Sandwich ELISA development

Sandwich Elisa quotationform

You need to detect small amounts of your target? Let us develop your own sandwich ELISA! We guarantee that your sandwich ELISA will detect your target even in complex samples (sera, tissue homogenates, cell lysates) because we master all the development steps from antigen design to the pilot kit production (validated with your own samples).

Why choose ProteoGenix for your
ELISA assay development?

Guaranteed Sandwich ELISA

Guaranteed
Sandwich ELISA

We guarantee that you will get a fully functional Sandwich ELISA.

Sandwich ELISA tested in real conditions

Your sandwich ELISA
tested in real conditions

Provide us hundreds of samples (sera, tissue homogenates, cell lysates). We optimize your sandwich ELISA in real conditions!

ready-to-use ELISA sandwich

Get your ready-to-use
pilot kits

Our fully integrated solutions include the whole development of your sandwich ELISA from antigen design to pilot kit production.

Sandwich ELISA development experts

Sandwich ELISA
design experts

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

Polyclonal or monoclonal antibodies

Monoclonal or
polyclonal antibodies

We generate polyclonal or monoclonal antibodies depending on your requirements.

Hybridoma or phage display

Hybridoma or
phage display

Our sandwich ELISA service is fully custom. It can include monoclonal antibody generation by hybridoma or phage display.

Our sandwich ELISA development process

Services: Process Details

Sandwich ELISA development process

Having helped a large number of corporations, laboratories and individual researchers with our 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

Case study: development of a sandwich ELISA for the detection and quantification of an early stage prostate cancer biomarker

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

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.

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.

Antibody pair determination

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 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.

Custom sandwich ELISA validation

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.

Sandwich ELISA FAQ

When should I go for a sandwich ELISA? What are the advantages of using a sandwich ELISA?

Sandwich ELISA differs from classical ELISA techniques by the adsorption of a capture antibody directly on the plate. This confers to sandwich ELISA the capacity to detect or to measure the concentration of an analyte in an impure sample (contrary to other ELISA techniques where the antigen coating is made by unspecific adsorption).

Which type of detection method should I use for my sandwich ELISA?

Sandwich ELISA allows for either direct or indirect detection.

Direct detection implies the use of two primary antibodies, a non-labeled one as capture reagent and a second labeled primary antibody that will be used both for capture and detection.

Indirect detection implies the use of two non-labeled primary antibodies, each recognizing a different and non-overlapping epitope of the same antigen and a secondary antibody detecting the second primary antibody (the one non coated to the plate). Indirect detection offers many advantages over direct detection as:

  1. It does not require to label a primary antibody. Consequently, the primary antibody retains its maximum immunoreactivity.

  2. Secondary antibodies can bind to several epitope of a primary antibody leading to signal amplification.

  3. A wide variety of secondary antibodies are available commercially.

Which antibody clonality should I choose as capture reagent for my sandwich ELISA?

Polyclonal and monoclonal antibodies present different properties. While monoclonal antibodies are characterized by high specificity due to the recognition of a single epitope of an antigen, polyclonal antibodies are able to recognize several epitopes of a same antigen leading to higher cross-reactivity. Of course, this will affect the properties of your sandwich ELISA.

Choosing the appropriate clonality will depend on your goal:

  • If your goal is to detect a maximal amount of your target, polyclonal antibodies as capture antibodies will represent the right solution. In counterpart, the use of polyclonal antibodies as capture reagent will led to some unspecific binding. Thus, using polyclonal antibodies as capture reagent will remain the best solution if you try to detect a low concentration target in a simple sample.

  • If your goal is to detect a specific target in a complex sample, a monoclonal antibody as capture reagent will be the best solution.

Which buffers should I use for my sandwich ELISA assay?

The are many factors to consider when determining the optimal experimental conditions of your sandwich ELISA assay. Concerning the buffers, this includes choosing the right temperature, pH, ionic strength, detergent… ProteoGenix includes the definition of the best experimental conditions in its Sandwich ELISA development offer.

Am I sure my Sandwich ELISA will work in real conditions?

Basically, our sandwich ELISA development service comprises several phases:

  • Antigen design

  • Antibody generation (monoclonal and/or polyclonal)

  • Antibody production

  • Antibody conjugation

  • Identification of the best antibody pairs (capture and detection antibodies)

  • Validation of the antibody pairs in real conditions (with customer’s samples)

  • Optimization of your sandwich ELISA in real conditions

  • Sandwich ELISA final validation: assessment of sensitivity, accuracy, reproducibility, reliability and robustness (includes several parameters such as limit of detection, linerarity, intra-plate and inter-plate precision…)

  • Sandwich ELISA shelf-life stability testing

  • Sandwich ELISA kit pilot production

Our sandwich ELISA development service includes a validation and an optimization step with your own samples. This means that the sandwich ELISA kit we deliver is fully functional for your assays.

Sandwich ELISA development principle

Sandwich ELISA is usually a method to assay a protein (in blue in the chart below) in different kinds of samples (such as serum samples, tissue homogenates, cell lysates) using a pair of antibodies. The capture antibody (in green below) is adsorbed 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.  ELISA Sandwich allows measuring the concentration of an analyte in a complex sample thanks to the immobilized capture antigen which acts as a purification step and confers to the assay a very high specificity. 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.

Sandwich ELISA principle