Validation of Enzyme Immunoassay for Preclinical Pharmacokinetic Trials of Rituximab

An integral part of preclinical pharmacokinetic studies is the development of a bioanalytical method for determination of the drug in a biological fluid.The aim of the research was to assess the suitability of the test system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of rituximab in the blood serum of laboratory animals after intravenous administration of rituximab at a dose corresponding to the therapeutic dose in humans. Th test system was developed by the Scientific and Production Center Probiotech.Materials and methods: the determination of rituximab in biological samples was carried out using a two-stage sandwich-type ELISA, followed by detection based on horseradish peroxidase. The ELISA results were recorded using a microplate photometer at a wavelength of 450 nm.Results: the experiments helped to establish the detection limit (0.24 ng/mL) and the lower limit of quantitation (1.00 ng/mL) of rituximab in rabbit blood serum, they also demonstrated high selectivity of analyte determination in a multicomponent biological matrix. The mean rituximab concentration was within 14 % of the nominal value in the entire working range of the method. The within-run and between-run precision of the assay did not exceed 7.4 %, the total error of the method did not exceed 20.1 %. The linearity of dilution makes it possible to use the assay for the analysis of biological samples with a wide range of rituximab concentrations. The stability of the analyte in the rabbit blood serum was confirmed by storing samples for 6 hours at room temperature, for 50 days at —35 °C, and after 3 freeze-thaw cycles. The validated immunoassay was successfully used to determine the rituximab concentration in biological samples obtained in the rituximab pharmacokinetic trial in rabbits. The accuracy of the results was confirmed for the entire range of the determined concentrations; parallelism was demonstrated between the calibration curve and the results of analysis of serially diluted rabbit serum samples with the maximum concentration of rituximab.Conclusions: the proposed enzyme immunoassay test system can be used for quantitative determination of rituximab in the blood serum of laboratory animals, as it meets acceptance criteria for all validation parameters described in the international guidelines on validation of bioanalytical methods.

Download Full-text

Production of Antibodies and Development of Enzyme Immunoassay for Determination of Monensin in Biological Samples

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/70.2.201 ◽

1987 ◽

Vol 70 (2) ◽

pp. 201-205

Author(s):

Michael E Mount ◽

Douglas L Failla

Keyword(s):

Enzyme Immunoassay ◽

Immunosorbent Assay ◽

Biological Samples ◽

Enzyme Linked Immunosorbent Assay ◽

Coefficients Of Variation ◽

Highly Sensitive

Abstract Monensin is converted to monensin bromoacetate, which provides successful coupling to protein and allows production of monensinspeciflc rabbit antisera. A modified indirect enzyme-linked immunosorbent assay (ELISA) was developed, which is highly sensitive (2 ng/mL) to monensin determination. Monensin recovery was 53- 81% at 10 ppb in sera or urine, and 81-130% at 100 ppb in dichloromethane- extracted feces or water. Overall recovery was 98.8% with coefficients of variation from 6 to 52% over the range of monensin concentrations studied. This is the first immunoassay reported for the carboxylic ionophore antibiotics.

Download Full-text

The Development of a Turbidimetric Method for the Quantitative Determination of Antibiotics of the Aminoglycoside Group in Medications

Antibiotics and Chemotherapy ◽

10.37489/0235-2990-2020-65-7-8-37-41 ◽

2020 ◽

Vol 65 (7-8) ◽

pp. 37-41

Author(s):

E. N. Semenova ◽

S. I. Kuleshova ◽

E. I. Sakanyan

Keyword(s):

Quantitative Determination ◽

Quality Assessment ◽

Aminoglycoside Antibiotics ◽

Metrological Characteristics ◽

Streptomycin Sulfate ◽

Turbidimetric Method ◽

Validation Criteria ◽

Validation Parameters ◽

Validation Tests

A method for the quantitative determination of streptomycin sulfate in medicines by the turbidimetric method has been developedand validated. Based on the results of the experiments, it was found that the metrological characteristics of such validation parameters of the method as linearity, precision, and correctness do not exceed the validation criteria. Linearity was noted in the range of streptomycin concentrations from 3.75 to 8.43 μg/ml. The results of validation tests of the method for the quantitative determination of streptomycin indicate the prospects and feasibility of introducing the turbidimetric method into the domestic system for standardization and quality assessment of aminoglycoside antibiotics.

Download Full-text

Quantitative determination of lysozyme in the hemolymph of Mytilus edulis by the enzyme-linked immunosorbent assay (ELISA)

Marine Environmental Research ◽

10.1016/0141-1136(84)90080-1 ◽

1984 ◽

Vol 14 (1-4) ◽

pp. 229-243 ◽

Cited By ~ 3

Author(s):

S.A. Steinert ◽

G.V. Pickwell

Keyword(s):

Quantitative Determination ◽

Mytilus Edulis ◽

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay

Download Full-text

DEVELOPMENT AND VALIDATION OF IMMUNOENZYME TEST-SYSTEM FOR DETERMINATION OF 25-HYDROXYVITAMIN D IN BLOOD SERUM

Biotechnologia Acta ◽

10.15407/biotech9.02.028 ◽

2016 ◽

Vol 9 (2) ◽

pp. 28-36 ◽

Cited By ~ 1

Author(s):

A. O. Mazanova ◽

Keyword(s):

Blood Serum ◽

Test System ◽

Hydroxyvitamin D ◽

25 Hydroxyvitamin D ◽

Development And Validation

Download Full-text

Quantitative determination of clopidogrel and its metabolites in biological samples: A mini-review

Journal of Chromatography B ◽

10.1016/j.jchromb.2012.12.024 ◽

2013 ◽

Vol 917-918 ◽

pp. 48-52 ◽

Cited By ~ 9

Author(s):

Paul W. Elsinghorst

Keyword(s):

Quantitative Determination ◽

Biological Samples

Download Full-text

Quantitative determination of morphine in biological samples by gas-liquid chromatography and electron-capture detection

Journal of Pharmacy and Pharmacology ◽

10.1111/j.2042-7158.1975.tb09432.x ◽

1975 ◽

Vol 27 (3) ◽

pp. 172-176 ◽

Cited By ~ 64

Author(s):

BENGT DAHLSTRÖM ◽

LENNART PAALZOW

Keyword(s):

Liquid Chromatography ◽

Quantitative Determination ◽

Electron Capture ◽

Biological Samples ◽

Electron Capture Detection ◽

Gas Liquid Chromatography

Download Full-text

Prognostic value of levels of specific autoantibodies in patients of reproductive age with endometrial polyps

Medical alphabet ◽

10.33667/2078-5631-2021-26-33-36 ◽

2021 ◽

pp. 33-36

Author(s):

L. V. Tkachenko ◽

N. I. Sviridova ◽

I. A. Gritsenko ◽

S. N. Maksimov

Keyword(s):

Blood Serum ◽

Prognostic Value ◽

Reference Range ◽

Relative Content ◽

Reproductive Age ◽

Enzyme Linked Immunosorbent Assay ◽

Women Of Reproductive Age ◽

Endometrial Polyps ◽

Double Stranded Dna

The problem of endometrial polyps in women of reproductive age is one of the urgent problems of modern gynecology. The detection rate of PE according to the data of domestic and foreign scientists reaches 25–35%, and therefore they occupy a leading place in the structure of intrauterine pathology.The aim. To study the levels of specific autoantibodies (to double-stranded DNA; to TrM‑03 antigens and collagen) and to assess their prognostic value for the risk of PE formation and recurrence in patients of reproductive age.Materials and methods. Examination of 86 patients aged 18 to 45 years (average age was 34.1 ± 6.3 years), admitted for hysteroscopy, hysteroresection of PE. Study of the relative content of specific autoantibodies to double-stranded DNA; to TrM‑03 antigens and collagen in blood serum was carried out by the method of enzyme-linked immunosorbent assay using specialized reagent kits (ELI-P-Complex).Results. In the course of this study, it was found that in the overwhelming majority of cases (65.1%) PEs were manifested by various types of AMC. All patients with PE were diagnosed with a significant decrease in the level of autoantibodies to double-stranded DNA, which is a marker of apoptosis processes, as well as a statistically significant decrease in the levels of autoantibodies to platelet antigens TrM‑03. The profile of deviations in the level of autoantibodies to TrM‑03 from the reference range in the area of negative values correlated with an increase in the average level of autoantibodies to collagen.Conclusions. Determination of the levels of auto-ATs to double-stranded DNA in serum can be used as a marker for predicting the recurrent course of PE. Determination of the levels of auto-ATs to platelet antigens TrM‑03 and to collagen in blood serum can be used as markers for the development of AMC by the type of BMC or a combination of BMC and BMC in patients with endometrial polyps.

Download Full-text

P0301FIT FOR PURPOSE VALIDATION OF A CLINICAL ASSAY FOR THE DETECTION OF SERUM LEVELS OF GALACTOSE-DEFICIENT IMMUNOGLOBULIN A1 (GD-IGA1) IN PATIENTS WITH IGA NEPHROPATHY (IGAN)

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0301 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Laura Sponton ◽

Hulin Jin ◽

Markus Fluck ◽

Yusuke Suzuki ◽

Amy Kao

Keyword(s):

Quantitative Determination ◽

Human Serum ◽

Iga Nephropathy ◽

Calibration Curve ◽

Clinical Studies ◽

Serum Samples ◽

Freeze Thaw ◽

Healthy Donors ◽

Validation Parameters

Abstract Background and Aims Analysis of serum or plasma from patients with IgA nephropathy (IgAN) has confirmed the presence of elevated levels of circulating immune complexes containing Gd-IgA1 (Czerkinsky 1986). New sensitive and reasonably specific noninvasive tests are emerging to guide the therapeutic strategy that is applicable to all stages of IgAN (Suzuki 2014). Here we are reporting the fit for purpose validation of an ELISA method for the quantitative determination of Gd-IgA1 in human serum samples to support biomarker investigations in clinical studies of Merck KGaA, Darmstadt. Method The assay was developed based on a commercially available immunoassay kit. The dynamic range of the calibration curve was determined from 1.56 ng/mL (LLOQ) to 100 ng/mL (ULOQ). With a minimum required dilution of 200-fold and standard assay volume of 50.0 μL, the range of the method in matrix was from 312 ng/mL to 20, 000 ng/mL. In assay validation phase, multiple validation parameters were evaluated, which included minimum required dilution (MRD), calibration curve, matrix effect, Intra- & Inter run accuracy & precision, selectivity, and parallelism. Additional validation parameters include sample stability (short/long term, freeze-thaw) and batch-to-batch comparison. Results All samples measured for intra & Inter - assay precision, accuracy, fulfilled the specifications according to the acceptance criteria. The selectivity was assessed using blank serum matrix from 10 individuals: the result indicated that matrix components in serum did not interfere with the detection of Gd-IgA1. Parallelism assessment was performed successfully for both samples from healthy donors and IgAN patient samples up to dilution factor (DF) 3200 (serum samples from healthy donors were determined up to DF 1600). All DF-corrected results within the assay range were determined with %CV ≤ 30.0%. Batch to batch comparison was assessed successfully based on the known shelf life of the kit. Short term stability using QC samples were given for up to 24hrs at room temperature. Freeze-thaw stability was given for up to 3 cycles at -20°C±5°C and -75°C±15°C. The investigations were performed according to general guidelines for method validation and applicable regulations. The results of investigated validation parameters fulfilled the requirements and recommendations, generally accepted for bioanalytical projects. Conclusion The present validation qualified the method for the quantitative determination of Gd-IgA1 in human serum samples from clinical studies.

Download Full-text