Multiple immune cell autophagy marker detection kit

Abstract

The application discloses an immune cell autophagy marker detection kit and belongs to the technical field of biomedical detection, comprising two kinds of fluorescent coding microsphere mixtures coupled with anti-LC3B-II and p62 / SQSTM1 capture antibodies respectively, a biotinylated detection antibody mixture, a streptavidin-phycoerythrin conjugate, six-point calibrators (S0-S5), a quality control product, a cell lysis buffer containing non-ionic and zwitterionic surfactants and 4 mol / L urea, and data analysis software based on LC3B-II / p62 natural logarithm ratio output autophagy activity comprehensive score.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to an immune cell autophagy marker detection kit based on fluorescently encoded microspheres, used to simultaneously and quantitatively detect two core autophagy marker proteins, LC3B-II and p62 / SQSTM1, in immune cells and output a comprehensive score of autophagy activity. Background Technology

[0002] Autophagy is a core metabolic process in eukaryotic cells that degrades damaged intracellular organelles and abnormal proteins via the lysosomal pathway, playing a crucial role in maintaining immune cell homeostasis and regulating innate and adaptive immune functions. Abnormal autophagy function is closely related to the development and progression of various diseases, including sepsis, autoimmune diseases, tumor immune escape, and transplant rejection. Therefore, accurate assessment of immune cell autophagy status is of great value for disease mechanism research and clinical translation.

[0003] Current methods for autophagy detection mainly include Western blotting, immunofluorescence microscopy, and transmission electron microscopy morphological analysis. While Western blotting can detect the lipid transformation of LC3B-I to LC3B-II and the degradation level of p62 / SQSTM1 protein, it can only detect one or two target proteins per experiment, requires a large amount of cell lysis buffer, and the semi-quantitative analysis results are significantly affected by the subjective influence of gel loading volume normalization. Immunofluorescence microscopy can observe the dynamic changes in the number of LC3B positive fluorescent spots, but it is difficult to achieve high-throughput quantitative analysis. Although transmission electron microscopy is the gold standard method for autophagosome morphological identification, its high instrument cost, long sample processing cycle, and extremely low throughput cannot meet the needs of clinical translation and large-scale drug screening.

[0004] Multiplex immunoassay technology utilizes polystyrene microspheres with different fluorescent codes as solid-phase carriers. Capture antibodies targeting different molecules are conjugated to the surface of these microspheres, each possessing a unique spectral address. Flow cytometry is used to simultaneously identify multiple microsphere types and quantify the binding signals of each target molecule in a single-tube reaction. This technology platform has been widely applied in cytokine profiling, screening for infectious pathogen antibodies, and multiplex quantification of tumor markers. However, its specific application to the simultaneous quantitative assessment of core biomarkers of autophagy in immune cells has not yet been reported.

[0005] In existing technologies, multiplex microsphere immunoassays are mainly developed for secreted proteins or cell surface antigens. These target molecules are usually present in free form in serum, plasma, or culture supernatant, and their concentration range and matrix effects are relatively controllable. In contrast, autophagy marker proteins are intracellular proteins. LC3B-II is anchored to the autophagosome bilayer structure via phosphatidylethanolamine, while p62 / SQSTM1 binds to ubiquitinated substrates in a multimeric form to form insoluble aggregates. The specific characteristics of these two proteins in subcellular localization, solubility, and conformational state impose different technical requirements on the composition of lysis buffer, antibody-epitaxygen juxtaposition, and signal detection sensitivity.

[0006] LC3B-II and p62 / SQSTM1 are currently recognized as core indicators for assessing autophagic flux. The LC3B-II / p62 ratio can effectively distinguish between two distinct cellular states: enhanced autophagy induction (elevated LC3B-II and decreased p62) and autophagic flux blockade (simultaneous accumulation of both). However, current techniques for detecting LC3B-II and p62 rely on separate measurements using Western blotting, which is cumbersome, has low quantitative accuracy, and limited throughput. Therefore, developing a standardized detection tool that can simultaneously quantify LC3B-II and p62 in a single-tube reaction and automatically output the ratio score has significant technical and clinical value.

[0007] US10119967B2 discloses a multiplex immunoassay method based on fluorescent microspheres, which couples fusion proteins to the surface of microspheres via AGT enzyme for simultaneous detection of antibodies against multiple infectious pathogens. WO2013119377A1 discloses an autophagy-inducible peptide and its activity detection method, which assesses autophagy flux by measuring LC3-II and p62 levels separately using Western blotting. None of the above-mentioned prior art involves specifically applying a multiplex microsphere immunoassay platform to the simultaneous quantitative detection of autophagy marker proteins in immune cells, nor does it disclose optimized lysis systems targeting the specific subcellular localization and conformational characteristics of intracellular autophagy proteins, or a comprehensive autophagy activity scoring algorithm integrating the LC3B-II / p62 ratio. Summary of the Invention

[0008] The purpose of this invention is to provide an autophagy marker detection kit for immune cells. This kit is based on a fluorescently encoded microsphere immunoassay platform, which can simultaneously and quantitatively detect the expression levels of two core autophagy marker proteins, LC3B-II and p62 / SQSTM1, in immune cells in a single tube reaction. It also outputs a comprehensive autophagy activity score through a built-in algorithm, thereby achieving a rapid, standardized, and quantitative assessment of the smoothness of autophagy in immune cells.

[0009] To achieve the above objectives, the present invention provides an immune cell autophagy marker detection kit, comprising the following components: The coded microsphere mixture consists of two different fluorescently encoded polystyrene microspheres, each with its own unique red and infrared fluorescence ratio spectral address. The surfaces of the two microspheres are covalently coupled with anti-LC3B-II monoclonal capture antibody and anti-p62 / SQSTM1 monoclonal capture antibody, respectively.

[0010] The biotinylated detection antibody mixture contains biotinylated detection antibodies targeting LC3B-II and p62 / SQSTM1, respectively. Each detection antibody recognizes an epitope on the corresponding target protein that is different from that of the capture antibody.

[0011] The streptavidin-phycoerythrin conjugate achieves fluorescence amplification of the detection signal through the high affinity binding of streptavidin to biotin.

[0012] The calibrator gradient dilutions were precisely prepared into six concentration gradients (S0~S5) using a recombinant protein mixture of two target proteins. S0 is the zero calibrator, i.e., the blank matrix control, and S1~S5 are five incremental concentration levels, covering the clinically relevant detection range of each biomarker.

[0013] Quality control samples are divided into three concentration levels: high, medium, and low. In high-concentration quality control samples, the content of each marker is in the upper 20% to 30% range of the calibration curve; in medium-concentration quality control samples, the content is in the middle 40% to 60% range of the calibration curve; and in low-concentration quality control samples, the content is between the detection limit and the lower limit of quantitation in the lower part of the calibration curve.

[0014] The cell lysis buffer contains a nonionic surfactant, an amphoteric surfactant, a mixture of protease inhibitors, and a mixture of phosphatase inhibitors. The nonionic surfactant is selected from octylphenyl polyoxyethylene ether at a concentration of 0.5% to 1.5%, and the amphoteric surfactant is selected from 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid at a concentration of 0.1% to 0.5%. The two work synergistically to maintain the native conformation of membrane proteins while gently lysing the cell membrane and effectively dissolving p62 protein aggregates.

[0015] The data analysis software is used to automatically identify the spectral address of each coded microsphere, read the reporter fluorescence intensity value of phycoerythrin on each microsphere, calculate the concentration of each biomarker protein based on the calibration curve, and output a comprehensive autophagy activity score. This score is calculated based on the natural logarithm ratio of the LC3B-II concentration to the p62 / SQSTM1 concentration. The specific calculation formula is AAS=ln(C~LC3B-II~ / C~p62~), where C~LC3B-II~ and C~p62~ are the measured concentrations of the two biomarkers, respectively. The higher the AAS value, the stronger the autophagy activity. An AAS value close to zero or a negative value indicates that autophagy flux is blocked.

[0016] Compared with existing technologies, the beneficial effects of this invention are reflected in the following aspects. First, by coupling capture antibodies of two core autophagy marker proteins, LC3B-II and p62 / SQSTM1, to different fluorescently encoded microspheres, the core indicator pair reflecting autophagy flux can be simultaneously detected in a single 50 μL reaction system. The sample volume is only one-tenth that of traditional protein immunoblotting, significantly improving the detection throughput. Second, the synergistic ratio of nonionic and zwitterionic surfactants in the dedicated cell lysis buffer, combined with the denaturing and solubilizing effect of 4 mol / L urea, solves the problem of simultaneous and efficient extraction of membrane-anchored LC3B-II and insoluble p62 aggregates. Third, the comprehensive autophagy activity scoring algorithm is designed using the natural logarithmic ratio of LC3B-II / p62, which eliminates systematic errors caused by fluctuations in cell lysis efficiency and sample volume, and effectively distinguishes between two distinct cell states: enhanced autophagy induction and blocked autophagy flux, outputting intuitive numerical assessment results of autophagy status. Fourth, the six-point calibration system (S0~S5) conforms to the industry standards for clinical immunoassay testing, facilitating seamless integration with existing immunoassay analyzers and laboratory workflows. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the detection principle of the immune cell autophagy marker detection kit of the present invention.

[0018] Figure 2 The bar chart shows the effect of five different lysis buffer ratios on the extraction and recovery rates of two target proteins, LC3B-II and p62 / SQSTM1.

[0019] Figure 3 The four-parameter logistic regression six-point calibration curves for two autophagy markers, LC3B-II and p62 / SQSTM1.

[0020] Figure 4 A bar chart showing the autophagy activity composite score (AAS) of peripheral blood mononuclear cells under different treatment conditions.

[0021] Figure 5 A comparison of AAS scores for four immune cell subsets under basal culture conditions and rapamycin-induced conditions.

[0022] Figure 6 Line graph showing the effect of different urea concentrations on the signal retention rates of LC3B-II and p62 / SQSTM1. Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The core technical solution of this kit includes the preparation and antibody conjugation of two types of encoded microspheres, optimization of the cell lysis system, establishment of a six-point calibration system, and construction of a comprehensive autophagy activity scoring algorithm. For example... Figure 1 As shown, the basic principle of the detection process is as follows: two different fluorescently encoded polystyrene microspheres are coupled with capture antibodies targeting LC3B-II and p62 / SQSTM1, respectively. After specifically binding to the target proteins in the cell lysate samples, biotinylated detection antibodies and streptavidin-phycoerythrin conjugates are added sequentially to form a sandwich immune complex. Finally, a dual-laser detection system is used to identify the microsphere type (classification channel) and quantify the target protein binding signal (reporter channel), respectively, to achieve simultaneous quantitative detection of the two core autophagy marker proteins and calculation and output of the comprehensive autophagy activity score.

[0024] Regarding the epitope selection strategy for capture and detection antibodies, the core design principle of this invention is to ensure that capture and detection antibodies for the same target protein recognize two non-overlapping epitopes, forming a classic sandwich immunoassay configuration. For the detection of LC3B-II, the capture antibody selects clones that recognize the lipid-specific new epitope exposed after phosphatidylethanolamine modification of the C-terminus of the LC3B protein. The cross-reactivity rate of this antibody with the LC3B-I form (i.e., the cytoplasmic soluble form without lipid modification) is verified by enzyme-linked immunosorbent assay (ELISA) to be no more than 2%, thereby ensuring that the detection signal specifically reflects the true level of lipid-modified LC3B-II. The detection antibody recognizes a linear epitope in the conserved N-terminal region of the LC3B protein. This epitope is fully exposed in both LC3B-I and LC3B-II forms and does not affect its pairing with the lipid-specific capture antibody. For the p62 / SQSTM1 protein, the capture antibody recognizes the conformational epitope of the PB1 domain of the p62 protein, and detects the linear epitope near the UBA domain. This pairing scheme can simultaneously capture soluble monomeric p62 and p62 molecules that have been polymerized but released after being depolymerized by the lysis buffer.

[0025] Regarding the optimized design of cell lysis buffer, this invention develops a dedicated lysis system targeting the specific subcellular localization and solubility characteristics of two autophagy marker proteins, LC3B-II and p62 / SQSTM1. The base buffer components of this lysis buffer are 50 mmol / L Tris-HCl (pH 7.4), 150 mmol / L sodium chloride, and 5 mmol / L ethylenediaminetetraacetic acid. The surfactant system consists of 1% octylphenyl polyoxyethylene ether and 0.25% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid. The former acts as a nonionic detergent to effectively disrupt the phospholipid bilayer of the cell membrane and endometrial system, while the latter acts as a zwitterionic detergent to gently dissolve lipid-modified LC3B-II on the autophagosome membrane while maintaining the immunoreactivity of the p62 protein PB1 domain conformational epitope. The protease inhibitor component contains 1 mmol / L benzyl sulfonyl fluoride, 1 μg / mL leuprolide, 1 μg / mL aprotinin, and 1 μg / mL gasstatin A, used to prevent the degradation of target proteins by acidic hydrolases released from lysosomes during cell lysis. The phosphatase inhibitor component contains 1 mmol / L sodium orthovanadate and 10 mmol / L sodium fluoride, used to maintain the p62 protein Ser 403 The integrity of the phosphorylation modification state at the site was maintained. In addition, 4 mol / L urea was added to the lysis buffer as a denaturing solubilizer to dissociate the p62 multimer aggregates into monomeric or oligomeric forms that can be recognized by the capture antibody. This concentration of urea was verified not to affect the binding activity of LC3B-II to the corresponding antibody.

[0026] In the experiment to optimize the surfactant ratio of the lysis buffer, human peripheral blood mononuclear cells were induced to autophagy with rapamycin (100 nmol / L, treated for 4 h), and 1×10⁻⁶ cells were collected. 6 Cells were lysed using five different lysis buffers with varying ratios. Ratio 1 consisted of 1% octylphenyl polyoxyethylene ether alone; Ratio 2 consisted of 1% octylphenyl polyoxyethylene ether plus 0.1% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid; Ratio 3 consisted of 1% octylphenyl polyoxyethylene ether plus 0.25% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid; Ratio 4 consisted of 1% octylphenyl polyoxyethylene ether plus 0.5% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid; and Ratio 5 was a strongly denaturing lysis buffer containing 1% sodium dodecyl sulfate as a control. All lysis operations were performed on ice. After adding the lysis buffer, the cells were vortexed for 15 seconds and incubated on ice for 30 minutes, vortexing every 10 minutes. The supernatant was then collected by centrifugation at 14000g for 15 minutes. The protein extraction efficiency of each lysis buffer was verified by Western blotting. like Figure 2As shown, formulation 3 (i.e., 1% octylphenyl polyoxyethylene ether plus 0.25% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid) exhibits the best overall extraction efficiency for both target proteins. Specifically, under formulation 3, the recovery rate of LC3B-II reaches 92% of that under formulation 5, significantly better than the 78% of formulation 1 (due to the lack of zwitterionic detergent to aid in the dissolution of membrane proteins). For p62 / SQSTM1, the recoveries of formulations 1 to 4 are all in the range of 82% to 86%. Although the strong denaturing conditions of formulation 5 result in the highest recovery rate (100%), residual SDS severely interferes with the subsequent antigen-antibody reaction on the microsphere surface, thus making it unsuitable for this detection system. Considering both the extraction efficiency of the two target proteins and compatibility with the microsphere immunoassay platform, formulation 3 was selected as the final formulation of the lysis buffer in this invention.

[0027] Regarding the establishment of the six-point calibration system, six concentration gradient calibrators (labeled S0-S5) were prepared using a mixture of recombinant proteins of two target proteins in phosphate-buffered saline containing 1% bovine serum albumin. S0 was the zero calibrator, containing no target protein, used to determine the baseline signal of the standard curve and assess system background noise. S1 to S5 were five incremental concentration levels. The LC3B-II recombinant protein used was a full-length human LC3B-phosphatidylethanolamine conjugate modified by in vitro lipolysis to ensure the calibrator's immunoreactivity was consistent with that of LC3B-II in natural samples, with S1 to S5 concentrations of 0.15, 0.5, 2.0, 10, and 50 ng / mL, respectively. p62 / SQSTM1 used full-length human p62 recombinant protein, with S1 to S5 concentrations of 0.7, 3.0, 15, 60, and 200 ng / mL, respectively. Figure 3 As shown, regression analysis was performed on the six calibrator concentration points using a four-parameter logic (4-PL) fitting model, and the coefficient of determination R of the standard curve was [value missing]. 2The value should be no less than 0.99. This six-point calibration system (S0~S5) is designed in accordance with clinical immunoassay industry standards and is consistent with the calibration specifications of mainstream immunoassay reagents on the market, facilitating the integration of laboratory personnel's operational habits and the standardized management of instrument calibration procedures. The S0 zero calibrator provides a reliable anchoring reference point at the lower end of the standard curve, helping to accurately determine the lower asymptote parameters in the four-parameter logistic regression model, thereby improving the quantitative accuracy in the low concentration range. In practice, the median fluorescence intensity value of the S0 zero calibrator represents the inherent background signal of the system, including the combined contribution of non-specific adsorption on the microsphere surface and the fluorescence of the reagent itself; this value typically does not exceed 50 MFI units. The four parameters of the four-parameter logistic regression model are the lower asymptote (anchored by S0), the upper asymptote (determined by the signal saturation level in the S5 region), the inflection point concentration (corresponding to the maximum slope of the standard curve), and the slope factor (reflecting the steepness of the curve). The inflection point concentration of the LC3B-II channel is approximately 2.5 ng / mL, and that of the p62 / SQSTM1 channel is approximately 18 ng / mL. This concentration range precisely corresponds to the most common detection concentration range of these two biomarkers in immune cells under physiological and pathological conditions, ensuring that clinical sample testing is performed within the range of highest standard curve sensitivity. The standard curve for each assay plate is fitted independently, and the software automatically determines the goodness of fit. When R0... 2 The system will automatically alert you to a calibrator malfunction and recommend recalibrating if the residual value of any calibrator falls below 0.99 or exceeds 15% of the expected value.

[0028] Regarding the design of quality control samples, three concentration levels of quality control samples were prepared independently of the calibrators, using a mixture of lysates from autophagy-induced immune cell lysates, which were then lyophilized and aliquoted for storage. In the high-concentration quality control samples, the levels of each marker were located in the upper 20%–30% range of the calibration curve; in the medium-concentration quality control samples, they were located in the middle 40%–60% range of the calibration curve; and in the low-concentration quality control samples, they were located between the limit of detection and the lower limit of quantitation at the lower end of the calibration curve. Each batch of experiments required simultaneous testing of the three concentration levels of quality control samples. The experimental results for this batch were considered valid only if the detected values ​​of each quality control sample fell within ±15% of the preset target value.

[0029] Regarding the testing procedure, place the immune cells to be tested (1×10⁶ cells recommended). 5 ~1×10 6After washing once with phosphate-buffered saline, add 200 μL of pre-chilled cell lysis buffer and lyse under the above conditions. Collect the supernatant as the test sample. Add 25 μL of detection buffer and 25 μL of coded microsphere mixture working solution (containing approximately 2500 microspheres of each type) to each well of a 96-well plate, followed by 25 μL of the test sample, calibrator, or quality control. Seal the plate and incubate at room temperature in the dark with shaking (500 rpm) for 2 h. After incubation, remove the supernatant using a magnetic separator and wash the microspheres twice with 200 μL of washing buffer. Add 25 μL of biotinylated detection antibody mixture working solution to each well and incubate at room temperature in the dark with shaking for 1 h. After two washes, add 25 μL of streptavidin-phycoerythrin conjugate working solution and incubate at room temperature in the dark with shaking for 30 min. After the final wash, resuspend the microspheres in 125 μL of driving solution and read the values ​​on a multiplex analyzer. The instrument identifies the category of each coded microsphere using a classification laser, and detects the fluorescence intensity of the target protein-detection antibody-streptavidin-phycoerythrin complex captured on the microsphere surface by a reporting laser. At least 50 events are counted for each type of microsphere, and the median fluorescence intensity is used as the basis for quantification.

[0030] Regarding the design principle of the comprehensive autophagy activity scoring algorithm, one of the core innovations of this invention is the development of a quantitative scoring system based on the LC3B-II / p62 ratio. In traditional methods, researchers typically interpret the absolute levels or relative changes of LC3B-II and p62 separately, lacking a unified numerical evaluation standard. The comprehensive autophagy activity scoring (AAS) algorithm of this invention is constructed based on the following biological principles: The LC3B-II / p62 ratio is a core indicator reflecting the degree of autophagy flow: when autophagy induction is enhanced and autophagy flow is unimpeded, LC3B-II levels increase while p62 levels decrease due to autophagy degradation, and the ratio increases significantly; conversely, when autophagy flow is obstructed, LC3B-II and p62 accumulate simultaneously, with a smaller change in the ratio, or even a decrease due to the faster accumulation rate of p62. Using the natural logarithmic transformation AAS=ln(C~LC3B-II~ / C~p62~) to map this ratio to the continuous numerical domain has the following advantages: First, the natural logarithmic transformation transforms the multiplication and division relationship into an addition and subtraction relationship, making the statistical distribution of the AAS score closer to the normal distribution, which is convenient for subsequent parameter testing and analysis; Second, the ratio design eliminates the systematic errors caused by cell lysis efficiency and sample size fluctuations, making the results between different experimental batches directly comparable; Third, the positive and negative signs of the AAS value intuitively reflect the direction of autophagy (positive values ​​indicate enhanced autophagy activity, and negative values ​​indicate obstructed autophagy or reduced basal autophagy levels).

[0031] For the validation experiment of the AAS scoring system, peripheral blood mononuclear cells were isolated from the peripheral blood of healthy volunteers and prepared using density gradient centrifugation. Cell viability was confirmed to be greater than 95% by trypan blue rejection assay. Cells were divided into six groups: control group (no treatment), rapamycin-induced group (treated with 100 nmol / L rapamycin for 4 h), starvation-induced group (cultured in Earle balanced salt solution instead of complete culture medium for 2 h), chloroquine inhibition group (treated with 50 μmol / L chloroquine for 4 h), rapamycin plus chloroquine combined treatment group, and bafloxacin A1 treatment group (treated with 100 nmol / L for 2 h). After treatment, cells in each group were lysed and analyzed using the kit of this invention. Parallel validation was performed using traditional Western blotting as a reference method. Figure 4 As shown, the median AAS score of the control group was 0.08 (interquartile range -0.12 to 0.22), while the AAS score in the rapamycin-induced group significantly increased to 1.65 (interquartile range 1.32 to 1.93), and the AAS score in the starvation-induced group increased to 1.28 (interquartile range 1.05 to 1.54). The chloroquine inhibition group, due to autophagy flow blockade leading to simultaneous accumulation of LC3B-II and p62, with a greater degree of p62 accumulation, had an AAS score of -0.15 (interquartile range -0.38 to 0.08), lower than the control group. This indicates that the ratio algorithm of the AAS score effectively identified the autophagy flow blockade state and distinguished it from true autophagy-induced enhancement. The bafloxacin A1 treatment group showed a similar autophagy flow blockade pattern, with an AAS score of -0.08. The AAS score of the rapamycin plus chloroquine combined treatment group was 0.52, falling between the single induction and single inhibition groups, reflecting the contradictory state of enhanced autophagy induction signaling but blocked degradation pathways. The AAS score trends of the above six groups were highly consistent with the gray-scale quantitative analysis results of Western blotting (Spearman correlation coefficient rs = 0.96, P < 0.001), and the consistency between the two was even better than that of the multi-marker weighted model. This is because the dual-marker ratio scheme eliminates the noise contribution from the variation in the detection of other markers.

[0032] For system validation of detection performance, the analytical sensitivity of the kit of this invention is characterized by the lower limit of detection (LoD) and lower limit of quantitation (LoQ) of each biomarker. LoD is defined as the concentration value corresponding to the average signal value of 20 repeated determinations of the S0 zero calibrator plus twice the standard deviation. LoQ is defined as the lowest quantifiable concentration with a coefficient of variation not exceeding 20%. Validation showed that LC3B-II had a LoD of 0.08 ng / mL and a LoQ of 0.15 ng / mL; p62 / SQSTM1 had a LoD of 0.35 ng / mL and a LoQ of 0.72 ng / mL. These sensitivity levels cover the concentration range of the two biomarkers at both basal and induced autophagy levels in immune cells. Precision validation was performed using three concentration levels of quality control samples, repeated 20 times within each batch and between batches across five independent batches. The intra-batch coefficient of variation for both biomarkers did not exceed 7%, and the inter-batch coefficient of variation did not exceed 10%. Recovery validation was performed by adding high, medium, and low levels of recombinant protein standards to cell lysates of known concentrations. The recoveries of LC3B-II were 91%–108%, and those of p62 / SQSTM1 were 88%–112%, both within the acceptable range of 85%–115%. Linear dilution experiments showed that the linear regression ratio (R0) between measured and expected values ​​after serial dilutions of high-concentration samples from two to sixteen times was [value missing]. 2 All are greater than 0.98.

[0033] To verify the cross-reactivity of the dual-channel detection, a single high concentration of recombinant protein (twice the S5 concentration of each marker) was added to the reaction system, and the signal response of the non-target channel was detected. The results showed that when 100 ng / mL of LC3B-II recombinant protein was added, the signal value of the p62 channel was only 1.08 times the S0 background value of that channel, not exceeding 50% of the corresponding LoD signal value. Conversely, when 400 ng / mL of p62 recombinant protein was added, the signal value of the LC3B-II channel was 1.12 times the S0 background value, also below 50% of the LoD signal value. These results confirm that there is no significant cross-reactivity between the two detection channels. This is attributed to the fundamental differences in protein sequence, molecular weight (approximately 16 kDa and 62 kDa, respectively), and structural characteristics between LC3B-II and p62 / SQSTM1, as well as the rigorous pairing and screening of the capture and detection antibodies. It is worth emphasizing that the dual-channel design has a natural advantage over multi-channel detection systems in terms of cross-reactivity control: the fewer the number of channels, the fewer the potential interference combinations between channels, and the system complexity is reduced exponentially. The dual-channel system of this invention only needs to verify the cross-reactivity of one direction pair (LC3B-II→p62 and p62→LC3B-II), while the five-channel system needs to verify twenty direction pairs, which greatly simplifies the quality control process and improves the long-term stability of the detection system.

[0034] To validate the suitability of the kit for different immune cell subsets, CD4+ cells were isolated from peripheral blood of healthy volunteers. + T cells, CD8 + T cells, CD14 + Monocytes and CD56 + Natural killer cells, with purity greater than 90% in each subpopulation. The levels of two autophagy markers and AAS scores of each subpopulation were measured under basal culture conditions and rapamycin (100 nmol / L, 4 h) induction conditions. Figure 5 As shown, CD14 under basic conditions + The median AAS value for monocytes was 0.25, higher than that for CD4. + 0.05% of T cells and CD8+ + The T cell count was 0.07 (P<0.01), which is consistent with the biological characteristics of the monocyte-macrophage system, which relies on autophagy to maintain antigen presentation and intracellular pathogen clearance. Following rapamycin induction, CD4... + The increase in AAS was most significant in T cells (ΔAAS=1.72), while that in CD56 cells was significantly higher. + The relatively small increase in natural killer cells (ΔAAS=0.95) suggests that different immune cell subsets exhibit varying sensitivities to autophagy responses to mTOR inhibition. These results indicate that the kit of this invention can accurately capture the heterogeneity of autophagy states among different immune cell subsets.

[0035] Regarding the implementation of the autophagy flux index (AFI) module, when users need to assess the patency of autophagy flow, cell samples from the same source can be divided into two equal parts. One part is pretreated with 100 nmol / L bafloxacin A1 (a type V proton pump ATPase inhibitor that leads to autophagosome accumulation by blocking the fusion of autophagosomes and lysosomes) 30 min before lysis; the other part serves as an untreated control. Both samples are simultaneously detected using the kit of this invention after lysis. The autophagy flux index module in the data analysis software automatically calculates AFI = (C~LC3B-II, +The ratio of Baf~-C~LC3B-II,-Baf~) / C~LC3B-II,-Baf~ represents the autophagic flux. A higher AFI value indicates more unimpeded autophagic flux, meaning more LC3B-II is being continuously produced and consumed through lysosomal degradation. An AFI < 0.5 indicates significantly impaired autophagic flux, an AFI between 0.5 and 2.0 represents normal autophagic flux, and an AFI > 2.0 indicates significantly enhanced autophagic flux. The clinical value of this module lies in helping to distinguish between two distinct cellular states: enhanced autophagy induction (elevated AAS and AFI) and impaired autophagic flux (low or negative AAS but elevated absolute LC3B-II and decreased AFI), providing precise biological information for the development of subsequent intervention strategies.

[0036] Regarding the composition of the detection diluent and wash buffer, the detection diluent is used to prepare the working solution for the biotinylated detection antibody mixture and the streptavidin-phycoerythrin conjugate. Its composition is phosphate-buffered saline (pH 7.4) containing 1% bovine serum albumin, 0.05% sodium azide, and 0.1% polyoxyethylene sorbitan monolaurate. Bovine serum albumin acts as a blocking protein to reduce non-specific binding, while polyoxyethylene sorbitan monolaurate acts as a non-ionic surfactant to maintain the dispersion stability of the detection antibody. The wash buffer is a 10-fold concentrated solution, diluted with deionized water before use. The working solution composition is phosphate-buffered saline (pH 7.4) containing 0.05% polyoxyethylene sorbitan monolaurate.

[0037] Regarding the storage conditions and shelf life of the kit: the encoded microsphere mixture should be stored at 2–8°C protected from light, and the shelf life is twelve months from the date of preparation. Lyophilized calibrators and quality controls should be stored at 2–8°C, used immediately after reconstitution, or aliquoted and stored at -20°C, with no more than two freeze-thaw cycles. The biotinylated detection antibody mixture and streptavidin-phycoerythrin conjugate should be stored at 2–8°C and must not be frozen. The cell lysis buffer should be stored at -20°C, thawed on ice and thoroughly mixed before use. The concentrated wash buffer should be stored at room temperature (15–25°C).

[0038] Regarding the assessment of matrix interference effects, since the target proteins detected in this invention are derived from cell lysates rather than serum or plasma, interference factors such as hemolysis, lipemia, and jaundice in conventional immunoassays are not the primary considerations. However, genomic DNA released during cell lysis, high concentrations of intracellular free hemoglobin (for peripheral blood samples contaminated with erythrocytes), and detergent residues in the lysate can all potentially cause nonspecific interference with the antigen-antibody reaction on the microsphere surface. Therefore, this invention conducted a systematic interference assessment experiment. Different concentrations of salmon sperm DNA (simulating genomic DNA release, concentration gradients of 0, 50, 200, and 500 μg / mL) and hemolysin (concentration gradients of 0, 0.5, 2.0, and 5.0 mg / mL) were added to standard lysate samples containing two target proteins at known concentrations. The results showed that salmon sperm DNA at a concentration of 500 μg / mL produced approximately 8% positive bias to the p62 / SQSTM1 channel, presumably due to nonspecific interactions between DNA-binding proteins and the PB1 domain of p62. To eliminate this interference, it is recommended that users selectively add 25 U / mL benzo[a]nuclease for 5 min after lysis to degrade the released nucleic acids. Hemolysin at a concentration of 5.0 mg / mL introduces approximately 6% negative bias to the LC3B-II channel, but this is below the methodologically acceptable ±15% range and does not affect the clinical interpretation of the results. The effect of detergent residue in the lysis buffer on the activity of the microsphere antibody has been effectively mitigated by the dilution step in the assay buffer (samples diluted at least four-fold).

[0039] To verify the effect of urea concentration on the immunoreactivity of LC3B-II, since 4 mol / L urea was added to the lysis buffer for the dissociation of p62 aggregates, it was necessary to verify that this concentration of denaturant did not significantly affect the immunoreactivity of LC3B-II with the corresponding capture antibody. Two recombinant protein standards were dissolved in buffer systems containing 0, 1, 2, 4, and 6 mol / L urea, respectively, and three levels of standard concentration (low, medium, and high) were prepared for each concentration condition. The relative signal retention rate under each urea concentration condition was calculated with the detection signal value under the urea-free condition as a 100% baseline. Figure 6As shown, the signal retention rate of LC3B-II was 97% under 4 mol / L urea conditions, confirming that this concentration of urea does not affect the lipidation-specific epitope conformation of LC3B-II. The signal retention rate of p62 / SQSTM1 was 88%. The slight decrease in signal is due to the altered exposure efficiency of the PB1 domain conformational epitope after urea dissociates the p62 polymer, but the concentration of monomeric p62 actually increases, and the overall detection efficiency remains within acceptable limits. Notably, when the urea concentration increases to 6 mol / L, the signal retention rate of LC3B-II drops to 85%, and p62 drops to 76%, indicating that high concentrations of urea begin to have a significant adverse effect on the immunoreactivity of both proteins. Therefore, 4 mol / L urea represents the optimal balance concentration for balancing p62 aggregate dissociation efficiency and target protein immunoreactivity.

[0040] Regarding the assessment of the detection dynamic range and hook effect, the competitive binding of target proteins to capture and detection antibodies in high-concentration samples may produce a hook effect, where the signal value initially increases and then decreases with increasing concentration, leading to false low readings. To eliminate this risk, recombinant protein standards for the two target proteins were prepared at ultra-high concentrations exceeding the S5 concentration by 2, 5, and 10 times, respectively, for detection. Results showed that in both detection channels, the signal value of the 10-fold above-limit sample still maintained a monotonically increasing trend, with no signal reversion observed. This result indicates that within the recommended sample dilution range (at least four-fold dilution), the hook effect does not affect the accuracy of the detection results. However, for the p62 / SQSTM1 channel, intracellular p62 can accumulate significantly under prolonged autophagy inhibition conditions (e.g., chloroquine treatment for more than 24 hours). In such cases, it is recommended to appropriately increase the sample dilution factor and retest to ensure the reading falls within the effective range of the standard curve.

[0041] Regarding the application validation of the kit in peripheral blood immune cells of sepsis patients, sepsis is one of the most common causes of immune dysfunction in intensive care units. Patients' immune cells undergo an initial excessive inflammatory response followed by immunosuppression, and autophagy plays a crucial role in regulating immune cell survival and functional recovery. This example collected peripheral blood samples from twelve sepsis patients (within 24 hours of admission to the ICU) and eight age- and sex-matched healthy volunteers. Peripheral blood mononuclear cells were isolated and analyzed using the kit of this invention. The results showed that the median AAS score of peripheral blood mononuclear cells from sepsis patients was -0.22 (interquartile range -0.51 to 0.05), significantly lower than the 0.08 in the healthy control group (P<0.01), indicating that autophagic flux of immune cells was significantly impaired in the early stages of sepsis. Further analysis of the trends of the two biomarkers revealed that the LC3B-II level in the sepsis group was 38% lower than that in the control group, while the p62 level was 56% higher. Both exhibited a typical pattern of autophagic flux obstruction (reduced LC3B-II production and impaired p62 degradation leading to accumulation). The median AFI value calculated simultaneously was 0.32 (compared to 1.25 in the control group), further confirming the significant decrease in autophagic flux patency. The negative direction of the AAS score was significantly negatively correlated with the SOFA score (r=-0.72, P<0.01), suggesting that the degree of autophagic flux obstruction can reflect the severity of immune function impairment in sepsis patients. This application example demonstrates that the kit of this invention can rapidly and quantitatively reveal the abnormal autophagic state of immune cells in sepsis patients.

[0042] This study explores the application of the kit in monitoring the efficacy of tumor immunotherapy. Autophagy regulation is closely related to the functional state of immune effector cells in the tumor immune microenvironment. In this example, peripheral blood samples were collected from six non-small cell lung cancer patients receiving anti-PD-1 immune checkpoint inhibitor therapy before treatment, two weeks after treatment, and six weeks after treatment. CD8+ cells were isolated. + Longitudinal monitoring was performed using the kit of this invention after T-cell therapy. Results showed that, prior to treatment, patients' CD8 levels... + The median AAS score for T cells was 0.03, lower than that of the healthy control group (0.07), suggesting a lower basal autophagic flux of effector T cells in cancer patients. Two weeks after treatment, the AAS score increased to 0.35, and further increased to 0.58 at six weeks, a trend positively correlated with the patients' clinical efficacy assessment (RECIST criteria). In the four patients who responded to treatment (partial or complete remission), the six-week AAS score was greater than 0.45; while in the two patients whose disease progressed, the six-week AAS scores were 0.12 and 0.18, respectively, failing to recover effectively. This preliminary exploration suggests that peripheral blood CD8... +T-cell AAS score may serve as a candidate biomarker for predicting early efficacy of immunotherapy, but its clinical value still needs to be validated by larger-scale prospective cohort studies.

[0043] Accelerated aging tests were conducted on the stability of the encoded microspheres. The two types of antibody-conjugated microspheres were stored at 4°C (recommended storage conditions), 25°C (room temperature accelerated storage), and 37°C (high temperature accelerated storage), respectively. Samples were taken at days 0, 7, 14, 28, 60, and 90 to detect the capture antibody activity (assessed using the signal response value of the medium-concentration standard S3) and spectral encoding stability (assessed using classification accuracy). The results showed that after 90 days of storage at 4°C, the signal retention rate of both microspheres was above 92%, and the classification accuracy remained above 99%. After 28 days of storage at 25°C, the signal retention rate of the LC3B-II microsphere decreased to 81%, below the acceptable threshold of 85%, indicating that storage at room temperature for more than four weeks would affect detection performance. Signal attenuation was even more rapid under the 37°C high-temperature accelerated storage condition; after 14 days of storage, the signal retention rate of both microspheres was below 75%. These results support the light-protected storage conditions of 2–8°C specified in the product instructions and validate the reasonableness of the twelve-month shelf-life claim under the recommended storage conditions. The stability of lyophilized calibrators and quality control samples at 2–8°C has been verified to be maintained for at least eighteen months.

[0044] Regarding the in vitro stability verification of lysed samples, the stability of target proteins in the lysis buffer directly affects the detection window and the flexibility of experimental arrangements. In this example, peripheral blood mononuclear cell lysates induced by rapamycin were divided into multiple equal aliquots and incubated at 4°C and room temperature (25°C) for 0, 2, 4, 8, and 24 hours before detection. The 0-hour detection value was set as 100% baseline, and the retention rates of each marker at each time point were calculated. The results showed that within 8 hours of incubation at 4°C, the retention rates of LC3B-II were 98% and p62 / SQSTM1 were 93%, indicating that the protease inhibitor system in the lysis buffer effectively prevented degradation by endogenous proteases. However, after 24 hours of incubation at 4°C, the retention rates of LC3B-II decreased to 88%, and p62 decreased to 82%, indicating that non-enzymatic degradation or aggregation of target proteins began after prolonged incubation. At room temperature, the stability decay rate was even faster; after 4 hours, the retention rate of p62 had decreased to 87%. Therefore, it is recommended that users complete sample loading within 4 hours after cell lysis (this can be extended to 8 hours under temporary storage conditions at 4°C). For long-term storage, the lysis products should be aliquoted and stored at -80°C, with no more than two freeze-thaw cycles.

[0045] Regarding the traceability of batch-to-batch calibrator values, to ensure the comparability of test results from different batches of reagent kits and the consistency of long-term longitudinal monitoring data, the calibrator values ​​of this invention adopt a step-by-step transfer strategy. The primary reference is a high-purity recombinant protein stock solution of each target protein, which is assigned a mass concentration value (in mg / mL) through dual determination by amino acid analysis and ultraviolet absorbance. The secondary working calibrators are prepared by diluting the primary reference in a phosphate-buffered saline matrix containing 1% bovine serum albumin. After multiple repeated measurements, consensus target values ​​and allowable deviation ranges are assigned for each concentration point (S0~S5). Each newly produced batch of commercial calibrators must be parallel-validated using the secondary working calibrators as a reference. The deviation between concentration points between two batches must not exceed 10% before release. This traceability system ensures the standardization and comparability of test data from different time periods within the same laboratory and between different laboratories.

[0046] Regarding the output report format of the data analysis software, after completing the calculation of each biomarker concentration and the output of the AAS score, the software automatically generates a structured detection report. The report includes the original median fluorescence intensity values ​​of the two biomarkers, the parameters and coefficient of determination of the standard curve fitted by the four-parameter logistic regression (based on six-point calibration from S0 to S5), the calculated concentration values ​​of each biomarker and their reportable range determination, the percentage deviation of the detected values ​​of the three quality control samples from the target values ​​and their pass / fail determination, the AAS score value and its percentile position relative to the reference population distribution. When the user simultaneously uses the AFI function module, the report additionally outputs the autophagy flux index and its corresponding classification determination (blocked, normal, or enhanced). The software also has a built-in trend analysis function; when there are multiple longitudinal detection data for the same subject, it can automatically plot the time trend of LC3B-II concentration, p62 concentration, and AAS score, assisting researchers in assessing the dynamic trajectory of autophagy status.

[0047] Regarding key precautions and quality assurance measures for detection operations, the following technical points must be strictly observed during operation to ensure the reliability of the test results: The coded microsphere mixture must be vortexed for 30 seconds before use to ensure uniform dispersion of the microspheres and avoid imbalances in the ratio of the two types of microspheres due to sedimentation. Cell lysis should be performed entirely on ice. The lysis buffer must be pre-cooled to 4°C before adding the cell pellet to maximize the inhibition of endogenous protease and phosphatase activity. Light protection during incubation is a fundamental requirement for ensuring the stability of the phycoerythrin fluorescence signal, especially during the streptavidin-phycoerythrin conjugate incubation step and the final reading stage. After resuspending the microspheres in the driving solution before instrument reading, it is recommended to let the mixture stand for 2 minutes to allow air bubbles to dissipate, avoiding abnormal event counting caused by air bubbles interfering with the laser detection path. A blank control well (containing only the detection buffer and microsphere mixture, without sample) should be set up for each experiment to monitor the background fluorescence level. The median fluorescence intensity of the blank well should be less than 10% of the signal value of the lowest concentration S1 calibrator. In addition, it is recommended to set at least two sets of S0~S5 calibration curves and two sets of three-level quality control samples in each 96-well plate for intra-plate repeatability evaluation. When the deviation of any concentration point of the two calibration curves in the same plate exceeds 15%, the experimental results of that plate should be retested.

[0048] Comprehensive mechanistic analysis reveals a close synergistic relationship among the various technical features of the kit of this invention. The selection of the LC3B-II and p62 / SQSTM1 combination focuses on the core indicator pair for autophagy flux assessment. LC3B-II reflects the level of autophagosome formation and maturation, while p62 reflects the degradation efficiency of selective autophagy substrates. Changes in their ratio can accurately distinguish three key cellular states: enhanced autophagy induction (LC3B-II↑ / p62↓, increased positive AAS value), autophagy flux blockage (LC3B-II↑ / p62↑, low or negative AAS value), and basal autophagy level (steady-state equilibrium between the two, AAS close to zero). The ternary combination of the dual surfactant system and 4 mol / L urea in the dedicated lysis buffer ensures that both membrane-anchored LC3B-II and insoluble p62 aggregates can be efficiently extracted to a detectable state, eliminating detection bias caused by the differences in the physicochemical properties of the two proteins. The AAS ratio design of ln(CLC3B-II / Cp62) eliminates systematic errors caused by fluctuations in cell lysis efficiency and sample size, making results directly comparable between different experimental batches and between different laboratories. The six-point calibration system (S0-S5) conforms to the industry standards for clinical immunoassay, ensuring accurate quantification of test results and standardized quality control. The synergistic integration of the above technical features enables the kit of this invention to achieve a complete technical closed loop from simultaneous quantification of dual biomarkers to comprehensive assessment of autophagic flux status, providing the core biological information required for clinical use while maintaining the practical operability of the detection protocol.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A kit for detecting autophagy markers in immune cells, characterized in that, include: The study included: a mixture of encoded microspheres composed of two different fluorescently encoded polystyrene microspheres, with anti-LC3B-II monoclonal capture antibody and anti-p62 / SQSTM1 monoclonal capture antibody covalently coupled to the surfaces of the two microspheres, respectively; a mixture of biotinylated detection antibodies containing biotinylated detection antibodies targeting the two target proteins, LC3B-II and p62 / SQSTM1; a streptavidin-phycoerythrin conjugate; a calibrator gradient dilution buffer prepared with a mixture of recombinant proteins from the two target proteins to six concentration gradients, including one zero calibrator and five incremental concentration levels; and quality control samples at high, medium, and low concentrations. Three concentration levels were used: the high-concentration quality control sample contained each marker in the upper 20%–30% range of the calibration curve; the medium-concentration quality control sample contained in the middle 40%–60% range of the calibration curve; and the low-concentration quality control sample contained in the lower limit of detection to the lower limit of quantitation. Cell lysis buffer was used, containing both nonionic and amphoteric surfactants. Data analysis software was used to automatically identify the spectral addresses of the encoded microspheres, calculate the concentrations of each marker protein based on the calibration curve, and output a comprehensive autophagy activity score, which was calculated based on the natural logarithmic ratio of the LC3B-II concentration to the p62 / SQSTM1 concentration.

2. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The mixture of encoded microspheres contains two different fluorescently encoded polystyrene microspheres with particle sizes ranging from 5.0 to 6.5 μm. Each microsphere possesses a unique spectral address due to the different ratios of red and infrared fluorescent dyes doped within it. The surface capture antibody density of each microsphere is (0.8–2.5) × 10⁻⁶. 5 One antibody molecule / microsphere.

3. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The anti-LC3B-II monoclonal capture antibody specifically recognizes the lipid-specific epitopes exposed after phosphatidylethanolamine modification of the LC3B protein, and the cross-reactivity rate with the LC3B-I form does not exceed 2%.

4. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The anti-p62 / SQSTM1 monoclonal capture antibody recognizes the conformational epitope of the PB1 domain of the p62 protein, and can simultaneously capture soluble monomeric p62 and p62 molecules released after depolymerization by lysis buffer.

5. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The cell lysis buffer contains octylphenyl polyoxyethylene ether as a nonionic surfactant at a concentration of 0.5%–1.5%, 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid as an amphoteric surfactant at a concentration of 0.1%–0.5%, and also contains a mixture of protease inhibitors, a mixture of phosphatase inhibitors, and 4 mol / L urea. The pH of the buffer is 7.2–7.

6.

6. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The calibrator gradient dilutions were prepared with six concentration gradients, labeled S0 to S5, where S0 is the zero calibrator and S1 to S5 are five increasing concentration levels. The concentration ranges for each target protein are as follows: for LC3B-II, S1 to S5 are 0.15, 0.5, 2.0, 10 and 50 ng / mL, respectively; for p62 / SQSTM1, S1 to S5 are 0.7, 3.0, 15, 60 and 200 ng / mL, respectively.

7. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The formula for calculating the comprehensive autophagy activity score is AAS=ln(C~LC3B-II~ / C~p62~), where AAS is the value of the comprehensive autophagy activity score, C~LC3B-II~ is the measured concentration of LC3B-II in ng / mL, C~p62~ is the measured concentration of p62 / SQSTM1 in ng / mL, and ln is the natural logarithm function.

8. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The data analysis software also includes an autophagy flux index module. When a user tests bafloxacin A1 pretreated and untreated control groups of the same source cell samples, this module automatically calculates the autophagy flux index AFI = (C~LC3B-II, + Baf~-C~LC3B-II,-Baf~) / C~LC3B-II,-Baf~; where AFI is the autophagic flux index, C~LC3B-II,+Baf~ is the measured concentration of LC3B-II in the bafloxacin A1 pretreatment group (ng / mL), and C~LC3B-II,-Baf~ is the measured concentration of LC3B-II in the untreated control group (ng / mL).

9. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The cross-reactivity rate between the LC3B-II detection channel and the p62 / SQSTM1 detection channel in the mixture of coded microspheres does not exceed 50% of the corresponding channel's lower detection limit signal value.

10. The immune cell autophagy marker detection kit according to claim 1, characterized in that, The coded microsphere mixture has a shelf life of twelve months under light-protected conditions at 2-8°C, while the lyophilized calibrators and quality control samples have a shelf life of eighteen months under the same conditions.

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