preparation of serum samples for stability evaluation of human adenosine deaminase 1 enzyme using microplate assay

<meta charset="utf8"></head><body>Valutazione della stabilità sierica di HsADA1: un approccio basato su micropiastre

The following method allows a user to quantitatively assess an enzyme&#39;s ability to retain its activity when exposed to conditions that mimic what it will encounter following intravenous injection. The in vitro method mimics such in vivo conditions and consists of the incubation of the enzyme in pooled human serum at 37 &#176;C and time-coursed analyses of retention of enzyme activity. We refer to an enzyme&#39;s ability to retain activity in these conditions as its serum stability, and the analysis method for enzyme activity takes advantage of differences in absorbance between an enzyme&#39;s substrate and the resulting product. The concept of serum stability is not just enzyme-specific and has been applied to several other treatment modalities as well. For example, the serum stability of RNA aptamers targeting COVID spike proteins has previously been assessed by monitoring their degradation post-incubation with fetal calf serum1. Antibacterial peptides have also been assessed for their ability to suppress bacterial growth post-incubation with pooled human serum2.
HsADA1 is an enzyme that catalyzes the conversion of adenosine or 2-deoxyadenosine into inosine or 2-deoxyinosine, respectively3. Adenosine has an absorption peak of 260 nm, while inosine absorbs strongest at 250 nm4. This shift in absorption peaks can be detected on a microplate reader by a decrease in absorption intensity at 260 nm when HsADA1 is added to adenosine. The HsADA1 enzyme has important implications in the human body, and its deficiency causes a severe combined immunodeficiency (ADA-SCID)5. The bovine homolog of HsADA1, BtADA1, can be utilized as an enzyme replacement therapeutic for the treatment of ADA-SCID, and we have previously shown that wildtype HsADA1 loses its activity when incubated with pooled human serum, potentially hindering its use as a therapeutic6. Therefore, we selected the wildtype HsADA1 enzyme to demonstrate a procedure to determine an enzyme&#39;s serum stability. A detailed purification method for HsADA1 has been described previously6.
In the following protocol (as detailed in Figure 1), we demonstrate how to co-incubate wildtype HsADA1 in pooled human serum at 37&#160;&#176;C. During this time, test samples are taken at defined time points and flash-frozen for future analysis. Once all samples have been taken, a microplate assay is run whereby each sample is combined with the substrate, with the resulting absorbance decrease being a correlation for the retained activity of the enzyme. Representative results illustrating the serum stability of HsADA1 are shown, and because this metric may be relevant to determining the potential therapeutic value of other enzymes, we also discuss considerations to adapt this protocol to an engineered Homo sapiens kynureninase enzyme (HsKYNase) and any enzymes more generally. HsKYNase is an enzyme involved in the metabolism of tryptophan and is able to degrade the tryptophan-byproducts kynurenine and hydroxy-kynurenine (OH-Kyn) into anthranilic acid and hydroxy-anthranilic acid (OH-AA), respectively. Enzyme-mediated modulation of tryptophan metabolism may be of therapeutic relevance7.

<meta charset="utf8"></head><body>Autore in primo piano: Progressi nell'ingegneria cellulare e proteica per controllare le funzioni biologiche e sviluppare nuove terapie

Determinazione della stabilità sierica dell'enzima adenosina deaminasi 1 umana

Preparazione di campioni di siero per la valutazione della stabilità dell'enzima adenosina deaminasi 1 umana mediante saggio su micropiastra

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Research

JoVE Journal

Bioengineering

Preparation of Serum Samples for Stability Evaluation of Human Adenosine Deaminase 1 Enzyme Using Microplate Assay

40 Views. Per iniziare, preparare un stock 10x di HsADA1 in 1x PBS a pH 7,4 per ottenere una concentrazione finale di 10 micromolari. Preriscaldare un'aliquota scongelata da 15 millilitri di siero umano a 37 gradi Celsius. Quindi aggiungere 100 microlitri del brodo 10x HsADA1 a 900 microlitri di siero umano aggregato e PBS prelevati in due provette da microcentrifuga separate a basso legame.Diluire 100 microlitri di ogni miscela enzimatica e non enzimatica uno a...

Video: Preparazione di campioni di siero per la valutazione della stabilità dell'enzima adenosina deaminasi 1 umana mediante saggio su micropiastra

Determining the Serum Stability of Human Adenosine Deaminase 1 Enzyme

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature increases. For a therapeutic enzyme, other measures of stability may also be critical, particularly its ability to retain function in human serum at 37 &#176;C, which we refer to as serum stability. Here, we describe an in vitro assay to assess the serum stability of the wildtype Homo sapiens adenosine deaminase I (HsADA1) enzyme using an absorbance-based microplate procedure. Specifically, this manuscript describes the preparation of buffers and reagents, a method arranging for the coincubation of HsADA1 in serum, a method to analyze the test samples using a microplate reader, and an accompanying analysis to determine the fraction of activity that an HsADA1 enzyme retains in serum as a function of time. We further discuss considerations to adapt this protocol to other enzymes, using an example of a Homo sapiens kynureninase enzyme, to help aid the protocol's adaptation to other enzymes where serum stability is of interest.

In this article, we detail methods to characterize an enzyme&#39;s ability to retain function when incubated at 37&#160;&#176;C in human serum, a pharmacological property referred to as its serum stability. This ability may be a key factor in predicting an enzyme&#39;s pharmacokinetic profile and its suitability for therapeutic use.

The concept of enzyme stability is typically used to refer to an enzyme's thermostability - its ability to retain structure and activity as temperature ...