It is becoming increasingly apparent that histopathology is the cornerstone of preclinical research.  Therefore, the ever-growing field of novel biomedical technologies and therapeutics awaiting to enter the clinical trial testing stage oftentimes calls for accelerated assay development solutions.  

Such studies can increasingly benefit from custom histopathology assays, enabling deeper insights into the identification of the target protein within the tissue of interest. Below, we have summarised the key things you need to consider before commencing an antibody assay development to meet your research objectives and outlined the benefits of outsourcing this key part of your research. 


1. Aim of your Project  

Histopathology assessments are traditionally performed to identify tissue-morphological changes in studies evaluating in vivo disease progression, response to novel therapy, basic research, and/or nonclinical safety assessments. Target or biomarker identification in tissues of interest serves to evaluate disease pathophysiology in animal models of diseases and the biological processes in response to a novel therapeutic intervention.  

As the scope of research studies can be broad, it is important to thoroughly discuss and consult experts within the field to make informed decisions, helping you progress in your research. While discussing the overall goal of the study it is equally important to discuss the study hypotheses and expected results. Identifying the expected target localization (I.e., is it a sub-cellular or nuclear protein), as well as the expected level of target expression, drive the antibody assay development process forward. Additionally, determining the expected level of target protein helps with the selection of the antibody type; for proteins with low expression, we recommend polyclonal whereas high-expression proteins are best detected with monoclonal antibodies.  

Lastly, discussing any protocol specifics beforehand significantly increases time savings, as the tissue type (FFPE or frozen tissue), fixation times, storage of the fixed tissue, etc., can subsequently influence the binding of your antibodies of choice.  


2. Availability of Validated Antibodies  

One of the most important steps in assay development is ensuring that you are working with primary antibodies that have been validated to be both sensitive and specific to your target protein. Clonality of the primary antibody, the production and purification methods of the vendor, and different isotypes can lead to unspecific binding, resulting in too much background signal or alternatively false-positive results.  

We are continuously expanding our validated list of antibodies for various research applications. Antibodies for application include but are not limited to, oncology studies (immune cell markers, proliferation markers, cytokines) and analysis of apoptosis (caspase proteins), fibrosis (Collagen III, smooth muscle actin, vimentin), and detection of specific reporter proteins for gene expression studies (e.g., green fluorescent protein [GFP]). In addition, our laboratory experts have ample experience in validating novel antibodies; and are happy to optimize the best conditions for your antibody of choice. 


 3. Chromogenic or Fluorescent Detection?  

Enzyme-linked secondary antibodies are suitable for the identification and visualization of one target protein, with some room to optimize protocols for multiple protein co-localization. Additionally, chromogenic detection of the signal is more suitable for tissues that show high autofluorescence which would make the interpretation of immunofluorescent signal challenging. However, immunofluorescent detection of target proteins allows you to perform extensive protein co-localization studies. Accordingly, the selection of fluorophores must be carefully done to minimize the bleeding of the fluorescence signal through the different channels.  

Connected-Pathology has extensive experience in advising you on how to select the best method for detecting your target within your tissue of interest for both single- and multiple-protein localization studies. Thanks to our collaboration with KU Leuven, we additionally offer extensive immunophenotyping studies using multiplex immunohistochemistry, allowing you to view up to 80 targets.  


4. Correct Positive and Negative Controls  

When validating your primary antibody of choice for the first time, antibody validation needs to be performed against both positive and negative controls. Positive biological controls are tissues that express your protein of interest, whereas negative controls are performed without primary antibodies or using isotype controls in the same tissue, ensuring the specificity of your antibody. Isotype controls match the class (for e.g., IgG, IgM…) and type (monoclonal or polyclonal) of the primary antibody of choice, but lack target protein specificity as they are raised against proteins that are not present in the species’ target tissue. Lastly, if your target protein is expressed ubiquitously, tissues with both low and high target protein expression can additionally serve as positive and negative controls, respectively.  

Having access to the correct tissues can be difficult, therefore, you need to make sure that your outsourcing laboratory has access to multiple tissue types to ensure confidence in your results. At Connected-Pathology, we assist you with providing relevant species-specific positive control tissues, according to the needs of your experiment. Moreover, our in-house veterinary pathologists often make use of additional H&E stains to thoroughly validate the observed IHC or IF signal at the biological level. For example, if cells are shown as Caspase-3 positive on an IHC-stained tissue, H&E serves as a valuable reference to confirm that the cells are indeed undergoing apoptosis.  


5. Target Species  

The choice of your experimental model (i.e., rodents, non-human primates [NHPs], poultry, bovine) ultimately also affects the choice of the primary antibody. This means that it is very likely that you need to select antibodies raised in different species other than that of your experimental model which would otherwise lead to unspecific bindings and/or cross-species reactivity. Most commercially available primary antibodies show species reactivity towards human and mouse tissue, however, preclinical research models are not limited to mouse models, and other in vivo models such as rats, guinea pigs, NHPs, etc., are required.  

The number of target proteins to be detected also affects the choice of antibody (both primary and secondary) to prevent cross-reactivity issues. This can be prevented either by selecting various antibodies raised in different species (e.g., mouse, rat, rabbit) or using different isotype-specific (e.g., IgG1, IgG2b) antibodies.  

Ideally, a laboratory has a validated antibody library with antibodies showing species-specific reactivity and produced in several different species, helping you avoid the issue of cross-reactivity. Experts at Connected-Pathology have years of experience in designing customized histopathology assays and are available to give you scientific advice on panel design.  


6. Protocol Optimisation and Development 

Although correct antibody selection is a critical step in the assay development process, optimization of the subsequent steps to obtain the correct expression pattern should not be underestimated. This calls for optimization every step of the way; possible tissue permeabilization steps, antibody concentration, incubation time, and the antibody diluent. Additionally, as we previously indicated, the fixation method of the tissue calls for further consideration of whether you need to perform antigen retrieval to uncover the epitopes of the target protein preventing false-negative results, or alternatively the blocking of other proteins to prevent non-specific signals.  

It thus becomes obvious that optimizing any experiment requires significant expertise, whereby having hands-on experience can significantly speed up the entire process. At Connected-Pathology we have ample experience with advising our clients, troubleshooting, and collaborating by looking at protocols together to suggest and test improvements. Our laboratory capacity allows us to run multiple protocols to isolate the issue, allowing you to reach the highest quality and accuracy of experiments.  

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Optimised IHC staining for detection of Cytokeratin (panCK) positive epithelial lung carcinoma cells, allowing the differentiation of epithelial from non-epithelial tumours. 

7. Biological Relevance 

Following an extensive antibody validation process, your results need to be put into perspective in line with your research goal and therapeutic area. The knowledge about your target protein expression (I.e., is it a nuclear protein or is it localized in subcellular compartments) helps you validate your experiments both at the quality-assurance level and the biological level. Additionally, you can gain deeper insights into their target protein function by combining gene and protein expression co-localization within a single tissue.   

We frequently make use of our DISCOVERY ULTRA from ROCHE to perform gene and protein co-localization studies on a single tissue slide. DISCOVERY ULTRA combines the detection of a target protein (via IHC) and the target gene (via in situ hybridization [ISH]). Additionally, the DISCOVERY ULTRA from ROCHE allows us to extensively modify and customize protocols, in comparison to routine protocols performed on the BenchMark ULTRA from Roche. This allows you to gain deeper insights into the molecular regulation of your target protein, providing you with valuable information such as the post-translational regulation of your target protein.  


8. Access to State-of-the-art Technologies  

The selection of an appropriate antibody together with associated optimization experiments put an end to the process of assay development. However, the developed assays will most likely be used in studies whereby hundreds of slides may need to be stained and analyzed. To avoid any human errors associated with high-volume benchwork and to ensure standardization, your research may benefit from novel state-of-the-art technologies that allow streamlining many of the mundane laboratory tasks. Automating both routine staining such as H&E as well as IHC protocols improves the turn-around times for tissue processing and staining.  

As the cost of running such machines is increasingly high, outsourcing your histopathology studies to a reliable histopathology provider allows you to take advantage of state-of-the-art technologies. While having access to state-of-the-art automation equipment is important, it is equally important to have control over the processes with track-and-trace automation systems. In addition, you can keep your time investments and costs minimal by taking advantage of our cloud-based LIS, Tx-Connect, which has embedded quality checks and end-to-end traceability to ensure simplicity and accuracy when ordering and tracking your research progress. 


9. Analysing the Results  

Correctly interpreting your histopathology results is up to board-certified veterinary and medical pathologists, ensuring and maintaining the highest care of both research and pathologic assessments. Our in-house veterinary pathologists possess relevant expertise in pathology across different animal species in oncology, medical devices, cardiometabolic, vaccine research, inhalation studies, and such. 


Your study may additionally benefit from the implementation of digital pathology workflow that is implemented at Connected-Pathology. Digital pathology is described as one of the most efficient and promising techniques within the histopathology field. This is because the use of artificial intelligence in analyzing your results leads to consistency, and accuracy, and allows our in-house pathologists to make an objective diagnosis (for e.g., automated cell quantification, integrated scoring systems, structured reporting, etc.).  



10. Viewing and Archiving the Results  

In line with our previous point, your antibody assay development process does not end with an analyzed set of tissue slides; viewing and archiving your obtained results is of crucial importance. This final stage allows you to effectively evaluate and document your findings, ensuring transparency, reproducibility, and traceability throughout your research. Additionally, archived results serve as a valuable resource for future research and collaborations. They provide you with a foundation for building upon previous work, facilitating the exploration of new research questions, and enabling the comparison of findings across different studies. 

Our V-Connect viewer allows our pathologists to assess histopathological results quickly and accurately, through dynamic and thoughtfully designed features. Thanks to the access to the V-Connect and our digital archives, we ensure that you do not lose ownership of your research, which is always made available to you through secure and reliable digital storage options. 


Take-away messages

Antibody assay optimization is both a time-consuming and costly process. Preclinical research already faces a lot of difficulties, therefore, outsourcing your histopathology assays allows you to better focus on progressing your own ground-breaking research. By outsourcing this key part of your research, you not only gain novel insights by working alongside our pathologists and laboratory technicians, but most importantly you uncover key results to allow progression of your research. 

With our flexible entry points, you can outsource as much (I.e., whole antibody assay development process), or as few processes (I.e., validation of novel antibodies in target tissue) as you deem necessary, resulting in the most cost-effective processes. Ultimately, we ensure that your research deadlines are met, accelerating the time to clinical trials, while reducing the hidden costs of extensive antibody optimization work.  

Outsource your assay development solutions with Connected-Pathology now: