms used in research, and how they relate to the intended use of reagents. [Read more…]

Hyaluronic acid – size matters!

HA or Hyaluronan measurement | tebu-bio

Hyaluronan (HA) Competitive ELISA standard curve (cat. nr 117K-1200-1ea)

Hyaluronic acid (HA), or hyaluronan, is an ubiquitous, very high molecular mass polysaccharide that has applications in a variety of fields, including cosmetics, some types of surgery (e.g. opthalmic) and regenerative medicine. It can even be present as a contaminant in some bio-production processes. HA has also been suggested as a possible biomarker for Alzheimer’s disease (AD).

HA acts as a molecular shock-absorber and stabilizer for cells. Its visco-elastic properties, biologically speaking, are valuable for separating tissue and maintaining shape. It is a key in tissue lubrication, and it may play a role in wound repair. It is the ideal choice for some implants, as it does not usually cause an immune response (contrary to what may happen with some biomaterials). Size of the HA used in therapy has an impact on its success. Usually, higher weight forms usually render longer benefits. For bio-production, however, smaller HA forms are usually the main concern.

So, depending on the reason why you are studying this marker, keep size in mind in order to choose the best assay to measure the HA levels in your experimental model. It is of key importance that whatever product you use, analysis of the HA sizes detected with it are clearly mentioned in the technical documentation.

HA or Hyaluronan measurement | tebu-bio

Hyaluronic acid (HA) sandwich ELISA standard curve (direct assay cat. nr 117K-4800-1kit)

A post by my colleague Dr. Philippe Fixe is of great help for choosing the right assay!

It’s not that these assays will allow you to discriminate between high- and low-weight HA. What is intended here is that these assays will allow you to detect all or part of the HA forms depending on its weight. And that may be crucial, as you may be using the assay that does not detect the HA sizes relevant for your experiment.

It is also possible now to outsource HA measurements to an external lab performing regularly validated Hyaluronic acid-specific immuno-assays. As an example, Echelon’s Competitive or Sandwich HA ELISAs outsourcing by tebu-bio’s lab in cosmetology or drug discovery.

Any comments? Feel free to share them below!

 

Sample interference in ELISA tests

A previous post explained how samples obtained from patients treated with mAbs can show interference with WB & ELISA methods. More rarely, we can also find other interference effects caused by different factors.

Immunoassay interference is typically caused by non-specific interactions between antibodies within the assay itself and endogenous antibodies present within a patient’s sample. Any type of interference can cause an assay to report a false result. This can be avoided by using a blocking reagent, whicHTS - Blog Thumbnailh is a product designed to prevent interfering antibodies from adversely affecting immunoassay results. [Read more…]

WB & ELISAs using mAb-treated samples

Biologicals are increasingly being used in therapy, helping to cure thousands of patients, or at least improving their quality of life.

Research continues to move forward in this area, as proved by 7 400 citations in 2014 only when using “biologicals” as a search term, or about 200 citations when using “therapeutic mAb”. Many research groups, especially those working on Oncology, continue to investigate on the mechanism of action of biologicals, specially mAbs, as well as on any possible resistances found in non-responsive patients.

For these mechanistic or functional studies, antibody-based techniques such as ELISAs, antibody arrays, flow cytometry, etc. are routinely performed, using samples from patients being treated with therapeutic mAbs. [Read more…]

6 Tips for a successful ELISA

ELISA are immunoassays widely used in biomarker detection and validation. They have been used in research and clinical settings for more than 40 years, and they allow to quantify, in a simple way, thousands of biomarkers. New ELISAs are being developed every day, either for new biomarkers or new species. They can also be used to detect contaminants in food, water or industrial processes.Raybiotech ELISA Kits by tebu-bio

Technology-wise, there are several types of ELISAs, depending on whether they are based on double (or sandwich) antibody detection, direct detection, competitive detection, etc. It is not the scope of this post to enter into details about the different formats that an ELISA can have, but if you think that you need this info, feel free to suggest that I do a post on that!

Nevertheless, the aim of this post is to give some hints on how to reach the best result for your ELISA tests, based on some troubleshooting experience with researchers and clinicians all over Europe. [Read more…]

Antibody pairs – the rebirth of a classic

Antibody pairs have been used for years to detect biomarkers in biological samples. They are a convenient and price-wise alternative to ready-to-use ELISAs, especially when they are used on a routine basis (for sporadic use, ELISA kits may be a better choice).

[Read more…]

Adipokine monitoring during Hunger and Satiety balance

Cytokines and growth factors that are produced by or have an effect on adipocytes and related peptide neuro-transmitters are central players in hunger vs. satiety balance and in eating behavior (1). Recent evidence shows that adipokines, and more generally mediators or indicators of inflammation (2), play roles in the development of insulin resistance (3), diabetes (4) and many other concomittant health problems associated with obesity, including hypertension, dyslipidemia and atherosclerosis (5).

[Read more…]

Host Cell Protein Analysis in Biologics

In a previous post, we looked at innovative methods to assess immunogenicity in biotherapeutics and biosimilars.

The objective though, is to have the least immunogenicity possible (exception made for vaccines). Some factors, especially those which are patient-related, cannot be always controlled but some can definitely be monitored during the production processes of biotherapeutics and biosimilars. Let’s take the case of  Host Cell Proteins (HCPs).

[Read more…]

New glycated hemoglobin antibody as long term diabetic biomarker

Glycation of proteins in blood circulation occurs naturally when circulating sugar is metabolized in the body. Glucose is known to attach to hemoglobin in the bloodstream to form glycated hemoglobin (HbA1c).

Glucose and Glycated hemoglobin (Bb1Ac) as diabetic biomarkers

Hemoglobin is a long-living blood protein complex (around 120 days). Such stability can be used to measure blood HbA1c and thus, indirectly monitor average blood glucose levels during a longer period of time than daily glycemia measurements.

A possible allegory would be to compare Hb1Ac measurements with a movie covering a period of several weeks (average hyperglycemia),  and glucose blood testing with instant photography (punctual glycemia with possible fluctuation over the day).

Glycemia and Hb1Ac measurements are complementary data. They can help diagnose, monitor and stratify patients with diabetes (eg. American Diabetes Association, World Health Organization…). It might also help in the design of personalized therapies. Diabetes.co.uk mentions 2 clinical studies which show that improving HbA1c by 1%, might reduce risks of microvascular complications by 25% for patients with type 1 diabetes or type 2 diabetes.

Direct ELISA with anti-HbA1c clone 75C9

Direct ELISA with anti-HbA1c clone 75C9

Immunoassays are robust antibody-based quantitative methods. They are used to measure glycated hemoglobin HbA1c concentration expressed as a percentage of HbA1c to total hemoglobin.

These immuno-assays require well characterized and reliable primary antibodies.

A new hybridoma clone (75C9) specific for HbA1c has been released.

This 75C9 clone has been validated to measure Hb1Ac as a diabetic biomarker by direct and sandwich ELISA and complements the clone Hb6 which reacts with both unmodified and glycated hemoglobin.

Want to develop hemoglobin-specific ummunoassays with reliable monoclonals?

HbA1c calibration curve with Hb6 and 75C9 clones in fluoro-immunoassay.

HbA1c calibration curve with Hb6 and 75C9 clones in fluoro-immunoassay.

This is something the experts at tebu-bio can help with, you might like to get in touch with them for solutions regarding your research projects.