key: cord-0023290-ptzkfha4
authors: Sun, Tao; Qu, Shufang; Huang, Tiancha; Ping, Ying; Lin, Qinyan; Cao, Ying; Liu, Weiwei; Wang, Danhua; Kong, Piaoping; Tao, Zhihua
title: Rapid and sensitive detection of L‐FABP for prediction and diagnosis of acute kidney injury in critically ill patients by chemiluminescent immunoassay
date: 2021-10-15
journal: J Clin Lab Anal
DOI: 10.1002/jcla.24051
sha: 162697d78f52bf42d6abb1c1bd6892fbba731cbb
doc_id: 23290
cord_uid: ptzkfha4

BACKGROUND: Acute kidney injury (AKI) was a common clinical complication among critically ill patients in Intensive Care Unit with high morbidity and mortality. Human liver fatty acid‐binding protein (L‐FABP) as a renal tubular injury biomarker was considered a predictor of AKI; however, high‐throughput and sensitive detection methods were still urgently needed. We constructed a sensitive and rapid detection method for detecting L‐FABP and for exploring the clinical application of L‐FABP as a predictor for AKI. METHODS: We developed an automated detection method of chemiluminescent immunoassay to measure L‐FABP and evaluated the analytical performance of the new methodology including analytical selectivity, analytical sensitivity, linear range, the minimum limit of detection (LOD), repeatability, and accuracy. One hundred patients were enrolled in this study to explore the predictive and diagnostic ability for AKI. RESULTS: The chemiluminescent immune‐based L‐FABP assay had outstanding analytical sensitivity including the detection limit of 0.88 ng/ml, and a wide linear range of 2 ng/ml to 160 ng/ml. It also exhibited excellent repeatability with intra‐analysis CVs of 8.73%, 4.72%, and 3.79%, respectively, and the inter‐analysis CVs of 13.47%, 7.28%, and 5.94%, respectively. The recovery rate assay exhibited a good accuracy with three L‐FABP concentration of 99.76%, 102.27%, and 96.92%, respectively. The reference interval of L‐FABP was between 0.88 ng/ml and 5.98 ng/ml. The evaluation of predictive and diagnostic performance showed that higher concentration of L‐FABP indicated higher risk of AKI occurrence and disease progression. CONCLUSIONS: The clinical application of rapid and sensitive detection method of L‐FABP based on the newly developed chemiluminescent immunoassay could offer benefits for patients. L‐FABP was a potentially predictive and diagnostic biomarker for AKI.

Acute kidney injury (AKI) is a common comorbidity of critically ill patients, which refers to a clinical syndrome characterized by a rapid decrease in renal excretory function, with the accumulation of products of nitrogen metabolism such as urea and creatinine and other clinical unmeasured waste products. 1 According to research statistics, 7% of hospitalized patients could have AKI, and the morbidity of AKI in the intensive care unit (ICU) was as high as 25%. 2, 3 However, there were no effective treatments currently except for supportive renal replacement therapy such as dialysis. 4 AKI is a common and important diagnostic and therapeutic challenge for clinicians. 5, 6 Although with the deepening of AKI research and new technologies and treatments are also emerging, such problems continue to appear. At present, the main diagnostic biomarkers of clinical reuse were serum creatinine (Scr) and urine volume. These two biomarkers had been used for many years. When kidney injury occurred, Scr due to the kidney's own compensation mechanism could not reflect the change of kidney very sensitively and would lag behind the progress of the disease. 7 The urine volume would be affected by clinical drugs, diuretics, and so on, which also could not truly reflect the progress of the kidney disease. 8, 9 The novel diagnostic technologies and biomarkers may help with early diagnosis. 10 Therefore, it was urgent and significant to find early sensitive and specific diagnostic biomarkers to improve the longterm survival rate and postoperative recovery of patients with AKI.

Human liver fatty acid-binding protein (L-FABP) was located as a cytoplasmic protein in proximal renal tubular cells of the human kidney, 11 renal L-FABP expression was upregulated, and urinary excretion of renal L-FABP was increased by various stressors, such as urinary protein, hyperglycemia, tubular ischemia, toxins, and salt-sensitive hypertension, which led to the progression of kidney disease. 12 L-FABP could be checked to indicate whether there was kidney damage. 13 A number of studies had shown that L-FABP had a good diagnostic value in kidney diseases including AKI, 14 chronic kidney disease, 15 and diabetic nephropathy. 16 However, few indicated whether L-FABP had the predictive value of AKI. 17, 18 So, it was vital to explore the predictive value of L-FABP for AKI. Early prediction of AKI was important for clinical practice. The aim of this research was to assess whether L-FABP could be a predictor for AKI.

Advances in enzyme-linked immunosorbent assay (ELISA) technology have enabled the high-throughput detection of L-FABP, making it possible for analyzing in many studies conveniently. 19, 20 However, common ELISA-based methods cannot satisfy the rapid detection requirements. The sufficient sensitivity and specificity of methodology are required for complicated procedures. Therefore, based on the platform of our laboratory, we constructed the new methodology of chemiluminescent immunoassay for detecting L-FABP and explored the predictive value of L-FABP for AKI in critically ill patients.

A photomultiplier instrument for chemiluminescent signal detection was developed by our laboratory. Chemiluminescent immunoassay instrument (Robust i1000) was provided by Hangzhou AiKang company. The incubation procedures were carried out using a constant temperature incubator. A magnetic separation device was purchased from Thermo Fisher Scientific (Invitrogen). Human liver fatty acidbinding protein (L-FABP) in its pure form used in this study was pur- Streptavidin-modified paramagnetic particles were purchased from Thermo Fisher Scientific (Invitrogen). N-hydroxysuccinimide biotin, acridinium ester, bovine serum albumin (BSA), phosphate-buffered saline (PBS), Tween-20, dimethylsulfoxide (DMSO), and anhydrous dimethyl formamide (DMF) were purchased from Sigma-Aldrich Ltd (USA). We prepared hydrogen peroxide and sodium hydroxide reagent. Washing buffer solution was made by PBS containing 1 mg/ml Tween-20 and 1 mg/ml BSA. Double-distilled water was prepared using water purified with an ultrapure water system (Zhejiang university second affiliated hospital). All other chemicals were standard commercial products of analytical reagent grade.

The inclusion criteria of this study was according to Kidney Disease Improving Global Outcomes (KDIGO) criteria patients were not diagnosed AKI when ICU admission. The exclusion criteria were patients having chronic renal diseases or patients who had undergone renal replacement such as dialysis or kidney transplant. The exit criterion was patients stayed in ICU less than 2 days. According to the Excluding 12 patients who stayed in ICU less than 2 days. Finally, 100 patients were included in the study. AKI was defined according to the KDIGO guidelines as renal function was suddenly decreased within 48 h and Scr increased at least 0.3 mg/dl, or Scr increased more than 1.5 times higher than baseline within 7 days, or urine volume less than 0.5 ml/Kg per hour for 6 hours. AKI was classified as stage 1 (Cr at peak was 1.5 to 1.9 times baseline), stage 2 (Cr at peak was 2 to 2.9 times baseline), and stage 3 (Cr at peak was 3 times baseline or greater). Ethical approval for this study was obtained from the research ethical committees, Zhejiang University School K E Y W O R D S acute kidney injury, chemiluminescent immunoassay, diagnosis, L-FABP, prediction of Medicine Second Affiliated Hospital. Blood samples were collected for all participants after they were admitted into ICU. Samples were collected every 24 h for 7 days. If patients were stayed in ICU less than 7 days, samples were stopped when out of ICU. All blood samples were centrifugated at 3000 rpm for 15 min to obtain plasma samples. Scr was measured by creatine oxidase method with Beckman Coulter AU5800 and L-FABP was measured by chemiluminescent immunoassay with Robust i1000. Plasma samples were stored for a maximum of 2 h at 4℃ before analysis. For L-FABP assay, all measurements were repeated twice. 

First, antihuman L-FABP antibody solution was diluted to 0.25 mg/ ml by 0.1 M PBS (PH:7.4). Then, the antibody solution was reacted with 10 μl acridinium ester solution (0.5 mmol/L) previously dissolved in anhydrous DMF. The mixed solution was reacted at room temperature for half an hour. The acridinium ester-labeled antibody solution was also purified by sephadex G-75 chromatography column. The purified antibody solution containing 0.2 mg/ml sodium azide and 2.5 mg/ml BSA was stored at −20℃.

L-FABP was detected using a newly developed methodology of paramagnetic particle-based sandwich chemiluminescent immunoassay, which specifically recognized L-FABP. As is shown in Figure 1 , 

The research data were analyzed by SPSS20.0, Graphpad prism 7.0, and R software 3.6.2. Continuous variables were presented as means (SD) when data satisfied normally distributed or as medians

The schematic diagram of the proposed chemiluminescent immunoassay with skewed distribution. Categorical variables were presented as percentages. Continuous variables which conformed to the normal distribution were used a student's t-test for statistical differences, and which conformed to the skewed distribution were used Mann Whitney U test for statistical differences. Categorical variables were used Chi-square test. p < 0.05 was considered statistically significant.

Receiver operating characteristic curve (ROC) was used for assessing the predictive value of occurrence risk of AKI; logistic regression was used for establishing models, which assessed the risk by measuring biomakers before AKI happened. The rms package was used for assessing the relationship between the change of L-FABP concentration and the risk of AKI with R software 3.6.2 (https://cran.rstud io.com/).

The clinical characteristics of the participants in this study were shown in Table 1 . At last, 100 patients were recruited for this study.

Among the patients, 15 patients were diagnosed with AKI and 85 patients with non-AKI. According to KDIGO criteria, there were 10 patients with AKI stage I, 3 patients with AKI stage II, and 2 patients with AKI stage III ( Figure 2 ). The incidence of AKI in ICU was 15%.

The baseline characteristics were analyzed between AKI cohort and non-AKI cohort. There were no significant differences in gender, age, BMI, hypertension, diabetes, smoking, drinking, K, Na, Cl, PLT, WBC, INR, and baseline creatinine. The purified L-FABP analyte (100 μg/ml) was diluted to a range of concentrations of antibody pair with 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μg/ml, the optimal antibody pair concentration was selected as 0.8 μg/ml according to the maximal signal-to-noise ratio, which not only could satisfy the clinical needs for detection but also could save the volume of L-FABP ( Figure 3A ). We also evaluated the incubation time of antibody pair, which was set as 15, 30, 45, and 60 min.

The RLU was increased with increased incubation time for 45 min.

After 45 min, the RLU leveled off with increasing incubation time.

Therefore, 45 min was selected as the optimal reaction time for further experiment ( Figure 3B ). Next, we explored the capture time of avidin-coated paramagnetic particles, which was set as 5, 10, 15, 20, and 30 min. The chemiluminescent efficiency was significantly high at 10 min than other times. Then, the optimal incubation time for further experiments ( Figure 3C ) was 10 min. The limit of detection was 0.88 ng/ml.

The precision of this methodology was estimated based on repeated measurements of different concentrations of L-FABP standards solution, which was prepared by adding L-FABP standards into healthy human plasma. Three different concentrations of L-FABP standards (15 ng/ml, 50 ng/ml, and 100 ng/ml) were measured ten times a day for three days by chemiluminescent immunoassay. The results showed that the intra-analysis CVs of the three samples enrolled were 8.73%, 4.72%, and 3.79%, respectively, and the interanalysis CVs of the three samples enrolled were 13.47%, 7.28%, and 5.94%, respectively ( Figure 4B ). The repeatability results were highly acceptable and revealed the excellent analytical performance of this methodology. 

To evaluate the influence of different matrixes to reactive system, the L-FABP standards (50 ng/ml) were added into samples containing three matrixes of plasma, urine, and BSA solution, respectively. L-FABP in different matrix solution was measured for three times. 

The reference interval of L-FABP based on healthy population was constructed by chemiluminescent immunoassay. In order to explore the reference interval of L-FABP, 120 participants were enrolled in this study by detecting L-FABP of plasma samples, and calculating the 95% confidence interval to obtain the results as 0.88 ng/ml to 5.98 ng/ml.

To explore the clinical application of L-FABP in critically ill patients, we analyzed L-FABP concentration between 15 AKI patients and 85 non-AKI patients and found that the median L-FABP concentration was significantly higher in AKI patients (median (IQR):10.65(6.74-14.56)) than non-AKI patients (median (IQR): 5.43(3.20-7.60)) ( Figure 5A ). When 5.95 ng/ml was selected as a cut-off value, the diagnostic sensitivity and specificity were 71.8% and 92.5%, respectively ( Figure 5B ).

Non-AKI (n = 85) p 

We used Restricted Cubic Spline plots to evaluate the association between the dynamic changes of L-FABP concentration and risk F I G U R E 6 (A-C) Comparision of L-FABP concentrations among patients with AKI and those without AKI before AKI occurrence. (D-F) The ROC curve analysis of L-FABP in patients with AKI compared with non-AKI before AKI occurrence. (G) The association between L-FABP concentration and different stages of AKI. * represented there was significantly different in patients with AKI stage I, AKI stage II, AKI stage III compared with those without AKI, p < 0.05. # represented there was significantly different in patients with AKI stage II, AKI stage III compared with AKI stage I, p < 0.05. H: The association between L-FABP concentration and the risk assessment of AKI assessment of AKI occurrence. The higher L-FABP concentration was significantly associated with increased risk of AKI, p < 0.05 ( Figure 6H ). When L-FABP concentration was more than 5.7 ng/ml, the hazard ratio of AKI was more than 1, which was needed to attach great importance clinically. L-FABP was an excellent predictor for risk analysis of AKI. L-FABP levels were associated with the risk of AKI. Higher quartiles of L-FABP levels were independently associated with increasing risk of AKI. The highest quartile of L-FABP on 1 day before AKI was associated with increased odds for AKI by 92-fold compared with the lowest quartile. The highest quartile of L-FABP on 2 days before AKI was associated with increased odds for AKI by 65-fold compared with the lowest quartile. The highest quartile of L-FABP on 3 days before AKI was associated with increased odds for AKI by 65-fold compared with the lowest quartile;

the results were shown in Table 2 . When L-FABP levels were analyzed as a continuous variable, higher L-FABP was also associated with the development of severe AKI. The L-FABP level of stage II was associated with increased odds for AKI by 12-fold compared with stage I. The L-FABP level of stage III was associated with increased odds for AKI by 14-fold compared with stage I; the results were shown in Table 3 .

Chemiluminescent immunoassay was a newly developed immuneanalytical-based detection technique, which combined the advantages of enzyme immunoassay and radioimmunoassay. 22, 23 The outstanding advantages of double antibody sandwich based chemiluminescent immunoassay high sensitivity and specificity, excellent analytical selectivity, good repeatability and accuracy were being increasingly applied to material analysis. [24] [25] [26] In this study, we constructed a rapid, sensitive, and practical chemiluminescent immunoassay detection method, which exhibited highly sensitive analytical performance, with a lower detection limit of 0.88 ng/ml compared with ELISA, where the limit of detection was 2.4 ng/ml. 27 Highly automated and high-throughput analytical assay based on our chemiluminescent immunoassay replaced complicated operating procedures, giving a fast and convenient application in clinical practice and were suitable for large-scale sample measurements. Remarkably, biotin can bind avidin with high specificity, and biotin-avidin system has been widely applied in biomedical fields. Biotin-streptavidin system (modified biotinavidin system) was used in our novel developed detection method to improve sensitivity by multistage amplifying function and to achieve the purpose of detecting trace antigen by streptavidinenzyme color reaction.

Recently, increasing attention had been placed on AKI. Many diseases could cause AKI such as sepsis, multiple injury, and hepatic and renal insufficiency. 28 AKI could easily progress to CKD without any intervention. 29 To construct a newly useful detection method and explore the clinical application of biomarkers were the direction of our future efforts.

We developed a new, rapid, sensitive double-antibody sandwichbased chemiluminescent immunoassay to identify the plasma con- 

All authors have agreed to the consent of the manuscript.

The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

The authors declare that they have no conflict of interest. All authors have read and approved the final manuscript.

All patients have written informed contents, and this study was approved by the Second Affiliated Hospital of Zhejiang University School of Medicine.

All authors have agreed to the consent of the manuscript.

Zhihua Tao https://orcid.org/0000-0002-0906-7143

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