ELISA assay development

Custom Assay form

You work on a new target and no ELISA kit exists? You try to detect a target at very low concentration? It’s time to try our ELISA assay development service! With 1500+ antibodies produced, our unique expertise in antibody development and ELISA design is your best guarantee to get the most specific and sensitive ELISA assay!

Why choose ProteoGenix for your
ELISA assay development?

guaranteed ELISA assay

Satisfaction
guaranteed!

We guarantee that your ELISA assay will work following your requirements.

sensitive ELISA assay development

From small molecule
to large protein

Our unrivalled know-how in antibody generation is our best tool to offer highly sensitive and specific ELISA assays

ELISA assay design experts

ELISA assay
design experts

You get access to our unique expertise and technical capabilities in ELISA assay design for various applications.

ELISA assay development experts

PhD account managers

As the development of an ELISA assay is highly technical, we put PhD account managers at your disposal to guide you along the process

antibody production leader

Antibody production leader

Benefit from the expertise of an antibody production leader and from all the latest antibody generation technologies: hybridoma development, phage display…

Custom ELISA assay

One-stop solution

From gene synthesis to ELISA kit development

Guaranteed ELISA assay

We guarantee to deliver the antigen and a pair of antibodies that detect your target in your samples. At the end of the process, we provide you with samples of the antibodies and antigen to verify the conformity towards the initial specifications. If you are satisfied, you pay the total amount. Otherwise, we charge 50% of the total cost only if we have been able to develop a sandwich ELISA against the antigen. Therefore, we take on the major part of the risk of your R&D or diagnostic project.

Guaranteed colorimetric immunoassay service

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Our ELISA assay development process

Services: Process Details

Custom assay development process

Having helped a large number of corporations, laboratories and individual researchers with our custom ELISA assay development services, we have managed to establish and fine-tune a thorough custom assay development procedure that takes into account every aspect of your research to deliver adaptive assay solutions for research, CE or IVD purposes. Here’s the blueprint of our custom assay services:

Assay Specifications Study

Once we have determined that existing ELISA kits, ELISA sandwiches or antibody pairs aren’t sufficient to meet your requirements (specificity, sensitivity, cross-reactivity, etc.), we design a fully customized strategy for assay development, in consultation with you. This is a very important step that determines the success of the projet. There are many possible strategies to develop an assay that meets specific requirements and every step of the process is critical.

Custom Assay Strategy Design

Once we have determined that existing ELISA kits, ELISA sandwiches or antibody pairs aren’t sufficient to meet your requirements (specificity, sensitivity, cross-reactivity, etc.), we design a fully customized strategy for assay development, in consultation with you. This is a very important step that determines the success of the projet. There are many possible strategies to develop an assay that meets specific requirements and every step of the process is critical.

Assay Development Strategy

Gene Synthesis

Our custom assay development strategy starts to materialize with double-stranded cDNA synthesis. With enhanced quality controls to guide our system, we guarantee 100% accurate sequencing, with an overall efficiency exceeding 99.9%. We further perfect our gene synthesis protocol with a run of gene mutagenesis to remove any traces of errors. Over the years, we have established a formidable reputation of being among the best service providers when it comes to the successful production of custom reagents starting with accurate gene optimization and synthesis. Gene optimization and construction design are very important to produce at the next step the most suitable antigen.

Protein/Peptide Production

Precision driven production of recombinant protein chains or peptide with sufficient yield factor to sustain further antibody development is a vital step, as far as custom assay development is concerned. With an extensive expertise in this regard, we ensure that our custom Competitive ELISA, custom Sandwich ELISA or other assay formats’ development processes are built on a solid base of antibodies developed with efficient antigen production of high purity.

Protein/peptide production, depending upon your project requirements and research goals, can also be expressed for sample delivery in best-fitting systems, including E.coli, B.subtilis, yeast, insect cells (baculovirus) and mammalian cells, to diversify downstream procedures in assay development. If need be, our team of scientists will also express recombinant protein or peptide in all these systems in parallel, so as to find the best expression conditions and improve the end-result validity of the antibodies used in the custom assay commissioned by you.

Primary Antibodies Development

We go about great care while developing primary antibodies from the antigen sample generated in the earlier step. As you must be aware, this is perhaps the most sensitive process in the custom assay development project, and that’s why, we have developed along the years an expertise in monoclonal and polyclonal antibodies that is peerless. Along with the cutting-edge techniques and equipment available in our labs, it’s also noteworthy that all of our animal facilities are AAALAC International accredited.

 

Generation of Monoclonal Antibodies

  • Hybridoma development with genetic immunization or simple protein antigen immunization
  • Advanced phage display technique with naïve or immune library screening
  • High-level of guarantee
  • Standalone delivery of monoclonal antibodies available if needed

 

Generation of Polyclonal Antibodies

  • Multiple animal systems for accurate generation of polyclonal antibodies with genetic immunization or simple protein antigen immunization
  • High-level of guarantee
  • Standalone delivery of polyclonal antibodies available if needed
Biomarker Discovery Services

Analytical Services

Upon successful production of desired primary antibodies, we set in motion our analytical services. Prior to conjugation, we offer three advanced, label-free techniques of antibody affinity measurement to gauge the levels of biomolecular interactions.

  • Surface Plasmon Resonance (SPR by Biacore™)
  • Surface Plasmon Resonance Imaging (SPRi by HORIBA Scientific®)
  • Rapid Extended Detection (Octet®RED by PALL Fortébio)

Antibody Conjugation for sandwich ELISA assay

The next step in our custom assay development services is the conjugation of antibodies. Thanks to the purity of antibodies produced in the earlier step, we can guarantee that the end-result of antibody conjugation will contain no stray protein chains or amine groups that can have a bearing on the conjugation process. Using the latest advanced techniques with a choice of multiple labelling methods, we leave no stone unturned to successfully accomplish this vital step towards accurate assay development.

  • Fast conjugation
  • Single step process to enhance accuracy
  • Minimum of 10 antibodies conjugation

Antibody Pair Matching for Sandwich ELISA assay

Working in symmetry with primary antibody development processes (see step 5), we determine and develop the best matched pairs of antibodies (coating antibodies and detection antibodies) for the custom sandwich ELISA assay. We carry out at least 90 pair matching tests to make sure that the most suitable antibody pair is selected. The accuracy of a Sandwich ELISA kit will wholly be a direct function of successful matching, and hence, it’s important to go with an experienced custom assay development supplier like ProteoGenix who masters all the process from gene synthesis to kit pilot productions.

  • Accurate matching of antibody pairs
  • Time saving process
  • Advanced matching techniques for unique pairs
  • Extensive matching tests (in excess of 90)
Antibody Matched Pair Screening

ELISA Protocol Development and Optimization

Based on the uniqueness of the target antigen and the desired antibody sensitivity, we proceed to develop a fully optimized ELISA protocol (Sandwich ELISA, Competitive ELISA or other format). The development of Sandwich ELISA or Competitive ELISA protocol drives the custom assay development project towards completion. The purpose of this step is usually to optimize the signal, sensitivity and reproducibility while reducing the noise/background to the minimum.

Thus developed custom Sandwich ELISA or Competitive ELISA assay protocol is then subjected to multiple process adjustments in order to optimize the following against the antigen:

  • The antibody pair concentration
  • The diluent concentration
  • The enzyme conjugate concentration
  • The buffer concentration
  • The signal intensity/accuracy
  • The blocking method

Assay Validation

Towards the conclusion of the custom ELISA development process, the developed custom assay is validated on the assigned biological samples to ensure that the detection of endogenous protein(s) in question is successful. Assay validation also provides veritable assurance that the target detection is consistent with recombinant protein/peptide developed.

ELISA Kit Pilot Production

Once the custom assay is fully developed and validated, we seek our customers’ approval to go ahead with the pilot production of kits. Keeping in line with our thorough approach regarding all procedures that custom assay development consists of; we deliver all the necessary reagents in fully developed and optimized states to facilitate the use of the custom assay in your laboratory. We eventually deliver dozens or hundreds of ready to use kits including pre-coated plates and all necessary reagents to perform the tests.

Elisa Kit Pilot production

What should you consider when choosing
the most relevant ELISA assay technique?

There are many factors to consider when choosing the most appropriate ELISA technique for your project. Here are some of the elements you should consider before making the decision:

  • The nature of the entity you want to detect: Sandwich ELISA requires two antibodies specific for two different and non-overlapping regions. Therefore, it will not be adapted for the detection of small antibodies. For hapten detection, competitive ELISA will be considered as more relevant.
  • The number of antibodies available: Consider preferentially direct or competitive ELISA if there is only one antibody available on the market for your target antigen.
  • The type of measurement: Sandwich or competitive ELISA is more adapted to the detection and quantification of an analyte whereas direct or indirect ELISA is more suitable to measure an immunological response.

As a leader in antibody production, ProteoGenix is able to develop all types of ELISA assays, whatever your requirements. If you would like to know more about our ELISA assay development service or if you have any doubt about the best format to use, feel free contact our PhD account managers.

The different types of ELISA assay formats

Direct ELISA

In direct ELISA, the protein antigen is non-specifically adsorbed to the well and washed up to suppress the amount of non-adsorbed antigen. Then, the antigen is directly detected by a labeled antibody.

There are two main advantages of using direct ELISA compared to other ELISA techniques:

  1. Its simple process as it requires less steps than other ELISA techniques

  2. It prevents the secondary antibody cross-reactivity

However, there are also some disadvantages such as:

  1. The unspecific protein immobilization which leads to the coating of the plate with all the proteins of the sample and therefore to higher background noise

  2. This technique requires a labeled primary antibody inducing a necessary conjugation for each target antigen

  3. Direct detection leads to lower sensitivity due to the lower labeled antibody density (maximum one/antigen)

Indirect ELISA

In indirect ELISA, the protein is non-specifically adsorbed to the well. This technique is called indirect ELISA because the antigen is detected indirectly through the use of a secondary antibody. Thus, the detection occurs in two steps. In the first one, an unlabeled antibody specific of the antigen is added. Then, a secondary antibody (polyclonal or monoclonal) detecting the primary antibody is added.

The main advantages of using indirect ELISA are:

  1. The possibility to use several primary antibodies with a unique secondary antibody

  2. The high sensitivity thanks to the high labeled secondary antibody density achievable leading to signal amplification (more than only one per antigen)

However, indirect ELISA also suffers from some drawbacks:

  1. The presence of a secondary antibody that could lead to cross-reactivity

  2. The longer process as compared to direct ELISA

Sandwich ELISA

Sandwich ELISA differs from direct and indirect ELISA as a capture antibody is bound to the plate instead of the antigen. Sandwich ELISA is based on the development of matched antibody pairs. A matched antibody pair is composed of two antibodies: the first one is used as a capture reagent and is directly coated to the plate, the second one is used as detection reagent. It is important that each of these antibodies detect different epitopes of the same antigen. Both direct and indirect detection can be used in a sandwich ELISA. Direct sandwich ELISA involves a labeled primary antibody as detection reagent whereas indirect ELISA uses a labeled secondary antibody detecting the primary antibody.

Sandwich ELISA offers several advantages over the other ELISA techniques:

  1. Thanks to the use of two primary antibodies, each specific of a different epitope of the same antigen, sandwich ELISA is highly specific.

  2. Sandwich ELISA even demonstrates a higher sensitivity than indirect ELISA due to the presence of a capture antibody only grabbing the antigen of interest. All the unwanted entities are washed up allowing for an accurate and sensitive detection of the antigen of interest.

  3. It is possible to make indirect or direct detection.

However, sandwich ELISA requires two antibodies targeting a different epitope of a same antigen. Thus, a custom antibody development might be necessary.

Competitive ELISA

Competitive ELISA is a complex technique which is particularly suitable for small antigens. Competitive ELISA is mainly used to measure the concentration of an antigen (or antibody) in a sample. The principle is to detect interferences in a signal output induced by the competition between the sample antigen (or antibody) and a labeled binding reference (antigen or antibody). As the signal is induced by the labeled reference, the rule is the following: “the weaker the signal, the higher the concentration of the analyte in the sample”.

Competitive ELISA can be based on direct, indirect or sandwich ELISA. Therefore, the advantages and drawbacks are related to the selected technique.

Let’s develop Your ELISA assay - Together!

With the best-in-class equipment, highly qualified and experienced scientists on board and a reputation that precedes our name, we, at ProteoGenix, are fully committed to providing nothing but the very best custom assay development services to you – all at a fair price, and with extensive guarantees, end-to-end quality control and quick turnaround times.

One step in the right direction saves you a hundred in the wrong one. So, let us bear the responsibility of developing and validating custom assays for you, so that you can drive your research further – much, much further!

Click ‘Contact US’ on your right to get in touch with us, ask us questions or request a quote. Keep reading on to learn more about how our custom assay development services can add that much-needed time, effort & cost effectiveness to your project.

Our ELISA Sandwich Development Expertise

Having developed hundreds of assays since 2003, ProteoGenix has indeed established emphatic expertise in a variety of fields. Here’s where we stand taller than our competitors when it comes to custom assay development::

      • Custom assay development from scratch (target accession number)
      • End-to-end custom assay solutions, from gene synthesis to pilot kit delivery
      • All intrinsic processes guided by strict quality control guideline
      • Expertise in unique targets and high performance/ high sensitivity tests

At ProteoGenix, we have on board a team of eminent scientists with illustrious academic, research and industrial backgrounds. Having at helm such a wealth of experience, we possess incomparable expertise in molecular biology, protein engineering, antibody & immunology and peptide synthesis.

Why Go for Custom Assay Development?

While ProteoGenix offers a number of multipurpose custom ELISA services including Direct ELISA assay services, Indirect ELISA assay services, Sandwich ELISA assay services and Competitive ELISA assay services, custom assay development often emerges as the best solution when you require validation / production kit for projects project involving very specific targets or markers. We work with automated Sandwich ELISA and Competitive ELISA techniques to develop the best-fitting assay solutions for you. According to the requested sensitivity and technology, we can develop colorimetric assays as well as fluorescent assay..
Our services include assay development for drug discovery but also for diagnostic or research purposes.

ELISA Assay Development Services at a Glance

With a long-standing commitment to the academic and industrial research community all over the world, we, at ProteoGenix, know what it takes to synergistically complement the countless hours you put into your research. Keeping the requirements of researchers in mind, we have developed a comprehensive custom assay development system that will take the pressure of validation and repetition of results off your shoulders.

SALIENT FEATURES

      • Unrivalled guarantees of success
      • Assay development strategy design to perfectly match your research goals
      • Wide variety of assays formats
      • High degree of customization, in accordance with the target accession number or biomarker specifications
        that you provide
      • Biomarker discovery through DNA-protein interaction studies (Yeast one-hybrid-system)
      • All-inclusive package, from gene synthesis to pilot kit delivery
      • Competitive costs, no hidden fees
      • Thorough post-delivery support from our team of veteran scientists

WHAT YOU GET WITH OUR CUSTOM ASSAY DEVELOPMENT SERVICES

      • Complete assay development strategy design
      • Timeline projections for the entire project
      • Custom assay development, from gene synthesis to relevant pilot kit delivery (Sandwich ELISA / Competitive ELISA / Other)
      • Assay validation
      • Delivery of all reagents
      • Assay transfer support
      • Documentation
      • Implementation support
      • Extensive quality control
      • Pilot kits delivery

ELISA Assay Development:

Common Areas of Application

A basic yardstick to decide whether your project requires a custom assay developed or a standard, off-the-shelf assay kit will do just fine is to assess:

      • The uniqueness of the target in question.
      • The quality of existing kits
      • The capacity of the existing kit to meet your specifications in terms of sensitivity, assay format, species specificity or cross reactivity.
      • The need to have your own assay to create a competitive advantage towards competitors and reduce the risk of copy.

If you can’t decide, feel free to get in touch with us and we will be more than happy to advise.

Here are some of the most common areas of application in which custom assay development comes to the fore:

      • Biomarker for drug discovery and development
      • Immunoassays for vaccine efficiency determination
      • Drug delivery monitoring
      • Diagnostic or prognosis kits development
      • Process development and optimization
      • Basic industrial and academic research
      • Target expression validation on different cell lines or cell extracts
      • Bioassays for food and water analysis
      • Advanced toxicity screening
      • Industrial process control
      • Environmental samples analysis

ELISA Sandwich Development at Work:

A Case Study

The best way to demonstrate our expertise in custom assay development is to share a representative case study of a biomarker assay we developed.

GOALS: IDENTIFICATION OF EARLY STAGE(S) PROSTATE CANCER BIOMARKER AND DEVELOPMENT OF BIOMARKER ASSAY KIT VIA SANDWICH ELISA

The aim of this project was to develop and optimize a biomarker assay to detect high levels of a biomarker specific to the early stage(s) of prostate cancer previously identified. The sample set of serum donors for validation consisted of healthy individuals, individuals with breast cancer and target individuals, i.e. individuals with prostate cancer.

BIOMARKER DISCOVERY

Standard OHS screening protocol was deployed to identify the regulators of a common protein target of anticancer drugs. The following figure (FIGURE 1) illustrates the generic working principle behind OHS screening.

One Hybrid System Screening for Biomarker Discovery

Principle of OHS system

In vivo identification of DNA-Binding protein thanks to fusion with GAL4-AD and activation of a reporter gene

DEVELOPMENT OF SANDWICH ELISA PROTOCOL FOR THE TARGET BIOMARKER

The steps detailed below were followed to develop a Sandwich ELISA protocol for the biomarker discovered via OHS screening in the preliminary stage of the custom assay development project.

      • Recombinant protein chain production was carried out in sufficient volumes and at above-threshold purity.
      • 10 high affinity monoclonal antibodies were developed in a hybridoma system.
      • All 10 monoclonal antibodies thus developed were produced, purified and conjugated.
      • Thorough antibody pair matching exercise was adopted, with as many as 90 pairs tested for the most suitable match.
      • The most optimal Sandwich ELISA protocol was developed to reach a sensitivity threshold sufficient to detect the target in patient serum.
      • The pair with the most suitable matching results was evaluated on labelled biological samples from a sufficiently large set of donors (healthy donors as well as cancer patients).
      • Epitome mapping was followed by development of two-tiered cell banking setup consisting of a Master Cell Bank (MCB) and a Working Cell Bank (WCB).

PROCESS DATA: DETERMINATION OF THE BEST PAIR FOR SANDWICH ELISA

As described in the step 4 earlier, an antibody matching experiment resulted in a set of highly reliable data points that were tabulated, and graphically and empirically analysed to determine the most suitable pair match.

The following graph (GRAPH 1) details the matching experiment results derived from matching various antibody pairs.

ELISA Sandwich Development Pairing Results

GRAPH 1: Determination of the best pair for Sandwich ELISA

As can be clearly noticed, Detection Antibody 4B and Detection Antibody 5B exhibit remarkably good pairing with Coating Antibody 17. Among these two contenders, the pair of Detection Antibody 5B and Coating Antibody 17 was noticed to provide consistently better pairing. Thus, empirical and experimental analysis of over 90 pairs allowed us to zero in on the best pair match.

PROCESS DATA: SANDWICH ELISA OPTIMIZATION

Once the best pair of antibodies is determined, using a Sandwich ELISA approach, a specifically optimized detection protocol was created. This involved the choice and usage of appropriate blocking buffers, along with suitable incubation periods, antibody dilutions and other parameters.

The following graph (GRAPH 2) plots the concentration values against the associated OD, giving us a highly reliable and accurate empirical curve that can be used in the future for extrapolation needs as a part of the complete Sandwich ELISA protocol for this biomarker assay.

ELISA Sandwich Development and Optimization

GRAPH 2: Detection threshold determination in blood samples

Conclusion: this optimized Sandwich ELISA protocol allows detecting the biomarker at a concentration ≤0.2 ng/ml in patient blood samples.

PROCESS DATA: CUSTOM SANDWICH ELISA EVALUATION ON BIOLOGICAL SAMPLES

The evaluation of the custom ELISA Sandwich protocol was carried out using blood samples from healthy donors, breast cancer patients and prostate cancer patients. The optimized ELISA protocol was used to assay biomarker levels in samples compared to PSA levels.

The following graph (GRAPH 3) illustrates the high levels of prostate specific biomarker in prostate cancer patients, as detected by the custom Sandwich ELISA protocol developed at ProteoGenix, enabling the categorical discrimination between non-prostate cancer patients (healthy donors and breast cancer patients) and prostate cancer patients. This discrimination was much clearer and more conclusive than when assaying PSA biomarker.

ELISA Sandwich Development for Prostate-specific Biomarker

GRAPH 3: Determination of Prostate-specific antigen level in blood samples of healthy and cancer patients (Sandwich ELISA)

A huge increase of Prostate-specific biomarker level is detected in prostate cancer patients

CONCLUSION

This case study represents a lot more than just facts, figures and data. Such advancement and ability to customize the assay development process – otherwise a long, tedious and meticulous task – to this degree is a great asset to have at researchers’ disposal. Custom assay development not only provides solutions tailored for your projects, but it also saves precious time, energy and other resources that you would otherwise spend on trying to make standardized assays work.

ELISA sandwich development principle

ELISA sandwich is usually a method to assay a protein (in blue in the chart below) in different kinds of samples such as serum samples using a pair of antibodies. The capture antibody (in green below) is quoted to the plastic of 96 well plates whereas the detection antibody (in red in the chart below) will allow a detection signal to be released. In an ELISA sandwich, the detection antibody can be directly conjugated to biotin, HRP or AP (in yellow in the chart below) but it is also possible to use a conjugated secondary antibody to detect it if the coating antibody is from a different host species.

Elisa Sandwich development principle