Lot-to-Lot Variance in Immunoassays-Causes, Consequences, and Solutions

Abstract

Immunoassays, which have gained popularity in clinical practice and modern biomedical research, play an increasingly important role in quantifying various analytes in biological samples. Despite their high sensitivity and specificity, as well as their ability to analyze multiple samples in a single run, immunoassays are plagued by the problem of lot-to-lot variance (LTLV). LTLV negatively affects assay accuracy, precision, and specificity, leading to considerable uncertainty in reported results. Therefore, maintaining consistency in technical performance over time presents a challenge in reproducing immunoassays. In this article, we share our two-decade-long experience and delve into the reasons for and locations of LTLV, as well as explore methods to mitigate its effects. Our investigation identifies potential contributing factors, including quality fluctuation in critical raw materials and deviations in manufacturing processes. These findings offer valuable insights to developers and researchers working with immunoassays, emphasizing the importance of considering lot-to-lot variance in assay development and application.

Keywords: critical quality attributes; immunoassay; lot-to-lot variance; quality control.

Figure 1

The impact of antibody aggregates on the sandwich (A), competitive (B), and indirect immunoassays (C). AE: acridinium ester; ALP: alkaline phosphatase; FITC: fluorescein-5-isothiocyanate; GNP: gold nanoparticles; HRP: horse radish peroxidase.

Figure 2

The purity determination of a recombinant monoclonal antibody against CTX-III by SEC-HPLC (a) and CE-SDS (b). CTX-III: C-terminal cross-linked telopeptides of type III collagen; SEC-HPLC: size exclusion column combined with high-performance liquid chromatography; CE-SDS: capillary electrophoresis sodium dodecyl sulfate.

Figure 2

The purity determination of a recombinant monoclonal antibody against CTX-III by SEC-HPLC (a) and CE-SDS (b). CTX-III: C-terminal cross-linked telopeptides of type III collagen; SEC-HPLC: size exclusion column combined with high-performance liquid chromatography; CE-SDS: capillary electrophoresis sodium dodecyl sulfate.

Figure 3

The purity determination of HRP-labeled monoclonal antibodies by SEC−HPLC. (a) antibody was labeled with a rapid HRP labeling kit (batch 1); (b) antibody was labeled with a rapid HRP labeling kit (batch 2); (c) antibody was labeled with a regular HRP labeling kit. HRP: horse radish peroxidase; SEC−HPLC: size exclusion column combined with high-performance liquid chromatography.

Figure 3

The purity determination of HRP-labeled monoclonal antibodies by SEC−HPLC. (a) antibody was labeled with a rapid HRP labeling kit (batch 1); (b) antibody was labeled with a rapid HRP labeling kit (batch 2); (c) antibody was labeled with a regular HRP labeling kit. HRP: horse radish peroxidase; SEC−HPLC: size exclusion column combined with high-performance liquid chromatography.

Figure 4

Two major additives to minimize the aggregation of protein are kosmotropes (a) and chaotropes (b). Adapted from Cytiva online course: Tips and troubleshooting: recombinant protein production.

Figure 5

Two weeks 37 °C stability comparison of PRO-C3 (a), PRO-C6 (b), and CTX-III (c) antibodies stored in different buffers. The PBS groups were the benchmark, whereas the non-stressed groups were positive controls. CTX-III: C-terminal cross-linked telopeptides of type III collagen; PRO-C3: the C-terminus of procollagen III N-terminal propeptide; PRO-C6: the C-terminus of type VIa3 collagen. BBS: 0.1 M borate buffered saline, pH 8, 0.9% NaCl; PBS: 0.1 M PBS, pH 7.4, 0.9% NaCl; SP2: 0.9% NaCl; SPR: 37 mM citrate, 125 mM phosphate, pH 6.0; SPR6: 37 mM citrate, 125 mM phosphate, pH 6.0, 0.9% NaCl; SPRN6.5: 29 mM citrate, 142 mM phosphate, pH 6.5, 0.9% NaCl; SPTN5: 50 mM Na-citrate, pH 6.0, 0.9% NaCl; SPRNE1: 37 mM citrate, 125 mM phosphate, pH 6.0, 0.9% NaCl, 25% ethylene glycol; SPRNZ5: 37 mM citrate, 125 mM phosphate, pH 6.0, 0.9% NaCl, 0.05% sulfobetaine; SPTNE5: 50 mM Na-citrate, pH 6.0, 0.9% NaCl, 25% ethylene glycol; SPTNZ5: 50 mM Na-citrate, pH 6.0, 0.9% NaCl, 0.05% sulfobetaine; No-stressed: 0.1 M PBS, pH 7.4, 0.9% NaCl, stored at −20 °C until the test.