Fluorescent protein-based detection of unconjugated bilirubin in newborn serum

Increased serum levels of unconjugated bilirubin are associated with the development of brain damage in newborns. In current clinical settings, there are no methods for directly determining serum levels of unconjugated bilirubin. UnaG, a fluorescent protein from Japanese eel muscle that specifically binds to unconjugated bilirubin was used in this study. Linear regression analysis was carried out to compare unconjugated bilirubin levels measured by UnaG and conventional bilirubin oxidase methods. Unconjugated bilirubin levels in the serum of newborns who were untreated or treated with phototherapy were compared. Effects of interfering factors in the serum (conjugated bilirubin, hemoglobin, and lipid) on unconjugated bilirubin concentration measured by the UnaG method were also evaluated. Unconjugated bilirubin levels measured by the UnaG method were highly correlated with those determined by the bilirubin oxidase assay. Unconjugated bilirubin levels determined by bilirubin oxidase and UnaG assays were similar in serum samples containing conjugated bilirubin. The performance of the UnaG assay was unaffected by phototherapy and the presence of serum hemoglobin and lipid emulsion. These results demonstrate the clinical applicability of the UnaG method for direct measurement of unconjugated bilirubin levels in newborn serum.


Influence of interfering factors in serum.
Unconjugated bilirubin concentrations were similar across 14 serum samples with conjugated bilirubin concentration ≥ 1.0 mg/dl as determined by the UnaG and bilirubin oxidase methods (Table 1). There was no significant difference between values obtained with the bilirubin oxidase and UnaG methods (P = 0.31). Next, we investigated whether substances such as hemoglobin and lipid emulsion in serum affect measurement of unconjugated bilirubin by the UnaG method. Five serum samples (a-e) with different concentrations of unconjugated bilirubin were analyzed. Unconjugated bilirubin concentrations as determined by the UnaG method were unchanged in the presence of various concentrations of hemolysate and lipid emulsion ( Table 2). No correlation was found between hemoglobin or lipid emulsion concentrations and unconjugated bilirubin concentrations detected by the UnaG method (Samples a: r = − 0.32, P = 0.54, b: r = − 0.33, P = 0.53, c: r = 0.0032, P = 1.0, d: r = − 0.62, P = 0.26, and e: r = − 0.14, P = 0.82).

Measurement of unconjugated bilirubin concentration in whole blood by the UnaG method.
A total of 26 whole blood samples from 18 newborns without PT were analyzed (median gestational age: 37 weeks, range: 27-40 weeks; median birth weight: 2432 g, range: 464-3558 g; median age when blood sample was collected: 5 days, range: 1-48 days). Median serum conjugated bilirubin concentration as determined by the bilirubin oxidase method was 0.2 mg/dl (range: 0.2-1.1); the median hematocrit value was 45% (range: 30-66%). Unconjugated bilirubin concentration in whole blood as determined by the UnaG method was strongly correlated  with serum concentrations obtained by the bilirubin oxidase and UnaG methods (y = 0.76x + 0.30, r = 0.962 and y = 0.73x + 0.77, r = 0.962, respectively; P < 0.001; Fig. 4A,B); however, the slopes were not equal to 1 in either case.

Discussion
This is the first large-scale clinical study demonstrating the applicability of eel-derived fluorescent protein for measurement of unconjugated bilirubin concentrations in newborn serum samples. The concentrations measured by this method showed high correlation with those obtained by the conventional bilirubin oxidase method, which has itself shown good correlation with those obtained by high-performance liquid chromatography, considered as the gold standard 12,14,15 . The performance of the UnaG method was unaffected by PT and the presence of serum hemoglobin and lipid emulsion. Moreover, only a small volume of whole blood or serum was required for the assay, which is advantageous since samples are collected from neonates. The precision of the method was confirmed by intra-and inter-day assays, which is an important factor for the clinical applicability of this method. These results indicate that the UnaG assay is robust and can be used for direct measurement of unconjugated bilirubin in newborns.
PT is a standard and effective treatment for severely jaundiced newborns 16 and works by inducing the isomerization of trans-bilirubin into the water-soluble cis isomer, which is excreted from the body 17 . Detection of unconjugated bilirubin by the UnaG method was unaffected by PT. Importantly, UnaG bound the unconjugated but not the conjugated form of bilirubin 13 . There was no siginificant discrepancy between unconjugated bilirubin levels measured by the UnaG and bilirubin oxidase methods.
Hemolytic jaundice resulting from blood group incompatibility, glucose-6-phosphate dehydrogenase deficiency, or hereditary spherocytosis is a condition associated with a high risk of developing kernicterus 18 . In these cases, bilirubin levels in hemolysate must be measured. Parenteral alimentation with lipids is normally provided as nutrition to preterm infants or sickly newborns 19 . Although the bilirubin oxidase method is affected by the presence of serum hemoglobin and lipid emulsion 11 , we found in this study that hemolysate or serum containing emulsion did not affect unconjugated bilirubin measurement by the UnaG method. Additional studies are required to determine whether the various drugs used in clinical neonatal medicine interfere with UnaG-based detection.
The clinical applicability of the UnaG method depends on the ability to obtain UnaG in substantial quantities. His-tag DNA technology is often used to generate large amounts of specific proteins 20 . We thus generated UnaG-His-FLAG and tested its efficacy in the UnaG assay. The presence of the epitope tags had no effect on the affinity of UnaG for unconjugated bilirubin. These results demonstrate the feasibility of large-scale UnaG production. An automated measurement system for UnaG would also be useful for clinical application of this method.
We measured unconjugated bilirubin concentration in whole blood using the UnaG method with hematocrit value correction 13,21 and found that the concentration was highly correlated with those measured in serum by the bilirubin oxidase and UnaG methods; however, the slopes of the linear regression lines were not equal to 1. This may be attributed to the fact that we used a standard curve generated using serum bilirubin solutions. To obtain corresponding values in serum and whole blood, appropriate standards and corresponding coefficient values must be used.

Difference in values between bilirubin oxidase and UnaG methods.
In conclusion, we established a highly specific and sensitive assay for detection of unconjugated bilirubin in serum samples from newborns using the fluorescent protein UnaG. The UnaG method yielded results that were at least comparable to those obtained by the conventional bilirubin oxidase method, in addition, this method is unaffected by the presence of serum hemoglobin and lipid emulsion.

Methods
Blood sample collection. Blood samples were obtained from newborns who were born between June in 2014 to December in 2015 for routine laboratory tests for a variety of reasons, such as respiratory disorder, neonatal jaundice, and conditions associated with premature birth. Residual blood samples after sampling for laboratory tests were immediately centrifuged at 3000 rpm for 10 min in the dark, and the serum was stored at − 20 °C in the dark until it was used. The residual whole blood was stored at room temperature in the dark and used on the same day that the sample was collected. The study protocol was approved by the ethical committee of Kobe University Graduate School of Medicine (approval no. 1618). Informed consent was obtained from parents of newborns prior to collection of blood samples. The methods were carried out in accordance with the approved guidelines.
Preparation of UnaG. The bilirubin-inducible fluorescent protein from Japanese eel muscle, UnaG was used in this study as we have previously shown 13 . UnaG-His-FLAG was generated by adding a polypeptide tag to the protein as previously reported 20 .  concentration of 2 μ M) was prepared, with 5 μ l artificial unconjugated bilirubin solution or newborn serum added to 995 μ l of 0.1 M phosphate-buffered saline (PBS; 200-fold dilution) as we have previously shown (Fig. 1A) 13 . A microplate reader (SH-9000; Corona Electric Co., Hitachinaka, Japan) was used at 37 °C with fluorescence filters for excitation and emission wavelengths of 498 and 527 nm, respectively. Bilirubin concentrations ranging from 0.0 to 66.8 mg/dl were prepared using artificial bilirubin standard solutions including albumin (Arrows Co., Osaka, Japan) and were measured to generate a standard calibration curve (Fig. 1B,C). Serum samples were diluted 200 fold with PBS and fluorescence intensity was measured. Serum concentrations of unconjugated bilirubin were extrapolated from the standard curve. Detection in whole blood: Artificial unconjugated bilirubin solution or newborn whole blood was diluted 100 fold in PBS. The fluorescence intensity was measured and a standard curve was generated using artificial unconjugated bilirubin solution as described above. Fluorescence intensity of diluted whole blood samples was measured and unconjugated bilirubin concentrations were determined from the standard calibration curve with a correction of (1 − hematocrit value). This correction method is generally used to covert to the serum concentration from the whole blood concentration 13,21 . Precision of the UnaG method. Intra-day precision was determined by analyzing six replicates of the serum sample over the course of 1 day; inter-day precision was estimated by analyzing the same sample on 6 difference days. Mean, standard deviation, and CV were calculated 22 . Bilirubin oxidase method of serum unconjugated bilirubin detection. Total and conjugated bilirubin concentrations in serum were measured using IatroLQ T-bil and IatroLQ D-bil kits (Unitika Co., Okazaki, Japan) as previously reported 14,15  Comparison between bilirubin oxidase and UnaG methods. Serum unconjugated bilirubin concentrations without conjugated bilirubin (<1.0 mg/dl by the bilirubin oxidase method) as determined by the UnaG and bilirubin oxidase methods were compared by linear regression analysis. The correlation between serum unconjugated bilirubin concentrations determined using UnaG and UnaG-His-FLAG was determined to verify whether these two forms of UnaG have different affinities for unconjugated bilirubin. Serum unconjugated bilirubin concentrations in the same serum samples were also determined using UnaG and UnaG-His-FLAG, with a comparison of the results. Unconjugated bilirubin concentrations in the serum of newborns treated with PT (blue light-emitting diodes, irradiance with around 30 μ W/cm 2 /nm) or untreated were compared, since PT induces the photoisomerization of unconjugated bilirubin 17,23 . Influence of interfering factors in serum. Serum samples (with ≥ 1.0 mg/dl conjugated bilirubin by the bilirubin oxidase method) were obtained from newborns experiencing severe asphyxia with hepatic failure, congenital cytomegalovirus infection, or trisomy 18. Unconjugated bilirubin concentrations in these samples were measured by the UnaG or bilirubin oxidase methods. Interfering factors in the serum (hemoglobin and lipid) were evaluated to determine whether they affect unconjugated bilirubin detection by the UnaG method. Hemolysates were prepared as follows. Erythrocytes obtained from fresh blood of a volunteer adult were washed with saline and hemolysates with various concentrations of hemoglobin (0, 1, 2, 4, 8, and 16 g/dl) were prepared by adding appropriate volumes of distilled water, and were added to newborn serum samples to determine whether hemolysis can affect the measurement of UnaG fluorescence intensity. Similarly, serum samples with various concentrations of lipid emulsion (0, 0.02, 0.2, 2, and 20% intralipos; Otsuka Pharmaceutical Factory, Naruto, Japan) were used to assess whether these factors affect measurement of unconjugated bilirubin concentration.

Analysis of unconjugated bilirubin concentrations in whole blood. Serum unconjugated bilirubin
concentrations determined by the bilirubin oxidase and UnaG methods and the concentration in whole blood determined by the UnaG method were compared by linear regression analysis.