Principles of Ria Data Management
Fuad S. Ashkar, Lelio G. Colombetti in Radiobioassays, 2019
When the microscopic binding constants are not equal, i.e., α ≠ β, an interaction between binding sites on the same antibody molecule is indicated. Such an interaction is known as “cooperativity” and results in nonlinear Scatchard plots. Negative cooperativity occurs when K1/K2>4 and results in the second binding site having decreased affinity. The result is a Scatchard plot which is concave upward (see Figure 15). Positive cooperativity occurs when K1/K2<4 and tends to create a plot that is concave downward. Positive cooperativity is thought to be responsible for the “hook” effect that occurs at low concentrations of ligand.45–48 The hook effect, which has been observed at high ligand concentrations24 is probably due to ligand heterogeneity.
The laboratory and imaging approaches to thyroid disorders
David S. Cooper, Jennifer A. Sipos in Medical Management of Thyroid Disease, 2018
Despite a trend toward assay standardization, the variability of results using differing Tg assays remains at at least 25% due to variations in the antithyroglobulin antibodies used and the molecular heterogeneity of Tg. Occasionally, immunometric assays may fail to detect very high serum Tg concentrations due to the so-called hook effect, in which the high concentrations of Tg bind to one antibody, preventing the formation of the two-antibody sandwich upon which the assay depends. If this effect is suspected, the sample should be reanalyzed after dilution. Another cause of a false-negative Tg in patients with differentiated thyroid cancer can be tumor production of variants of Tg that fail to be recognized by the antibodies used in an assay (86). Recently, thyroglobulin LC-MS/MS assays have been introduced that purport to circumvent the problem of anti-Tg antibody interference (87). However, recent data suggest that these assays are still capable of generating falsely low serum Tg levels in patients with known residual disease (88).
Drug Monitoring: Modern Approaches to Quality Assurance
Steven H. Y. Wong, Iraving Sunshine in Handbook of Analytical Therapeutic Drug Monitoring and Toxicology, 2017
The design of the drug testing system and selection of reagent components determine the extent of imprecision. Both the antibody concentration and the affinity constant used in the type of reagent system have some bearing on the precision. For a system using antibody coated for the solid phase, the amount of antibody coating will determine the extent of antigen binding. The coating process for the competitive assay (limited amount of antibody coated) may contribute to imprecision; thus, uncoated or partially coated tubes will contribute to falsely low values. Sometimes, the antibody conjugate contributes to the amount of signal generated, which in turn affects the sensitivity and precision of the assay. The type of substrate used may also contribute to the enzymatic reaction and the final color development or fluorescence. The actual assignment of the calibrator’s value and stability is another dimension that may contribute to imprecision. Other testing components—such as type of separation technique, incubation temperature, diluents, wash solutions, quench solutions, QC materials, pH, and ionic strength—may affect the precision. The detecting device component may also introduce imprecision (e.g., spectrophotometry and fluorometry). The curve-fitting models for data management systems could affect the total precision. Sometimes, the use of the logit-log model to linearize the calibration curve could have imprecision at both ends of the curve. Sample-related, nonspecific interference factors, as well as specific interferences such as heterophilic antibodies, may affect both precision and accuracy. Other factors are sample carryover and high-dose hook effect.
Data-driven quality assurance to prevent erroneous test results
Published in Critical Reviews in Clinical Laboratory Sciences, 2020
Bridgit O. Crews, Julia C. Drees, Dina N. Greene
Analytes with the physiologic capacity to span a large dynamic range have the potential to cause what is known as a hook effect, resulting in falsely low results when the analyte is present in high concentrations [23]. This can occur for some assays when the analyte present in a sample exceeds the number of binding sites available on the solid phase and/or detection antibodies, thereby minimizing the potential for each of the antibodies to bind the same analyte molecule. Susceptibility to the hook effect depends on assay design: homogenous assays are most prone to hook effect whereas heterogeneous assays are less likely to suffer from the hook effect since they often include a wash step to remove excess antigen prior to the introduction of the signal antibody. The mechanisms resulting in the hook effect have been extensively reviewed [23–25]. Methods for evaluating the hook effect do not require sophisticated data analysis, but do require a sample with a very high concentration of the analyte to evaluate. Specimens affected by the hook effect are often identified when the ordering provider contacts the laboratory with concerns over the result. Serial dilution of a specimen leading to an apparent increase in the concentration of analyte confirms the hook effect.
Mechanistic insights into the rational design of masked antibodies
Published in mAbs, 2022
Carolina T. Orozco, Manuela Bersellini, Lorraine M. Irving, Wesley W. Howard, David Hargreaves, Paul W. A. Devine, Elise Siouve, Gareth J. Browne, Nicholas J. Bond, Jonathan J. Phillips, Peter Ravn, Sophie E. Jackson
Some hook effect was observed at high concentrations (full raw data is presented in Figure S14). This can be caused by excessively high concentrations of the primary antibody simultaneously saturating both HER2 and the secondary antibodies. The high-dose hook effect occurs mostly (but not exclusively) in one-step immunometric (sandwich) assays, giving a decrease in signal at very high concentration of primary antibody.44,45,46 Therefore, the signal obtained is probably caused by insufficient washes between the incubation of the primary and secondary antibodies. The higher concentration points were therefore excluded from the fitting as they are not representative of the actual binding to HER2 and are artifacts of the protocol followed. The other data points were fitted to an agonist versus response fit using GraphPad Prism 9 and Equation 1:
Application of thyroglobulin and anti-thyroglobulin antibody combined with emission computed tomography in the adjuvant diagnosis of differentiated thyroid carcinoma
Published in Annals of Medicine, 2023
Nan Jiang, Benzheng Jiao, Laney Zhang, Jialong Li, Yungeng Li, Chenghe Lin
Considering that Tg levels are affected by TgAb, some scholars have proposed to reverse the influence of TgAb using the Tg recovery test. The results, however, are not satisfactory due to the pathophysiological changes of patients and experimental techniques. The hook effect, a common feature of most immunoassays, occurs when the antigen level is high, which results in a solid support for the binding capacity of the antibody, leading to false negative results [31]. Therefore, in 2006, the National Academy of Clinical Biochemistry (NACB) Guidelines proposed to abandon the recovery test and directly test TgAb instead [32]. At this stage, how to eliminate the interference of TgAb on Tg level detection remains a conundrum to decipher. The thyroid gland is known to have the function of absorbing iodine and concentrating iodine. Radioactive iodine, which is mostly distributed in the thyroid gland after entering the human body, can not only display the thyroid gland morphology, but also measure the iodine absorption rate of the thyroid gland. However, studies have found that some TCs have poor 131I uptake function [33]. Therefore, based on the principle of radionuclides, 131I with poor uptake should be avoided so as not to affect the diagnostic performance. Through cubital vein injection of radioisotope 99mTc, the ECT technique enables the formation of radioactive concentration differences between the lesion and the surrounding normal tissue, thereby providing imaging evidence for the diagnosis and differentiation of benign and malignant thyroid lesions. It is shown that due to the difference in radionuclide capacity between thyroid nodules and surrounding normal thyroid tissues, TCs have relatively low uptake capacity and are mostly manifested as cold and cool nodules [34]. In this study, we found that ECT alone was in good consistency with the pathological results in the diagnosis of DTC, with diagnostic sensitivity, specificity, and accuracy of 88.1%, 75.9%, and 83.8%, respectively, indicating that ECT can provide a reference for clinical diagnosis of DTC. Nonetheless, many studies have confirmed [35,36] that cold nodules with defective radioisotope distribution are usually malignant tumors, though not in all cases. Besides, ECT is unable to distinguish the cystic and solid nature of benign and malignant lesions, with some certain limitations in the diagnosis of TC.
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