Pharmacokinetic (PK) Bridging ELISA Measuring Free Drug

Introduction

The PK bridging ELISA allows for the measurement of free drug in plasma using a kinetic format. Free drug is an important parameter when evaluating drug absorption, distribution, metabolism and excretion (ADME) pharmacokinetic properties. The PK bridging ELISA workflow begins by preparing standard curves with serial dilutions of a free drug stock solution to generate concentration-response curves in plasma samples spiked with known amounts of free drug and then comparing these curve fits with those obtained from standards containing different levels of free drug to determine if any reaction products interfered with the assay.

PK bridging ELISA workflow

ELISA plate coating:

• Coat ELISA plates with 100 µl of working solution per well in a 96-well plate (nonspecific binding). The working solution should be prepared by diluting the capture antibody in PBS with 0.1% BSA (a buffer used to keep the antibodies in solution).
• Incubate at room temperature for 1 hour or overnight at 4°C. Be sure not to disturb during incubation because any movement may cause air bubbles that could interfere with subsequent steps.

ELISA plate incubation:

• Dilute sample (drug plasma) and standards approximately 1:10-1:500 in drug-free plasma or buffer containing no drugs for background control. It is important that all samples be diluted equally so that each well receives an equal amount of drug from each sample and standard dilution curve can be obtained from these data later on. Do not use too much drug-free plasma/buffer because this will make it difficult for your machine to detect low amounts of drug present in samples later on!

Notably, if using human serum instead of rat serum as a diluent for your standards or samples then remember that human serum contains high concentrations of albumin which binds strongly to IgGs like those found on ELISA plates so it must be removed first before adding other reagents like antibodies used during assay development phase (e.,g., rat monoclonal antibodies). To remove albumin from human serums simply add one volume 10x blocking buffer per two volumes diluted serum volume(s); incubate gently at room temperature overnight (*do not shake vigorously as this may cause hemolysis*) after which centrifuge at 2000 g for 20 minutes; discard supernatant; resuspend pellet gently using same volume 10x blocking buffer again; centrifuge once more 20 minutes @ 2k rpm before resuspending pellet once more into appropriate amount ei

Materials

For this experiment, you will need:

• Plasma samples. Plasma samples for PK analysis must be collected from healthy volunteers. We recommend using 30 ml of plasma from each volunteer if the volume of your sample is too small or if you are unsure how much sample to collect. You can use blood collection tubes (e.g., EDTA or lithium heparin) to collect plasma samples and store them at 4°C until they are needed for testing; once they have been used in any part of your experiment, however, they should not be re-used.
• Free drug standard preparation. Prepare a stock solution of free drug by dissolving 1 mg/ml free drug (dissolved in DMSO) into 0.5 ml water then diluting this solution with 50 ml water to make 100 µg/ml free drug stock solution; store the stock solution at 4°C until it is needed for analysis (e.g., use within 2 weeks).

Free drug standard preparation

• Prepare a standard curve
• Prepare a blank and a sample
• Preparation of samples for ELISA

Free drug ELISA

The free drug ELISA uses a polyclonal antibody that reacts with the free drug, and a monoclonal antibody that reacts with the conjugated drug.

Data analysis

Data analysis was done using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA). The maximum concentration (C max ) and area under the curve from time 0 to 24 hours post-dose (AUC(0-24)) were calculated by linear interpolation of individual plasma concentration versus time data for each animal at each time point. The terminal elimination rate constant (K el ) was estimated using nonlinear regression of C(t) versus t data. AUC(0-∞) , which reflects the total exposure over a period of time, including both absorption and elimination phases and is a measure of drug availability to tissues or target sites, was determined as described above.

The PK bridging ELISA allows for the measurement of free drug in plasma

The PK bridging ELISA allows for the measurement of free drug in plasma, which is important to know because this is the active form of the drug.

Free drug is also called unbound or free-fraction bioavailable. It's important to know how much free drug you have in your system because it's the part that binds to plasma proteins and is available for therapeutic use by target organs.

Why do we need free drug measurement?

The need to measure free drug is a result of the fact that drugs are absorbed into the body in ways that are unpredictable. Drugs may be metabolized differently, distributed to different tissues and organs, or excreted through different routes.

How is free drug measured?

How is Free Drug Measured?

The assay is performed in a high-throughput format and can measure free drug in multiple samples at once. The assay measures free drug in plasma/serum, which is the body fluid that contains most of the drug at any given time. In order to measure free drug, we use an enzyme-linked immunosorbent assay (ELISA). An ELISA is like a test strip for drugs; it allows you to measure a specific substance using antibodies that bind specifically with it, much like how an antibody will attach itself to viruses and bacteria so they can be detected by your immune system.

Determining if an assay is suitable for a study.

When you are looking to measure free drug in your study, it is important to know whether an assay can measure free drug. If the assay cannot measure free drug, then it will not be suitable for your study.

You should be aware that some assays do not have the ability to measure free drug directly; they must first measure bound drug and then deconvolute this value into a percentage of bound drug versus unbound (free) therapeutic agent. These assays require complex calculations that may take significant time and resources at both the preclinical stage and clinical stage.

What should you expect?

You should expect the PK bridging ELISA to be a reliable and accurate method. The assay should be used in a way that is consistent with the manufacturer's instructions and the study protocol, including sampling frequency and volume, sample storage conditions, and assay procedure. As for any analytical method, you should also expect to receive good training before using it on your own samples.

Conclusion

The PK bridging ELISA is an important tool for measuring free drug in plasma. It is a sensitive and quantitative assay, which has been shown useful in determining if patients are taking their medications as prescribed. This method can be used to monitor compliance with a variety of drugs including antibiotics, antivirals, and antihypertensives.