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Development of a rapid enzyme immunoassay for the detection of retinol-binding protein^1,2,3

¹ From the Program for Appropriate Technology in Health, Seattle (JH, JM, and MT); the Institute of Nutrition for Central America and Panama, Guatemala City (CM); Vista Diagnostics, Kirkland, WA (IB); the Johns Hopkins School of Medicine, Baltimore (AS); and Helen Keller Worldwide, Jakarta, Indonesia (AS).

² Supported by the US Agency for International Development under the Technologies for Health (HealthTech) project, Cooperative Agreement no. HRN-A-00-96-90007. The monoclonal antibody cell line was provided by Samuel Black (University of Massachusetts).

³ Address reprint requests to J Hix, Program for Appropriate Technology in Health (PATH), 1455 NW Leary Way, Seattle, WA 98107-5136. E-mail: jhix{at}path.org.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: Retinol-binding protein (RBP) was chosen as a surrogate marker for retinol because of the close correspondence between retinol and RBP.

Objective: To meet the need for rapid, cost-effective determination of vitamin A status in populations, a quantitative enzyme immunoassay (EIA) for detection of RBP was developed.

Design: The resulting RBP EIA, a competitive assay, uses RBP adsorbed to microtest strip wells to compete with RBP in serum. The assay takes 40 min.

Results: With a reference panel of sera, test accuracy was found to be within 4% of expected values through the calibrated range of 0.48–1.92 µmol RBP/L (10–40 µg RBP/mL). Intraassay and interassay variability averaged 6.7% and 8.9%, respectively. Specificity testing showed no interference from other serum proteins, prealbumin, rheumatoid factor, bilirubin, estrogen, or C-reactive protein. The RBP EIA provided linear results between 0.43 and 1.80 µmol RBP/L (9 and 38 µg RBP/mL). Preliminary laboratory evaluations indicated that the RBP EIA correlates well with radial immunodiffusion for RBP and with HPLC for retinol, the current reference standard. A field evaluation in a population at risk for vitamin A deficiency (VAD) resulted in close correlation between RBP EIA measures and retinol measures by HPLC (R² = 0.82).

Conclusions: The RBP EIA is as reliable in estimating VAD as is HPLC retinol. After successful validations, the test should enable public health authorities to rapidly monitor VAD and track vitamin A status in populations.

Key Words: Retinol-binding protein • vitamin A deficiency • enzyme immunoassay • vitamin A status • serum retinol

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Micronutrient malnutrition has emerged as one of the most significant public health problems in the world today. Recent estimates indicate that vitamin A deficiency (VAD) is a public health problem in 118 countries, with an estimated 100 million children suffering from VAD; of these, some 250 000–500 000 become blind each year, mostly in developing countries (1-5). In children, VAD causes visual impairment and blindness and increases the risk of severe illness and even death from diarrhea and measles.

Several strategies exist for the control of VAD through both short-term and long-term interventions (3, 6, 7). Global efforts supported by multiple donors have relied on periodic supplementation with high-dose capsules, food fortification, nutrition education, and food-based strategies to reduce VAD (3, 8). However, targeting and implementation of effective interventions require accurate and timely epidemiologic data on the magnitude and distribution of the problem. Generating information on VAD has been hampered by technological and cost constraints. The main biological indicator recommended by the World Health Organization for tracking progress in VAD-control programs is serum retinol. Current methods used for the assessment of serum retinol include HPLC and fluorometry (9-17), which require a functioning laboratory, expensive and delicate equipment, well-trained and highly skilled technicians, and significant investments of time and other resources (18).

There has been a need to develop rapid, inexpensive, and quantitative tools to reduce the programmatic burden of accurately determining vitamin A status (9, 18). Retinol-binding protein (RBP) was shown to be a useful surrogate marker for retinol because of the approximate 1:1 (molar) correlation between retinol and RBP in serum (6, 19-21), which implies that RBP may be used to assess and monitor VAD in populations.

This report describes the development and laboratory validation of an RBP enzyme immunoassay (RBP EIA) that was designed to quantify RBP from individual serum specimens. The RBP EIA was designed to analyze biological specimens rapidly and to reduce reliance on costly, centralized laboratory facilities. Such a test could provide an invaluable tool for field monitoring and recognition of VAD in at-risk populations. Acceptance and use of the test will allow health care workers to effectively assess the extent of VAD within populations and implement appropriate interventions.

	MATERIALS AND METHODS

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Assay development
The RBP EIA was developed as an antigen competition assay in a Maxisorb microwell strip (Nalge Nunc International, Rochester, NY) format to detect and quantify RBP from human serum. To develop this test, a murine hybridoma cell line secreting monoclonal antibodies (MAs) that were highly specific for RBP was licensed from the University of Massachusetts, Amherst. The MAs were purified from mouse ascites fluids by protein A (Prosep; Millipore Corporation, Bedford, MA) chromatography and stored at –70°C until used. The MAs were directly labeled with horseradish peroxidase (22) for use in the assay. The labeled MAs bind to the RBP found in serum and to the RBP adsorbed to microtest strip wells. RBP serum specimens used for the development of this assay were purchased from Sigma (St Louis), and RBP calibration standards were purchased from Dade Behring (San Jose, CA).

Assay protocol
For performance of the assay, 10 µL of each serum specimen and calibrator was first diluted 1:25 in assay buffer containing phosphate-buffered saline, 0.1% (wt:vol) bovine serum albumin, 0.1% (wt:vol) Tween 20, and 0.1% (wt:vol) thimerosal at pH 7.2. All dilutions were made in wells of a low protein-binding microplate (Costar, Cambridge, MA). After dilution, specimens and calibrators were gently mixed by pipetting, and 100 µL of each specimen or calibrator was transferred to the test wells. One hundred microliters of diluted MA, which was directly conjugated with horseradish peroxidase, was immediately added to the test wells. The wells were incubated at ambient temperature (18–25°C) for 15 min. After incubation, the contents of the test wells were emptied into a sink, and the wells were then rinsed 5 times with wash buffer (phosphate-buffered saline-Tween 20, pH 7.2). Two hundred microliters of 3,3,5,5-tetramethylbenzidine (Moss Inc, Pasadena, MD) containing hydrogen peroxide was added to each test well. The test wells were then incubated at ambient temperature (18–25°C) for an additional 10 min. One hundred microliters of stop solution [1% (vol:vol) HCl; Sigma] was added to each test well. The plates were immediately read with either an EIA plate reader or strip-well EIA reader fitted with a 450-nm filter, and the resulting absorbance was recorded. The results were calculated on the basis of values obtained from the calibrator sera by linear regression software (REVELATION software included with a model MRX Dynex plate reader; Dynex Technologies, Chantilly, VA) or by graphing the RBP concentration versus the absorbance obtained from the calibrator sera.

Proof of concept
Assay validation was facilitated by the analysis of 25 serum specimens that were provided by the Johns Hopkins University (Baltimore) and that were a subset of sera collected from children during a micronutrient study in Papua New Guinea (23). Retinol concentrations measured by HPLC were previously obtained but were not disclosed for this study until the RBP EIA measurements were completed. The specimens were stored at -70 °C until they were used for this study. They were analyzed in duplicate by using the RBP EIA, and the results were averaged. Approval for the use of these sera for micronutrient research was obtained through the ethical review boards at the Johns Hopkins University and the Program for Appropriate Technology in Health (PATH), Seattle.

Assay performance characteristics
Assay performance characteristics were established with serum specimens from 5 adult volunteers and a commercially available source (Analytic Control Systems Inc, Fishers, IN). After blood samples were collected by venipuncture from volunteers, the samples were transferred to clean tubes and allowed to coagulate for 1 h at 37 °C. The serum fraction was then separated from the clotted blood by centrifugation at 800 x g and 2–15°C for 15 min, and the serum was immediately removed, placed in glass tubes, and stored at 2–8 °C until testing was performed. Four investigators then tested the samples independently by using the RBP EIA, and the results were averaged. The performance characteristics of the RBP EIA, including accuracy, precision, detection limit, quantitation limit, linearity, range, analyte recovery, and intraassay variability were established in the laboratory at the PATH by using the guidelines referenced in Validation of Compendial Methods (24).

Interfering substances
Analytic interference testing was performed to determine the effect of endogenous and exogenous substances on test results. Interference testing protocols were designed according to guidelines in Validation of Compendial Methods (24). Substances tested included human serum with elevated C-reactive protein (catalog no. S2895; Sigma), human serum with increased rheumatoid factor (catalog no. S3145; Sigma), bilirubin (catalog no. B4136; Sigma), hemoglobin (catalog no. H7379; Sigma), red blood cells (catalog no. R0043; Sigma), triacylglycerols (as triolein, catalog no. T7140; Sigma), L-thyroxine (catalog no. T2376; Sigma), prealbumin (catalog no. P7528; Sigma), retinol (catalog no. V7763; Sigma), estrone acetate (catalog no. E7132; Sigma), and ß-estradiol (catalog no. E8875; Sigma). The concentrations of the potentially interfering substances that were tested are shown in Table 1.

Evaluations of sera collected in Nicaragua
Eighty-four serum specimens were randomly selected from a large panel of specimens collected from mothers and their children as part of a population-based study in Managua, Nicaragua, by the Micronutrient Operational Strategies and Technologies (Rosslyn, VA) project in 2000. Aliquots (250 µL) of frozen sera from whole blood specimens were delivered to the PATH and kept frozen at -80 °C until they were assayed. They were then thawed at ambient temperature, agitated gently, and immediately tested in duplicate by using the RBP EIA, and the results of the 2 measurements were averaged. The RBP concentrations from the RBP EIA analyses were then compared with RBP concentrations estimated by using the RBP radial immunodiffusion (RID) method (The Binding Site, San Diego) in 40 of these randomly selected specimens. To determine RID plate results, the instructions provided by the manufacturer were followed. Five microliters of each specimen was introduced into the wells cut in the agarose plate. The plates were incubated at ambient temperatures (18–25°C), and the results were read after 3 d. The degree of antigen-antibody binding was measured by determining the diameters of the resulting "halos" of precipitation around the wells into which the specimens had been introduced. The precipitin measurements were made with the aid of a calibrated jeweler's magnifying loupe and a fluorescent light box to provide incident lighting. In addition, the RBP concentrations were compared with retinol concentrations that were measured by HPLC with the Bio-Rad HPLC vitamin A/E determination kit (Bio-Rad, Hercules, CA). Of the 84 specimens, 14 were not used in the comparison between RBP EIA and HPLC because of either insufficient sample volume or baseline drift during the HPLC retinol analysis. Therefore, only 70 samples were used for this comparison. A Shimadzu (Tokyo) LC10A HPLC system with an SPD 10AV VP detector (Shimadzu), LC10ADVP pump (Shimadzu), and Rheodyne 7725i injector (Rheodyne LLC, Rohnert Park, CA) was used, and the data were registered by using a C-R5A chromatopac recorder (Shimadzu). The analytic 4.0-mm (inside diameter) x 250-mm (length) HPLC column supplied with the Bio-Rad vitamin A/E determination kit was used. All manufacturers' instructions were followed exactly when using the HPLC instrument and the commercial retinol detection kit.

Data analyses
SDs and CVs were calculated by using Microsoft EXCEL 2000 (Microsoft, Redmond, WA). Results from the retinol and RBP analyses were converted to µmol/L from either µg/dL or µg/mL. For comparisons, data were presented graphically as scatter plots, and linear regression models were developed to estimate the correspondence between RBP and retinol.

	RESULTS

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Proof of concept
To establish assay validity, the 25 serum samples from Papua New Guinea were assayed with the RBP EIA, and the resulting values were compared with serum retinol values. These results are summarized in Figure 1. The data were initially calculated both on the basis of values obtained from linear regression software and by hand graphing the assay results and the ODs obtained from the calibrator sera to obtain the RBP concentrations. We found that both methods could be accurately used, with the latter resulting in only a minimal loss in assay precision (data not shown). The area inside the box in the lower left corner of Figure 1 represents the calibrated range for the RBP EIA. This suggests a reasonable correlation of the RBP EIA results with the HPLC retinol results within the given range (R² = 0.86). The mean retinol concentration from the HPLC analysis was 1.14 µmol/L, and the mean RBP concentration from the RBP EIA was 1.36 µmol/L. From these data, concentrations <0.7 µmol/L in both assays were classified as representing VAD. The VAD prevalence was estimated to be 36% according to HPLC retinol and 32% according to the RBP EIA. Note that although 24 of the 25 samples showed a correlation, there was one data point in the figure that fell far outside of the regression line because of an unusually high retinol concentration. This sample was later shown to have been spiked with retinol for use as a blind control to demonstrate specificity.

View larger version (13K): [in this window] [in a new window]   FIGURE 1. Comparison of retinol-binding protein (RBP) concentrations obtained with the RBP enzyme immunoassay with retinol concentrations obtained with HPLC in a serum panel from Papua New Guinea (n = 25) (y = 0.6511x + 0.6232; R2 = 0.61). The data point farthest to the right was obtained from a sample that was spiked with free retinol as a blind control. Thus, a high retinol value was obtained with a low RBP value when the sample was assayed. Removing all values outside the assay's calibrated range or >40 µg RBP/mL produced the following relation: y = 0.9478x + 0.3593; R2 = 0.86.

Table 2

Correspondence between vitamin A indicators
Data from the 3 assay methods (RBP EIA, RBP RID, and HPLC retinol) were compared in two-way analyses. With an allowance for assay variation, there was a significant correlation in the 70 sera tested between RBP concentrations obtained with the RBP EIA and retinol concentrations obtained with HPLC analysis (R² = 0.82) (Figure 2). As seen in Figure 3, RBP concentrations obtained with the RBP EIA also correlated well with RBP concentrations obtained with the RBP RID (R² = 0.73). Note that in both comparisons, the RBP EIA appeared to be more closely correlated with the other 2 assays over the critical lower ranges, where VAD would need to be determined, than over the upper assay ranges (at and beyond the upper calibration limits) that would indicate vitamin A sufficiency.

View larger version (13K): [in this window] [in a new window]   FIGURE 2. Comparison of retinol-binding protein (RBP) concentrations obtained with the RBP enzyme immunoassay with retinol concentrations obtained with HPLC in a serum panel from Nicaragua (n = 70) (y = 0.6216x + 0.3233; R2 = 0.82).

View larger version (13K): [in this window] [in a new window]   FIGURE 3. Comparison of retinol-binding protein (RBP) concentrations obtained with the RBP enzyme immunoassay (EIA) with RBP concentrations obtained with RBP radial immunodiffusion (RID) plates in a serum panel from Nicaragua (n = 39) (y = 0.4999x + 0.4487; R2 = 0.73).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

To meet the need for a more efficient, simpler method for the assessment of VAD in populations, we developed a competitive immunoassay for the detection of RBP. In comparison with protocols for conventional or direct EIAs, our method is relatively simple. The test has now been optimized in the laboratory and is in the process of undergoing extensive field validation to fully determine its optimal applications. When performed correctly, the test can be completed and results obtained in as few as 35–40 min after the samples are prepared and loaded into test wells. As many as 96 or as few as 8 measurements can be performed, because each composite, 96-well plate includes 12 strips, each of which contains 8 test wells that can be individually added or removed from the frame for use as needed with the appropriate number of sera, calibrators, and controls.

It is preferable to routinely test samples in duplicate and to average the 2 results. Duplicate sample testing allows the operator to calculate the CV between the 2 measurements, which should be <10% according to established assay acceptance criteria. If the 2 measurements are found to vary by >10%, it is recommended that the specimen be assayed again. The use of duplicate analysis increases data confidence levels by compensating for pipetting or other operator-induced errors and allows the user to more accurately quantitate the RBP in a given sample.

To validate the assay, we conducted rigorous performance testing in which we followed the guidelines in Validation of Compendial Methods (24). The results suggest that the RBP EIA is both accurate and reproducible. The results are linear and reproducible in the key range of 10–40 µg RBP/mL that was originally targeted. Specimens producing results in the higher and lower extremes of the calibration curve may be less accurate. However, these lower and higher values usually represent a minor proportion of the population studied, and there is a high probability that these specimens will still be correctly classified as vitamin A deficient or sufficient.

In our laboratory-based evaluations of specimens collected from the field, results obtained with the RBP EIA were compared with serum retinol results obtained with HPLC, which is the reference standard for determining vitamin A status. A close correlation between the 2 methods was found (R² = 0.82). A somewhat lower, but still acceptable, correlation was found between the RBP EIA and RBP RID methods (R² = 0.73). A lower correlation was found when the RBP RID method was compared with HPLC measurement of retinol (R² = 0.71). Although we found that the RBP RID method was slightly less accurate than the RBP EIA method in capturing the variation in retinol, our findings support those of previous reports indicating that the RBP RID method could be a useful tool in identifying VAD in populations (14). We noted an inherent subjectivity in reading the RBP RID plates because the diameters of the test reactions are determined by measuring the rings of antigen-antibody precipitation with a calibrated jeweler's loupe and an indirect light source. In our opinion, in comparison with other methods, the RBP EIA method was simpler to use and much more rapid and generated a higher sample throughput. We are now finalizing the validation of the RBP EIA in larger studies conducted under typical field conditions, because the number of samples tested in this initial study was limited.

Because the RBP EIA is a competitive immunoassay developed in a microwell plate format, it has the potential to generate more data at significantly lower costs and in less time than does HPLC measurement of serum retinol. For example, one technician working <3 h analyzed the entire panel of 70 specimens from Nicaragua (each sample was tested in duplicate). By contrast, testing the same specimens by using the reference HPLC retinol method required >5 d to complete in our laboratory. The serum retinol measurements could have been done in less time if an autosampler had been used. Note that with an autosampler, 20–25 samples, on average, can be extracted and analyzed in 1 d.

The RBP EIA was developed to meet the need for a low-to-moderate-volume assay. This method can produce data rapidly, minimize reliance on centralized laboratory facilities, and provide an effective tool for vitamin A assessment in populations. We recently identified a manufacturer (Scimedx Corporation, Denville, NJ) who will provide the RBP EIA in bulk. This company has estimated that the user cost of the test for a single specimen (run in duplicate) will be <$3.00. The RBP EIA is a population-based tool that can be used to identify groups at risk of VAD and to track changes in the vitamin A status of populations but that is more straightforward and less technical to perform than is HPLC measurement of retinol. This method was specifically designed for use in laboratories at the provincial or district level or by epidemiologic surveillance teams in the field. It does require some training and previous laboratory experience to ensure that specimens are correctly collected and processed and accurately diluted and that the recommended testing protocol is followed. Although not a requirement, experience in the use of EIA technologies could also benefit the users.

The RBP EIA can potentially provide health care workers with a tool for more effective assessment of vitamin A status in populations by alleviating the major cost and time constraints inherent in evaluating specimens for retinol by HPLC, which has burdened micronutrient programs in the past. Application of the RBP EIA, with the use of minimal laboratory facilities, may allow health care workers in the future to more effectively assess the extent of VAD within communities or populations. With such data, it will be possible to design, implement, and monitor the most appropriate intervention activities to improve vitamin A status and control VAD.

	ACKNOWLEDGMENTS

 
We thank Samuel Black (University of Massachusetts, Amherst) for provision of the MA cell line and assistance in the development of the test method; and William Blaner and Jonathan Gorstein for their review of this manuscript. We also thank Omar Dary for the expert advice he provided on vitamin A during assay development.

JH was responsible for test development and validation. CM helped optimize the prototype assay through field use and feedback. IB provided input during test development and assisted with early assay development. JM provided expert advice on competitive EIAs and helped facilitate test development. MT was the senior technical advisor for assay development at the PATH. AS provided samples and expert advice on vitamin A during assay development. JH and MT are employees of the PATH. The other authors had no personal or financial interests in the PATH.

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