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Ann Thorac Surg 1999;68:2196-2201
© 1999 The Society of Thoracic Surgeons

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Original Articles: Cardiovascular

A new optical immunoassay for detection of S-100B protein in whole blood

^a BioStar, Inc, Boulder, Colorado, USA
^b AB Sangtec Medical, Bromma, Sweden
^c Johns Hopkins Medical Institutions, Baltimore, Maryland, USA

Address reprint requests to Dr Ettinger, BioStar Inc, 6655 Lookout Rd, Boulder, CO 80301
e-mail: a_ettinger{at}biostar.com

	Abstract

Top Abstract Introduction Material and methods Results Comment References

 
Background. S-100B is a protein mainly found in astroglial cells and only detected to a low level in blood. Serum levels of S-100B increase in patients with acute brain injuries. The aim of this study was to establish feasibility of a new Optical ImmunoAssay ([OIA], BioStar, Inc, Boulder, CO) test for determination of S-100B in blood.

Methods. We have developed a new, rapid, and sensitive OIA test to identify elevated levels of S-100B in whole blood. The OIA test for S-100B combines monoclonal antibodies specific for the B-subunit of S-100 with OIA thin film technology. Each sample was tested for S-100B by the OIA method and a commercially available immunoluminometric assay. Blood samples were drawn serially from 9 patients undergoing coronary artery bypass graft surgery and during the early postoperative period.

Results. The OIA test determination of S-100B protein correlated with immunoluminometric assay data (r = 0.8) with a detection limit of 0.25 ng/mL.

Conclusions. The sensitivity and feasibility of this rapid assay may be suitable for rapid evaluation of S-100B in urgent care settings (surgery, intensive care units, or emergency room).

	Introduction

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S-100B is a 21 kDa glial calcium binding protein synthesized by astrocytes, oligodendrocytes, and Schwann cells [1–3] and represents 0.2% of the total soluble brain protein [4]. To date, 17 different proteins have been assigned to the S-100 protein family [5]. The main forms of S-100 exist as homo or heterodimers consisting of two subunits, A1 and B. S-100B is predominating in the brain, glial, and Schwann cells [6]; S-100A1B is in glial, but not Schwann cells; S-100A is mainly in muscles, heart, and kidney [1, 3, 5].

The physiological function of S-100 is still under investigation. It may play a role in the development of the nervous system, maturation and function of glial cells, prolactin secretion, and protein phosphorylation [1]. Injury to the brain causes a selective leakage of S-100B from the brain tissue into the cerebrospinal liquid and then into the blood, which suggests increased permeability of the blood–brain barrier [7].

S-100B protein is detectable to a very low level in the blood of a normal human population [8]. S-100B is a highly sensitive and specific marker for central nervous system dysfunction, and elevated concentrations of S-100B in serum have been reported after head trauma [9, 10] and stroke [11–13]. S-100B protein can also be a marker for Alzheimer’s disease, Down syndrome, dementia, epilepsy [1, 5, 14, 15], and cerebral tumor [16]. Recently, elevated S-100B levels have been described after adult cardiac operations complicated, or not complicated, by neurologic injury [17], after intracardiac operations [18], after deep hypothermic circulatory arrest [19], and after out-of-hospital circulatory arrest [20].

The LIA-mat Sangtec 100 (AB Sangtec Medical, Bromma, Sweden) is a sensitive immunoluminometric test to measure S-100B in serum with high sensitivity (< 0.02 ng/mL). However, this test and other current S-100B immunoassay tests, take approximately 3 to 4 hours to perform and require a luminometer or scintillation counter for quantitation. For many applications, such as monitoring of the S-100B level during cardiac surgery, or assessing severity of head trauma, stroke damage, or consequences of cardiac arrest, there is a need for a rapid, simple assay, which can provide results in less than 20 minutes in a simple assay format.

In this report, we describe a rapid, simple and sensitive point-of-care test, the Optical ImmunoAssay ([OIA], BioStar, Inc, Boulder, CO), to identify elevated levels of S-100B in whole blood. This test is potentially useful for the assessment of patients with suspected brain injury resulting from cerebral ischemia due to stroke, vasospasm after subarachnoidal hemorrhage, head trauma, or complications of surgery. The purpose of this work was to compare the feasibility of this rapid OIA method to the commercially available immunoluminometric assay.

	Material and methods

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Clinical samples
The study population consisted of 9 high-risk patients undergoing elective coronary artery bypass graft (CABG) surgery who had predisposing risk factors (history of previous stroke, increasing age, history of diabetes mellitus, history of hypertension, and/or presence of carotid bruits). All patients were prospectively enrolled in the study.

Mean age was 69 ± 3 years, 6 of 9 patients were male. The multiple serum samples taken from each patient were analyzed to compare the S-100B determination by the OIA assay with the results obtained by LIA-mat Sangtec 100 assay. LIA-mat Sangtec 100 assay was performed at Johns Hopkins University (Baltimore, MD). The OIA assay was developed and performed at BioStar Inc (Boulder, CO).

The samples were taken serially during CABG surgery (induction, cross-clamping of the aorta, 30 minutes after cross-clamping, lowest body temperature, after removal of crossclamp, at 36°C body temperature, end of cardiopulmonary bypass (CPB), 30 minutes post-CPB, and at 6, 12, and 24 hours after CPB). The samples were centrifuged within 15 to 30 minutes after collection, and serum was kept frozen at -20°C prior to analysis. General anesthesia was similar for all patients. Combination of benzodiazepines (midazolam, diazepam, and lorazapam) served as premedication. Inhalation agents and opiates (fentanyl citrate and sufentanyl citrate) were used for induction. Anesthesia was maintained by additional boluses of midazolam and fentanyl. After cannulation of the aorta and right atrium, nonpulsatile CPB with a membrane oxygenator was instituted. For 7 patients, the CPB circuit was primed with Ringer’s lactate (1,542 mL ± 139 mL; range 1,300 mL to 1,700 mL). One CPB circuit was primed with 715 mL Ringer’s lactate and 885 mL of fresh frozen plasma, and one with 1,000 mL Ringer’s lactate plus 2 units of packed red blood cells. Flow rate was set at a mean of 3.6 ± 1.7 L/min. A roller pump (Cobe Cardiovascular Inc, Arvada, CO) was used for all patients. Mean arterial blood pressure was maintained between 60 to 90 mm Hg during CPB. The alpha-stat protocol for blood gas management was applied for all patients under conditions of mild hypothermia (29.1 ± 1.7°C).

The study was approved by the Joint Committee of Clinical Investigation of the Johns Hopkins Medical Institutions (RPN 96-01-31-06) and a consent form was signed by all enrolled patients.

Materials
Silicon wafers were coated with Si₃N₄ by a standard vapor deposition process. T-polymer (polydimethylsiloxane) was purchased from United Chemical Technologies, Inc (Bristol, PA). Tetramethylbenzidine (TMB) was from Kirkegaard & Perry Laboratories (Gaithersburg, MA). Anti S-100B monoclonal antibodies SMST 12, SMSK 25, and SMSK 28 were received from AB Sangtec Medical. Bovine S-100A1B was purchased from Calbiochem (San Diego, CA) or Sigma (St. Louis, MO). PD-10 columns were from Pharmacia Biotech (Uppsala, Sweden). Blood samples were obtained from healthy individuals with their verbal consent. Heparinized whole blood was collected using Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) with Hemogard (Becton Dickinson, Franklin Lakes, NJ) closure, stored at 4°C and used within 1 to 2 weeks. Serum was collected in siliconized Vacutainer tubes, allowed to clot for 20 to 30 minutes at room temperature, and centrifuged at 1,000 RPM for 10 minutes. All reagents were analytical grade purity.

Wash solution
To wash OIA surfaces, 0.05% Tween-20 in 10 mM phosphate buffer, containing 0.5% Proclin 300 (Rohm Haas Co, Philadelphia, PA), pH 6.5 was used.

Wafer antibody coating
T-polymer coated wafers were coated with Sangtec monoclonal antibodies SMSK 25 and SMSK 28 (2 µg of each antibody/1 mL in 0.1 M carbonate buffer, pH 9.3) overnight at room temperature (17 to 24 hours). Excess antibody solution was rinsed from the wafer with deionized water, wafers were dried under a stream of nitrogen, and the surface was overcoated with a sucrose casein preservative.

Preparation of anti-S-100B-HRP-conjugate
Anti-S-100B-HRP (horseradish peroxidase) conjugate was prepared using a standard conjugation procedure [21]. HRP (100 mg/ml in 0.05 M acetate buffer, pH 4.5) was activated with NaIO₄ in molar ratio 1:4, 30 minutes at room temperature in the dark. The excess NaIO₄ was removed using a PD-10 column in the acetate buffer. Activated HRP was mixed with antibody SMST 12 (1:1 weight ratio) at room temperature for 1 hour in 0.1 M carbonate buffer, pH 9.3. The reaction was quenched with NaBH₄ (2 mg/mL) 15 minutes in the dark, at 25°C. The conjugate was precipitated with 50% ammonium sulfate, 1 hour at 4°C. A pellet was obtained after 10 minutes centrifugation at 8,000g, dissolved in 1 x PBS to a final concentration of 1 mg/mL and stored at 4°C. The conjugate was diluted 1:100 in 0.05 M Mopso buffer, pH 7.5, containing 0.5% Proclin 300 preservative. Analytical experiments were conducted by diluting bovine S-100A1B in 1 x PBS containing 1% BSA.

Optical ImmunoAssay test
The Optical ImmunoAssay test for S-100B combines monoclonal antibodies specific for S-100B with OIA thin film technology. The OIA surfaces were coated with two anti-S-100B antibodies, SMSK 25 and SMSK 28, and antibody SMST 12 was conjugated to HRP. All incubations were performed at room temperature. To determine the limit of S-100B detection in whole blood, blood samples from different healthy individuals were spiked with bovine S-100A1B. The simultaneous assay format combines exposure of the sample to the antibody on the surface and to the HRP conjugate in solution. A 100 µL sample was diluted with methanol and incubated for 3 minutes. One drop of conjugate (35 µL) was added to the diluted sample, and 1 drop (35 µL) of the sample mixture was placed on the OIA surface. After 10 minutes of incubation, the surface was rinsed and 1 drop (35 µL) of TMB substrate was added and incubated for 5 minutes. The surface was rinsed, blotted, and the results were read visually and by ellipsometry. The whole procedure was completed in 18 minutes.

The amount of S-100B in clinical samples was estimated visually by the OIA, compared to a standard curve, and measured quantitatively using the comparison ellipsometer. A standard curve was constructed by adding bovine S-100A1B to the serum of healthy individuals, and the OIA test was performed as described above. To estimate the amount of S-100B in the clinical samples, the OIA results were compared visually to the standard curve results. All experiments were performed in duplicate.

LIA-mat sangtec 100 test
The commercially available LIA-mat Sangtec 100 assay was performed as described by the manufacturer. The LIA-mat Sangtec 100 assay is a monoclonal two-site immunoluminometric method (sandwich principle). Antibody-coated polystyrene tubes serve as solid phase. The LIA-mat Sangtec 100 assay discriminates between the A1 and B-subunit as defined by the three monoclonal antibodies. The light signal measured in relative light units is directly proportional to the amount of S-100B present in the standard and sample. With pipeting of samples, two incubation periods (total 3 hours), washing cycles, and measurements, the whole procedure takes about 4 hours.

Comparison ellipsometer
We used the comparison ellipsometer to quantitate the OIA signal. An ellipsometer measures thin film thickness in the angstrom range. The principle of the measurements is based on the change of polarization of light reflected from the thin film [22]. When linearly polarized light reflects from a thin film, it becomes elliptically polarized. The amount of ellipticity induced depends on the optical properties of the base material as well as the thickness and refractive index of the overlying films. The measured detector output is proportional to the concentration of analyte bound to the optical surface. The Comparison Ellipsometer Sagax-225 used in this work was manufactured by Sagax (Goteborg, Sweden).

Data analysis
All results are presented as mean value ± standard error. Data analysis included paired Student’s t tests to evaluate differences between S-100B protein concentrations defined by different methods. All p values are two-tailed. The significance level was set up at p less than or equal to 0.05.

	Results

Top Abstract Introduction Material and methods Results Comment References

 
Principle of the OIA method
The OIA principle is based on detection of a physical change in optical thickness of molecular thin films resulting from specific binding events. The OIA assay consists of a silicon wafer to which an optical coating and S-100B capture antibodies are attached. The interaction of S-100B with anti-S-100B antibodies produces a thin film on the antibody-coated silicon surface. Reflection of light through the thin film results in destructive interference of particular wavelengths of light. The qualitative result is determined visually by a color change, from gold to purple–blue, generated by an increase in thin film thickness on the optical support.

Analytical performance of the OIA test
We developed protocols to determine the level of S-100B in buffer and also in whole blood. The detection limit for S-100B in buffer was 0.125 ng/mL. The sensitivity level for S-100B determination in blood was 0.25 ng/mL, which is comparable with radioimmunoassay sensitivity. The methanol-based extraction reagent reduced the interference from whole blood in the assay, making detection of sub ng/mL quantities possible.

Comparison of the OIA test with the LIA-mat sangtec 100 test
S-100B protein was detected in serum of 9 patients before sternotomy after induction of anesthesia, 0.35 ± 0.1 ng/mL (range, 0.03 to 0.72 ng/mL) and 0.59 ± 0.3 ng/mL (range, 0 to 2.5 ng/mL) defined by LIA-mat Sangtec 100 and the OIA assays, respectively. None of the patients had a stroke after CABG, however the S-100B levels (measured by the OIA and LIA-mat Sangtec 100 methods) increased gradually from baseline toward the end of surgery. There was a peak of S-100B at the end of CPB (n = 5), at 30 minutes after CPB (n = 1), 6 hours after CPB (n = 3), 12 hours after CPB (n = 1), and 24 hours after CPB (n = 1). Two patients had two peaks of S-100B (one patient, at 6 and 24 hours; second patient, at 30 minutes after CPB and 12 hours after CPB). At 24 hours, the values of S-100B defined by both methods were close to baseline. The mean cross-clamp time was 73.1 ± 8.1 minutes, and CPB time was 106.0 ± 11.7 minutes for all patients. Time-profile and mean values of S-100B protein for different stages of surgery for all patients using the OIA and the LIA-mat Sangtec methods are presented in Figure 1 and Table 1.

View larger version (33K): [in this window] [in a new window]   Fig 1. Mean values of S-100B for all patients determined by Optical ImmunoAssay (OIA) (ellipsometry and visual) and LIA-mat Sangtec methods. (CPB = cardiopulmonary bypass.)

The S-100B levels were also measured using the comparison ellipsometer, and there was no statistically significant difference in all measurements, except one (Table 1). As an illustrative case, the visual and ellipsometric OIA results of S-100B analysis were plotted with the LIA-mat Sangtec 100 determinations for the patient who had S-100B peak at 6 hours after CPB was stopped (Fig 2).

View larger version (32K): [in this window] [in a new window]   Fig 2. Assay comparison for patient 1 (Optical ImmunoAssay [OIA] ellipsometry and OIA visual versus LIA-mat Sangtec methods) throughout coronary artery bypass graft (CABG). (CPB = cardiopulmonary bypass.)

	Comment

Top Abstract Introduction Material and methods Results Comment References

Levels of tissue enzymes released into body fluids as the result of pathological processes often indicate the presence of injury and may aid in assessing the patient’s clinical course and outcome. Determination of S-100B protein in serum has been suggested for evaluation of possible brain injury after CABG: Indeed this marker may differentiate gray and white matter damage because S-100B is present mainly in glial cells. Measurement of S-100B protein in serum, or in whole blood, may be slightly less sensitive than measurement in cerebrospinal fluid. However, the serum test has a high specificity, and good correlation between serum and CSF levels of S-100B has been reported [23]. Our results suggest that S-100B determination in whole blood may be a useful, direct, rapid, and less expensive alternative compared to the traditional radioimmuno- or luminoimmunoassays. The coincidental occurrence of other conditions with elevated S-100B protein levels, such as small-cell lung carcinoma or neuroblastoma, is rare enough not to interfere with prognostic assessment of brain injury. Potential release of S-100B protein from other sites is relatively insignificant compared to the brain [24].

Previous publications have demonstrated that S-100B peaks at the end of CPB [25]. Our data confirm these results. However, our findings demonstrated a gradual increase in S-100B (defined by the OIA and LIA-mat Sangtec 100 assays) after the induction of anesthesia toward the end of surgery with a potential additional S-100B protein peak between 6 to 24 hours after CPB. The reason for elevated levels of S-100B protein after the induction of anesthesia and before sternotomy is not apparent. Among the possible explanations could be an effect of anesthesia, age, and sex related changes of S-100B protein [8]. The S-100B protein dynamics revealed in this work suggest there may be a greater susceptibility of high-risk patients to subclinical injury. These findings stress the importance of detailed monitoring of these patients in the early postoperative period.

The time course and the importance of S-100B release early after extracorporeal circulation must be further characterized. The true specificity and sensitivity of the OIA determination of S-100B protein as an indicator of a cerebral complication will require studies involving neuropsychologic, neurologic, and neuroimaging methods.

	Acknowledgments

 
We are grateful to Dr Penelopa M. Keyl for the consultation regarding statistical analysis of our data, and Ms Nicol A. McBee for performing the statistical analysis. We also thank Dr Diana Maul for the critical reading of the manuscript, and Mr Tony Mershon for technical assistance in performing OIA experiments.

This work was supported by BioStar Inc, Boulder, CO and AB Sangtec Medical, Bromma, Sweden.

	Footnotes

 
Doctor Anna Ettinger, Doctor Rachel Ostroff, and Ayla Laumark are employees of BioStar. Doctor Jan Brundell is an employee of AB Sangtec Medical, and the LIA-mat tests for this study were provided by Sangtec to Johns Hopkins University.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): William A. Baumgartner Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ettinger, A. Articles by Razumovsky, A. Y. Search for Related Content PubMed PubMed Citation Articles by Ettinger, A. Articles by Razumovsky, A. Y.