Modification of Commercial Kit Assay Conditions May Be Necessary for

Increasing the Sensitivity of Biomarker Quantification: A Case StudyMatalin Shine, Deborah Martin and Masood Khan - KCAS, Shawnee, Kansas 66216 USA

Ghrelin is an appetite-stimulating peptide hormone. Several

commercial kits are available for quantification of Total and

Active Ghrelin. We set out to identify, select, and perform a Fit-

for-Purpose Validation of a commercial kit-based assay for

reliable quantification of Total Ghrelin in human plasma.

Appetite-stimulating peptide hormone Ghrelin exists in both

Active acylated [octanoyl group on serine-3] and Inactive un-

acylated forms. Metabolic effects of Ghrelin are mediated by

Active Ghrelin. Ratios of Total and Active Ghrelin are measured

as biomarkers in a variety of studies. In our quest for a reliable

kit for quantification of Ghrelin, we evaluated two commercial

kits from the same vendor. Though the kit for Active Ghrelin

worked well under assay conditions specified by the kit

manufacturer, significant changes in incubation conditions were

required to achieve optimum sensitivity of the ELISA kit for Total

Ghrelin. In this case study, a fit-for purpose modification of the

Total Ghrelin assay is described and analytical method

qualification characteristics of the assay are presented.

OBJECTIVE

INTRODUCTION

METHOD

RESULTS

Fig. 1: Ghrelin: An Appetite Biomarker

RESULTS (CONT.)RESULTS (CONT.)

CONCLUSION

Fig. 2: LLOQ Evaluation: Original Condition (A)

Fig. 3 : LLOQ Evaluation: Modified Condition (B)

Table 1: Dynamic Range of Quantification

Table 2: Precision of Measurements (Assay Controls)

Table 4: Assay Specificity

Table 7: Dilutional Linearity (Spiked Human Plasma)

Table 5: Short and Long-Term Stability in Human Plasma

• An ELISA kit for quantification of Total Ghrelin in human plasma

was used essentially as instructed by the manufacturer

(Condition A: 2 hours, RT). The kit manufacturer claimed a

theoretical minimum detectable concentration of 100 pg/mL.

However, assay sensitivity (LLOQ) at even 300 pg/mL could not

be achieved under these conditions.

• Alteration of the incubation conditions dramatically improved the

assay LLOQ to 200 pg/mL (Condition B: Overnight, 4 oC).

• Stabilized plasma control samples showed excellent

Inter- and Intra-assay precision of measurements.

• Concentrations determined in plasma control

samples were used as nominal concentration when

these samples were used for stability evaluation

experiments.

Table 6: Assay Selectivity (Addition Recovery)

• LQC and HQC samples were assayed in the absence (Baseline) and presence of Test Compounds (Glucagon and Insulin) at concentrations of 1200 and 4800 pg/mL, respectively.

Table 3: Precision of Measurements (Plasma Controls)

* Anchor Point

• Caution should be exercised in taking face value of assay

sensitivity claimed by the kit manufacturer. This may be a

theoretical value rather than the LLOQ of the assay.

• It may be necessary to have a clear understanding of the

assay design in order to make significant modifications in

assay conditions to achieve optimal method performance for

the intended use.

• The modified method must then undergo a formal analytical

method qualification (Fit-for-Purpose Validation) prior to use

for study sample analysis.

• Khan, M., Adaptation of fit-for-purpose biomarker assay validation using

commercial kits: A CRO perspective. Drug Research 11/12: 33-34, (2010).

• Lee, JW et. al., Fit-for-Purpose Method Development and Validation for

Successful Biomarker Measurements. Pharm Research 23 (2): 312-28

(2006).

REFERENCES

Plasma Sample Stabilization• Active Ghrelin is extremely unstable in blood / plasma.

• Blood samples are drawn into chilled EDTA Vacutainer tubes

preloaded with Aprotinin (500 KIU/mL).

• The blood is promptly centrifuged and the plasma separated and

acidified with 100 µL of 1 N HCl/mL. Aliquots of stabilized plasma

samples are stored frozen at a temperature ≤-20 oC.

• Condition A: (Original Condition): Lower Limit of

Quantification (LLOQ) could not be achieved even at

300 pg/mL.

• Condition B: (Modified Condition): LLOQ dramatically

improved to 200 pg/mL.

* Nominal Conc. = 1750.9 pg/mL

S-GHRL- Stomach Ghrelin

H-GHRL – Hypothalamus Ghrelin

R – Receptor

AgRP-Agouti-Related Peptide

NPY - Neuropeptide Y

LEPT - Leptin * % Baseline conc. (981.1 & 1750.9 pg/mL for Control-1 and Control-2, respectively)