Quantitative Range Research Articles

Development of Monoclonal Antibody-Based EIA for Tetranor-PGDM which Reflects PGD2 Production in the Body.

Tetranor-PGDM is a metabolite of PGD2. Urinary tetranor-PGDM levels were reported to be increased in some diseases, including food allergy, Duchenne muscular dystrophy, and aspirin-intolerant asthma. In this study, we developed a monoclonal antibody (MAb) and a competitive enzyme immunoassay (EIA) for measuring tetranor-PGDM. Spleen cells isolated from mice immunized with tetranor-PGDM were utilized to generate Ab-producing hybridomas. We chose hybridomas and purified MAb against tetranor-PGDM to develop competitive EIA. The assay evaluated the optimal ionic strength, pH, precision, and reliability. Specificity was determined by cross-reactivity to tetranor-PGEM, tetranor-PGFM, and tetranor-PGAM. Recovery was determined by spiking experiments on artificial urine. Optimal ionic strength was 150 mM NaCl, and optimal pH was pH 7.5. Metabolites other than tetranor-PGDM did not show any significant cross-reactivity in the EIA. The assay exhibited a half-maximal inhibition concentration (IC50) of 1.79 ng/mL, limit of detection (LOD) of 0.0498 ng/mL, and range of quantitation (ROQ) value of 0.252 to 20.2 ng/mL. The intra- and inter-assay variation for tetranor-PGDM was 3.9–6.0% and 5.7–10.4%, respectively. The linearity-dilution effect showed excellent linearity under dilution when artificial urine samples were applied to solid-phase extraction (SPE). After SPE, recovery of tetranor-PGDM in artificial urine averaged from 82.3% to 113.5% and was within acceptable limits (80%–120%). We successfully generated one monoclonal antibody and developed a sensitive competitive EIA. The established EIA would be useful for routine detection and monitoring of tetranor-PGDM in research or diagnostic body fluids.

PF188 NGS COMBINED WITH ASO REAL‐TIME PCR BASED MRD DETECTION ASSAY FOR PEDIATRIC ALL PATIENTS

Background:Monitoring of minimal residual disease (MRD) has become routine clinical practice in frontline treatment of pediatric acute lymphoblastic leukemia (ALL) patients. MRD diagnostics has proven to be the strongest prognostic factor, allowing for risk group assignment into different treatment categories, ranging from significant treatment reduction to mild or strong intensification. MRD diagnostics is also guiding treatment decisions in relapsed ALL patients and patients undergoing stem cell transplantation.Aims:Our aim is to develop and apply a sensitive (≤ 10–4), universal, accurate, reliable and fast assay for MRD detection in ALL patients. To date, flow cytometry and polymerase chain reaction (PCR) analysis of rearranged immunoglobulin and T‐cell receptor genes (allele‐specific oligonucleotide [ASO]‐PCR) are claimed to meet these criteria, but classical flow cytometry does not reach a solid 10–4, whereas classical ASO‐PCR is time‐consuming and labor intensive.Methods:LymphoTrack® IGH + TRG Assay – MiSeq is applied on diagnosis sample using the Illumina MiSeq platform. This is used for the identification of clonal IGH VH‐JH and TRG V‐J rearrangements, DNA sequences and the distribution frequency of V and J segment utilization. The clone sequence is then compared against the germline sequences contained in the IMGT V‐QUEST databases for the identification of patient‐specific junctional regions following by the design of six different D‐N‐J specific ASOs primers for each clone according to UKALL protocol. Five point standard curve is used with triplicates at 5x10^‐5 and 1x10^‐5 for more reliable results at the critical lower level and six pooled negative controls for confirmation of the specificity of the assay.Results:Forty pediatric ALL patients have been tested at several time points. All informative patient sample dilutions reached 5x10^‐5, and most of the patients reached 1x10^‐5. The R2 of standard curves were within the expected range (≥0.98), and all slopes within range (3.1–3.9) with an average of 3.21 +/‐ 0.16. In most negative control samples no amplification was detected. In 8 assays amplification was detected in two negative control samples. In these cases the CT value was more than 3 CT from the Quantitative Range (QR).Our coupled NGS‐ASO‐PCR method sensitivity was compared to Flow cytometry and Fusion Transcript Real Time PCR methods. NGS‐ASO based MRD method has detected MRD populations in several samples were Flow cytometry or real time PCR failed. None of the patient tested were positive by other methods and was negative by ASO based MRD method.Summary/Conclusion:In the ALL field, MRD has become part of diagnostic patient care. Consequently, standardized MRD diagnostics should be available for assessment of treatment response in each individual ALL patient, to be used for personalized medicine such as accurate risk group assignment with risk‐adapted treatment. The coupled NGS‐ASO‐PCR method seams to fulfill a series of requirements for acceptance in the field, such as broad availability, easy implementation, applicability in the vast majority of patients, sufficient sensitivity (quantitative range of 5x10^‐5) and fast (short turn‐around time, particularly for follow‐up samples).

Simulation model for predicting limit of detection and range of quantitation of competitive enzyme-linked immunosorbent assay

The limit of detection (LOD) and range of quantitation (ROQ) of competitive enzyme-linked immunosorbent assay (ELISA) were determined from a model describing the calibration curve and precision profile of the assay. The calibration curve is given by solving the differential equations describing the change in the concentrations of an antigen-antibody complex and an enzyme-conjugated antigen-antibody complex by a Runge-Kutta method. The precision profile is described in terms of possible error sources such as the pipetting volumes of the analyte, enzyme-conjugated antigen, antibody and substrate solutions, calibration curve and inherent absorbances between the wells in an ELISA plate. An appropriate concentration of the enzyme-conjugated antigen that balances a low detection limit and sufficient color development was found to be in a narrow range, which is consistent with the empirical rule. The optimum conditions for competitive ELISA using an antibody with a kinetic property can be designed from our model.

Validation of a Method for Predicting the Precision, Limit of Detection and Range of Quantitation in Competitive ELISA

The mathematical model for predicting the precision, limit of detection (LOD) and range of quantitation (ROQ) in a competitive enzyme-linked immunosorbent assay (ELISA) proposed by Hayashi et al. (Anal. Chem., 2004, 76, 1295) was validated. The model describes the relative standard deviation (RSD) of concentration estimates by the RSDs of pipetting volumes of analyte, enzyme-conjugated antigen, antibody and substrate solutions, and the standard deviation (SD) of inherent absorbances between the wells in an ELISA plate. For 6 kinds of direct competitive ELISA kits, the LOD and ROQ predicted by the model agreed well with those obtained by experiments with real samples. It was also confirmed that the model is applicable to the prediction of uncertainty that depends on the pipetting error of the viscous antiserum solution. The model was demonstrated to be useful for estimating the LOD and ROQ of competitive ELISA.

Precision, limit of detection and range of quantitation in competitive ELISA.

This paper develops a mathematical model for describing the within-plate variation as the RSD (relative standard deviation) of absorbance measurements in a wide concentration range in competitive ELISA and proposes a method for determining the limit of detection (LOD) and range of quantitation (ROQ). The ELISA for 17 alpha-hydroxyprogesterone is taken as an example. The theoretical RSD description involves analyte concentration as an independent variable and error sources as parameters which concern the pipetting and absorbance measurement. Our model can dispense with repeated experiments of real samples, but the error parameters should be determined experimentally. The theory is in good agreement with the experiments. The most influential error sources at low and high sample concentrations are shown to be the pipetting of a viscous solution of antiserum and the absorbance inherent to the wells of a plate, respectively. The LOD and ROQ are defined as the concentration with 30% RSD and the region with <10% RSD, respectively, and are found in the theoretical plot of the RSD of concentration estimates vs concentration.