Accelerated in vivo Research with Assay Genie | Biomol Blog | Resources

Written by Emily Locke

These topics await you:

1) Multitasking - the diverse Applications of in vivo Antibodies

2) Highest Quality at a fair Price - this is what characterizes the mAbs from Assay Genie

3) Low and Ultra Low Endotoxin in vivo Antibodies

5) Simple Monitoring of Antibody Levels with ELISA Kits

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Removing the brakes of the immune system to fight tumors - this is the idea behind the so-called checkpoint inhibitors, which have been used in cancer therapy for several years now. The new class of drugs was developed based on the work of James Allison and Tasuku Honjo, who were awarded the Nobel Prize in Medicine in 2018 [1]. The two Nobel laureates have researched the above-mentioned brakes that prevent the tumor from being fought by the patient's own immune system in many cancers, thus enabling the development of a whole range of immunotherapeutics. The most important immune checkpoints inhibited by such therapeutics are the receptors CTLA-4 and PD-1 as well as the ligand PD-L1. Established checkpoint inhibitors are therefore usually monoclonal antibodies [2].

Immune checkpoint blockade is just one of the many examples of applications where researchers need functional in vivo antibodies. Such monoclonal antibodies (mAbs) are mainly used for translational and preclinical in vivo studies, but are also used in in vitro assays as well as in classical applications such as ELISAs, flow cytometry or Western blots. Sound interesting for your projects? Our partner Assay Genie supports your research with a wide range of high-quality in vivo antibodies. The Irish company's products are particularly attractive due to the highest quality standards in the industry and an incomparably cost-effective price range.

**Multitasking - the diverse Applications of in vivo Antibodies**

As already mentioned, functional in vivo studies are the most important application area for in vivo antibodies - hence their name, of course. They are used in translational and preclinical animal studies to effectively treat diseases or manipulate biological systems, such as the immune system [3]. Of particular interest is research into the effects of mAbs on tumor growth and cancer development in the context of immuno-oncology - as in the case of checkpoint inhibitors. Immunologists also use the antibodies to selectively deplete or reduce certain immune cell types such as T, B and NK cells. This enables researchers to understand the function and role of these specific cell types in cellular signaling pathways and subsequently, test potential therapeutic strategies based on these findings.

In addition to their use in vivo, the mAbs from Assay Genie can also be used in functional in vitro assays. In particular, the blocking of certain receptors, the neutralization of viruses or cytokines, for example, as well as the co-stimulation and activation of certain cell types play a role here. Such assays help to investigate important cellular signaling pathways and thus, to discover new therapeutic target structures. Blocking, neutralization and activation can also be performed in mouse models using Assay Genies in vivo antibodies. In addition, the mAbs are suitable for classic laboratory methods, including ELISAs, flow cytometry, immunohistochemistry and Western blots [3].

Highest Quality at a fair Price - this is what characterizes the mAbs from Assay Genie

What makes Assay Genie's in vivo antibodies so special is that they meet the highest quality standards in the industry and are still extremely affordable! The in vivo antibodies are produced in a cGMP and ISO-certified facility and purified in several steps (up to ≥98% purity). They are then subjected to strict quality control to ensure high reproducibility between different lots. The formulation of the mAbs is free of preservatives, stabilizers and carrier proteins [3]. In addition, the antibodies are tested for a range of murine pathogens to enable safe injection into animals. For this purpose, a comprehensive IDEXX IMPACT™ PCR screen for rodent pathogens (e.g. Mycoplasma sp. or murine hepatitis virus) is performed, protecting your valuable research animals from an infection outbreak [4].

Another aspect is the removal of endotoxins from the antibody formulation, which is particularly important in the production of in vivo biologics [5]. Endotoxins are bacterial, cell-associated toxins that are part of the outer cell membrane of gram-negative or cyanobacteria (Fig. 1). Chemically speaking, they are lipopolysaccharides (LPS), which are made up of a complex, hydrophilic polysaccharide and a lipophilic lipid component. While so-called exotoxins are toxins that are secreted by living bacteria, endotoxins are mainly released after the death or lysis of the bacteria (Fig. 1). They are biologically active even at very low concentrations and can trigger numerous physiological reactions in humans [6].

Endotoxine

Figure 1: Endotoxins are constitutive components of the bacterial membrane. They consist of lipopolysaccharides that are released when the bacteria die. This can occur through phagocytosis by macrophages or through lysis. The released endotoxins can then enter the bloodstream and cause an unwanted immune reaction (figure created with biorender.com).

Biological therapeutics and research reagents contaminated with endotoxins lead to adverse reactions in patients, which can be attributed to an overactivation of cells of the innate immune system, among other things. Thus, a low endotoxin content in biological products used in the preclinical setting for both in vivo and in vitro testing is essential. To limit LPS-associated toxicity, the endotoxin threshold for humans and preclinical animal models is 5 EU/kg (i.e. endotoxin units) [5]. With Assay Genie's in vivo antibodies, you don't take any risks: the mAbs have an even lower endotoxin level of only ≤1.0 EU/kg in the Low Endotoxin and ≤0.5 EU/kg in the Ultra Low Endotoxin product range [3]. This way you can be sure that you are testing your drugs for their actual biological effects and not for the effect associated with endotoxin contamination.

*Low and Ultra Low Endotoxin* in vivo Antibodies
Specifications	in vivo mAbs (Low Endotoxin)	in vivo mAbs (Ultra Low Endotoxin)
Binding Validation (WB, FC or ELISA)	Yes	Yes
Endotoxin Level (LAL Method)	≤1,0 EU/mg	≤0,5 EU/mg
Antibody Aggregation Screening (SEC)	≥95% monomer	≥98% monomer
Purity (SDS PAGE)	≥95%	≥98%
*Formulation for in vivo* use**	No preservatives No stabilizers No carrier protein Sterile PBS pH 7.2 - no K⁺ or Ca²⁺ Concentration: >5 mg/mL	s. Low Endotoxin
Murine Pathogen Screening	Not applicable	Pathogen tested (IMPACT1)
Applications	WB, FC, IF or IHC	WB, FC, IF or IHC
Storage and Handling	Manual defrost freezer, avoid repeated freeze-thaw cycles 1 month, 2 to 8 °C under sterile conditions, as supplied 12 months, -70 °C under sterile conditions	s. Low Endotoxin

*Functional grade preclinical antibodies are manufactured in an animal free facility using only in vitro protein free cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.

Assay Genie supports scientists involved in preclinical R&D or working with animal models with a selection of over 200 functional in vivo antibodies. Whether you are looking for antibodies against "classic" target structures such as CTLA-4, PD-1 and CD8 or whether you are investigating more unusual targets in your projects - Assay Genie offers you a suitable in vivo antibody at a fair price! Browse through the wide range now and accelerate your in vivo research with the excellent products from Assay Genie!

Monoclonal in vivo antibodies represent the latest addition to the Assay Genie portfolio. These functional grade antibodies can be used for co-stimulation, blocking, neutralization or cell depletion and are validated for ELISA, flow cytometry, immunoprecipitation, western blot and other applications. Take advantage from low and ultra low endotoxin antibodies that have at least 95% purity and that come without any preservatives, stabilizers or carrier protein.

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In vivo Antibodies by Assay Genie

Similar, but not the Same: Biosimilar Antibodies for your in vivo Research

Biosimilars have been a therapeutic alternative in Germany for over 15 years, including a steadily growing number of monoclonal antibodies for oncology [7]. A therapeutic antibody is used as part of an immunotherapy to recognize a cell-specific protein and then initiate an immune response in the patient. These include, for example, the checkpoint inhibitors Ipilimumab (anti-CTLA-4) and Nivolumab (anti-PD1). A biosimilar is an almost identical copy of such a therapeutic antibody with similar efficacy and structure, which is often produced when the patent of the original biopharmaceutical expires [8]. Due to their complexity and the way they are manufactured, they are subject to special quality and approval requirements [9].

In research, biosimilar antibodies are used particularly as reference standards in drug development and for preclinical in vivo animal studies. For example, scientists can test antibodies that are still in development for their quality, efficacy and safety and compare the results with a reference biosimilar. Our partner Assay Genie also offers a wide range of in vivo antibodies in the field of biosimilars, which have been extensively validated and tested for immunogenicity. The company produces the monoclonal antibodies using a revolutionary high-throughput technology that allows IgG sequences to be obtained directly from immunoreactive B cells without hybridoma fusion. This not only makes it possible to obtain mAbs from a range of animal species, but also to process and optimize the antibodies directly for humanization. This guarantees high specificity, sensitivity and affinity of Assay Genie's biosimilar products [8].

Simple Monitoring of Antibody Levels with ELISA Kits

In addition to the use of a biosimilar as a reference standard, the measurement of therapeutic, biosimilar and anti-drug antibody levels in blood and serum is of great importance for the optimization of drugs. Anti-drug antibodies, or ADAs for short, are antibodies that are directed against a previously administered biologic (Fig. 2) [10]. Since ADAs can have a negative effect on disease treatment, monitoring their concentration in the blood after drug administration is very important in order to increase the efficiency of the drug and develop appropriate dosing strategies. However, this often requires the use of outdated and expensive methods such as high performance liquid chromatography (HPLC) or mass spectrometry (MS). Our partner Assay Genie has recognized this problem and is providing a solution by offering 82 TDM (Therapeutic Drug Monitoring) ELISA kits. With these cost-effective kits, it is possible to test the pharmacokinetic and pharmacodynamic properties of a drug in humans, mice and other species in the shortest time possible! The optimized protocol requires only 10 µl of sample material, is easy to automate and enables simple analysis with a plate reader [11].

ADAs

Figure 2: The release of anti-drug antibodies. After drug administration and cellular uptake of the biotherapeutic agent through endocytosis, antigens are presented on the cell surface via MHC-II complexes. The antigens are recognized by naive CD4⁺ T cells via the T cell receptor (TCR), which leads to the activation of these T cells. These in turn activate naive B cells via the release of certain cytokines. The resulting plasma cells then produce and secrete anti-drug antibodies, which bind the administered drug in the blood. This can result in a loss of efficacy of the drug (figure created with biorender.com).