Valproic acid (VPA), chemically known as 2-propylpentanoic acid, is a fatty acid derivative and one of the most frequently prescribed anticonvulsant drugs. Approved in the 1970s, it is now used in the management of epilepsy, bipolar disorder, and as a migraine prophylactic. VPA works by increasing the concentration of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, thereby stabilizing neuronal activity. Its broad clinical use, however, is accompanied by significant challenges in dosing due to its narrow therapeutic index (i.e., the difference between effective and toxic doses).

VPA’s effectiveness in controlling seizures and stabilizing mood in patients with bipolar disorder or epilepsy is well-established. However, its use is complicated by its pharmacokinetic variability, which can be influenced by factors such as age, liver function, concurrent medications, and genetic variations in drug metabolism. Elevated serum levels can lead to toxic effects, including hepatotoxicity, pancreatitis, and central nervous system (CNS) depression, while insufficient levels result in poor seizure control or mood destabilization.

The therapeutic range of valproic acid typically lies between 50 and 100 μg/mL, but individual patients may experience clinical effects outside this range. It is crucial for clinicians to monitor plasma levels regularly to ensure that patients are within the desired range and avoid the risk of toxicity or suboptimal drug effects. VPA toxicity can present with symptoms ranging from drowsiness and tremor to severe complications such as coma and irreversible damage to liver function.

Factors such as liver enzyme activity, genetic polymorphisms, and drug-drug interactions can influence how VPA is metabolized in the body, necessitating frequent and accurate monitoring of blood levels to ensure proper dosing. In pediatrics, for example, metabolic rates may differ significantly from adults, influencing the drug's clearance and necessitating individualized therapy.

The accurate measurement of VPA is essential for both ensuring its therapeutic efficacy and minimizing adverse effects. Monitoring drug levels through serum measurements is the primary method for assessing whether the drug is within the therapeutic range. However, current methods, such as enzyme-linked immunosorbent assays (ELISA), liquid chromatography-mass spectrometry (LC-MS/MS), and gas chromatography (GC), are either resource-intensive, require specialized equipment, or lack the required specificity and sensitivity, leading to potential errors in result interpretation.

• Liquid Chromatography-Mass Spectrometry (LC-MS/MS): This method is considered the gold standard due to its high sensitivity and specificity. However, it is costly, time-consuming, and typically unavailable in routine clinical settings.
• Gas Chromatography (GC): While GC is highly accurate, it also requires significant sample preparation and expertise, making it less feasible for routine clinical use.

The need for a more reliable, and accessible method of measuring VPA levels has prompted interest in developing immunoassays based on monoclonal antibodies, which could overcome some of the challenges of traditional methods. Compared to LC-MS/MS and GC, monoclonal antibody-based assays are less technically demanding, more cost-effective, and can be easily adapted to high-throughput formats. Furthermore, they can be used in various testing environments, from centralized laboratories to point-of-care settings, making VPA monitoring more accessible to a broader range of patients. Although ELISA tests are more affordable and widely available, they suffer from cross-reactivity with structurally similar compounds, such as other fatty acids or metabolites, which can skew results.

Monoclonal antibodies (mAbs) are highly specific and can be engineered to recognize specific epitopes on a target molecule, such as VPA. The application of mAbs for the development of immunoassays offers several advantages over traditional analytical methods.

Since VPA is a non-immunogenic hapten, it must be previously conjugated to a carrier to trigger a humoral response. To keep the VP structure, a VP analogue (VPA) with a NH2 function was used to form the conjugate with the BSA or KLH carriers.

LOU/C rats (SYnAbs proprietary strain) were immunized intraperitoneously (IP) or in footpad (FP) with VPA-BSA or VPA-KLH using a confidential adjuvant (SYnAbs proprietary). The footpad response that is specific to the injected antigen permits to avoid the interferences of non-specific natural antigens that may reduce the response against the antigen of interest.

Immunization control (ELISA) of rats immunized with VPA-BSA were performed on VPA-KLH and vice versa.  The best immunized rats were rats immunized with VPA-KLH.

Fusion was carried out with the splenocytes of the best IP immunized rat and with the poplitea lymph nodes lymphocytes of the best FP immunized rat. The fusion was performed by electrofusion with the rat IR983 fusion cell line (SYnAbs proprietary myeloma).

Clones supernatants were first screened on VPA-BSA.

The positive clone supernatants were secondly screened in a competitive ELISA on VPA-BSA in presence of various concentration of free VP. The secondary mAb used for the screening was the mouse anti rat kappa light chain MARK-1 HRP (SYnAbs proprietary).

From 2000 screened clones, 45 clones deplaced by free VP were obtained.

3 clones (27, 30, 44) were selected for their capacity to be deplaced by the lowest free VP concentration. They were isotyped, developed and purified. To purify the rat mAbs, a rat specific MARK-1 chromatography column (SYnAbs proprietary) was used to avoid bovine IgG contaminants.

SYnAbs monoclonal antibodies offer unprecedented specificity in recognizing their target (lowest IC50 - 3.5µM). They have been designed to bind exclusively to VPA, without any cross-reactivity with similar substances. This point was crucial in maintaining the accuracy of the assay and ensuring that the results are not confounded by the presence of other compounds with similar molecular structures. Furthermore, since our rat-LOU hybridoma are stable, SYnAbs monoclonal antibodies can be produced in large quantities and stored for long periods, ensuring consistency and reproducibility in assays for our partners.

SYnAbs monoclonal antibodies have been used in a variety of assay formats for VPA detection, including:

• Immunoassays (ELISA): This method can offer high-throughput, affordable, and rapid testing with minimal sample processing.

• Biosensors: With the incorporation of SYnAbs mAbs in biosensor technology, point-of-care testing becomes feasible, providing rapid and convenient drug level monitoring, particularly in outpatient or home settings. Kinetic analysis performed using Bio-Layer Interferometry Technology (BLI). Rat/mouse monoclonal antibody coated in carboxylated sensor chip. VP-BSA increasing concentrations: 3.3 to 270nM (Sensorgram curves on the graph corresponds to 3.3nM of VP-BSA).

Recent advances in immunoassay technology have led to the development of highly sensitive and precise detection methods for VPA. These assays could revolutionize how clinicians monitor patient drug levels, offering a more accessible and less resource-intensive alternative to traditional techniques.

The structural similarity of VPA to other fatty acids presents a real challenge in generating monoclonal antibodies with high specificity. Cross-reactivity with metabolites or structurally similar compounds, such as valproic acid derivatives or other anticonvulsants, can lead to false readings. To overcome this, SYnAbs has designed particular hapten, modifying the structure of VPA to increase immunogenicity while minimizing cross-reactivity and identifying specific binding sites on VPA that are less likely to overlap with similar compounds, ensuring the generation of highly selective antibodies.

Therapeutic drug monitoring is critical for drugs like VPA, which exhibit significant interpatient variability. With the availability of monoclonal antibody-based assays, clinicians would be able to monitor VPA levels more efficiently and adjust dosages with greater precision, thus improving treatment outcomes and reducing the risk of side effects.

As personalized medicine continues to grow, the ability to measure drug levels in real-time using accessible immunoassays will enable healthcare providers to tailor treatments to individual patients' needs. This could be particularly valuable in pediatric and elderly populations, where the metabolism of drugs like VPA may differ substantially.

Integrating SYnAbs monoclonal antibody-based assays into wearable biosensors provide continuous monitoring of VPA levels in patients, ensuring that they remain within the therapeutic range. This is especially valuable for individuals with epilepsy, who need constant monitoring of drug levels to avoid breakthrough seizures or side effects.

Valproic acid is a vital therapeutic agent for managing epilepsy, bipolar disorder, and other conditions, but its narrow therapeutic range necessitates precise monitoring of blood levels to ensure optimal treatment. Traditional methods for measuring VPA levels have inherent limitations that can affect clinical outcomes. The development of monoclonal antibody-based immunoassays represents a promising solution to these challenges, offering high specificity, sensitivity, and ease of use. With continued innovation in antibody engineering and assay development, these methods will enhance patient care and allow for more personalized, effective treatment strategies. SYnAbs is proud to have been able to contribute to improving patient care thanks to its antibodies.

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