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Am J Physiol Renal Physiol 293: F956-F960, 2007. First published June 6, 2007; doi:10.1152/ajprenal.00090.2007
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INNOVATIVE METHODOLOGY

Novel sandwich ELISA for human angiotensinogen

Akemi Katsurada,1,* Yoshiaki Hagiwara,2 Kazuya Miyashita,2 Ryousuke Satou,1 Kayoko Miyata,1 Naro Ohashi,1 L. Gabriel Navar,1 and Hiroyuki Kobori1,*

1Department of Physiology, and Hypertension and Renal Center of Excellence, Tulane University Health Sciences Center, New Orleans, Louisiana; and 2Immuno-Biological Laboratories Co., Ltd., Fujioka, Gunma, Japan

Submitted 20 February 2007 ; accepted in final form 25 May 2007


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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 REFERENCES
 
We recently reported that urinary excretion rates of angiotensinogen (UAGT) provide a specific index of intrarenal renin-angiotensin (ANG) system (RAS) status in ANG II-dependent hypertensive rats. When this is shown to be applicable to human subjects, a diagnostic test to identify those hypertensive patients most likely to respond to an RAS blockade could provide useful information to allow a mechanistic rationale for selection of an optimized approach to treatment of hypertensive patients. However, simple and accurate methods to measure human angiotensinogen (hAGT) are unavailable. For future studies of human subjects, we developed antibodies and a sensitive and specific quantification system for hAGT using a sandwich ELISA. We raised two antibodies against hAGT: a mouse monoclonal antibody and a rabbit polyclonal antibody. The standard curve of this ELISA exhibited a high linearity (0.31–20 ng/ml). The correlation coefficient was >0.99. Plasma angiotensinogen concentrations of healthy volunteers ranged from 28 to 71 µg/ml (n = 10). The ratio of UAGT to urinary creatinine concentration ranged from 5.0 to 30 µg/g (n = 7). Intra- and interassay coefficients of variation ranged from 4.4 to 5.5% and from 4.3 to 7.0%, respectively. This ELISA system had no cross-reactivity with major proteins in proteinuric urine samples, such as human albumin, immunoglobulin, or transferrin. Moreover, the cross-reactivity of the system with angiotensin peptides was also negligible. This hAGT ELISA will be a useful tool to investigate the relationship of UAGT and reactivity to antihypertensive drugs in hypertensive patients.

renin-angiotensin system; plasma; urine

THE RENIN-ANGIOTENSIN SYSTEM (RAS) is well known to play an important role in blood pressure regulation and fluid and electrolyte homeostasis (18). In recent years, the focus of interest on the RAS has shifted to a main emphasis on the role of the local/tissue RAS in specific tissues (5). Emerging evidence has demonstrated the importance of the tissue RAS in the brain (1), heart (4), adrenal glands (15), and vasculature (3, 6), as well as the kidneys (18). There is substantial evidence that the major fraction of angiotensin II in renal tissues is generated locally from angiotensinogen delivered to the kidney, as well as from angiotensinogen locally produced by proximal tubule cells (7). Renin secreted by juxtaglomerular apparatus cells into the renal interstitium and vascular compartment also provides a pathway for the local generation of angiotensin I (17). Angiotensin-converting enzyme is abundant in the rat kidney and is present in the proximal and distal tubules, collecting ducts, and renal endothelial cells (2). Angiotensin I delivered to the kidney can also be converted to angiotensin II (14). Therefore, all the components necessary for generation of intrarenal angiotensin II are present along the nephron (18).

The presence of all components of the RAS in the kidney provides a great flexibility and independence in regulating intrarenal levels of angiotensin II, and the differential regulation of angiotensin peptide levels in plasma and kidney has been established (19). Recently, we reported that urinary excretion rates of angiotensinogen provide a specific index of intrarenal RAS status in angiotensin II-dependent hypertensive rats (8–11, 13). When this is shown to be applicable to human subjects, a diagnostic test to identify those hypertensive patients most likely to respond to blockade of the RAS could provide useful information to allow a mechanistic rationale for selection of an optimized approach to treatment of hypertensive patients. ELISA for human angiotensinogen has been reported (16, 21). However, simple and accurate methods to measure human angiotensinogen are unavailable. For future studies of human subjects, we developed antibodies and a sensitive and specific quantification system for human angiotensinogen using a microtiter plate-based sandwich-type ELISA.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Protocol. The experimental protocol of this study was approved by the Institutional Review Board of Tulane University. All samples were obtained from healthy volunteers, who provided written informed consent.

Preparation of expression vectors for human angiotensinogen. The full length, except for the signal peptide (1-33 aa), of the human angiotensinogen (34-485 aa) gene (gene identification no. 73622269) was amplified by PCR using a high-fidelity Pyrococcus furiosus DNA polymerase (Promega) with sense (5'-CGG GAT CCG ACC GGG TGT ACA TAC ACC CC-3') and antisense (5'-CGG TTG GGC GAC TCG TGT CGT GAG CTC GCC-3') primers from the human adult liver cDNA library (Clontech). Then this fragment was inserted into pGEX4T1 expression vectors (Promega) with the glutathione S-transferase (GST) tag at the 5'-end by the restriction enzymes BamH I and Sal I.

Preparation of recombinant proteins for human angiotensinogen. The recombinant constructs were transformed to a high-efficiency expression bacterial strain (Takara). Large-scale bacterial cultures were induced with isopropyl-beta-D-thiogalactopyranoside (Takara) and harvested for protein purification. GST-tagged proteins were purified using glutathione beads (Upstate) in native conditions.

Antibody preparation. We raised two antibodies for human angiotensinogen: a mouse monoclonal and a rabbit polyclonal antibody. The monoclonal antibody was raised in mouse against recombinant protein of human angiotensinogen (see above). The polyclonal antibody was raised in rabbit against synthetic oligopeptide corresponding to human angiotensinogen (72-89 aa). Both antibodies were affinity purified.

Western blot. Western blot analysis was performed as previously described (12, 20, 22) using the LI-COR Odyssey infrared imaging system.

Epitope mapping. The epitopes of these antibodies were determined by Western blot analysis using a variety of lengths of recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa).

Plate preparation. The ELISA plates were coated with the polyclonal antibody against human angiotensinogen (100 µl/well in 100 mmol/l carbonate buffer, pH 9.5) at 4°C overnight. The plates were washed with PBS and blocked with 1% bovine serum albumin (200 µl/well) in PBS containing 0.05% NaN3 at 4°C overnight. The plates were stored at 2–8°C.

Sample collections. Peripheral blood samples from healthy volunteers were collected into EDTA-containing tubes, and plasma samples were separated after centrifugation. First-morning-urine samples were also collected from healthy volunteers.

Development of sandwich ELISA. Highly purified angiotensinogen from human plasma was used as the standard. Human angiotensinogen standards (0.31–20 ng/ml diluted in ELISA buffer), plasma (1:8,000 dilution in ELISA buffer), and urine (1:8 dilution in ELISA buffer) samples (100 µl/well) were added to each well of the plates and incubated at 37°C for 1 h. Then the plates were washed a total of seven times with a washing buffer (PBS containing 0.05% Tween 20, pH 7.5). After the plates were incubated with horseradish peroxidase-labeled monoclonal antibody against human angiotensinogen (100 µl/well, 1:30 dilution in antibody solution) at 37°C for 30 min, they were washed a total of nine times with the washing buffer. Then the plates were incubated with 3,3',5,5'-tetramethylbenzidine solution (100 µl/well) under light-protected conditions at room temperature for 30 min. The reaction was stopped by treatment of the plates with sulfuric acid (100 µl/well, 0.5 mol/l). The absorbance values were measured at 450 nm.

Measurement of urinary creatinine. The urinary creatinine concentrations were measured by an automated machine (model DCA 2000+, Bayer) with microalbumin/creatinine reagent kits (Bayer). The urinary angiotensinogen concentrations were normalized to urinary creatinine concentrations.

Statistical analysis. Statistical analysis was performed using a one-way factorial ANOVA with post hoc Scheffé's F test. Values are means ± SE. P < 0.05 was considered significant.


   RESULTS AND DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
GRANTS
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Epitope mapping of antibodies. An equal amount (10 ng) of various lengths of recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa) was blotted on nitrocellulose membranes, and epitope mapping was done by Western blot. The polyclonal antibody against human angiotensinogen recognized GST-tagged recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa), but not mock protein or GST protein (Fig. 1A) . The monoclonal antibody against human angiotensinogen recognized GST-tagged recombinant proteins of human angiotensinogen (34-485 aa), but not GST-tagged recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, and 34-393 aa), mock protein, or GST protein. An antibody against GST protein recognized GST-tagged recombinant proteins of human angiotensinogen (34-124, 34-223, 34-303, 34-393, and 34-485 aa), as well as GST protein, but not mock protein. These data clearly indicate that the epitope for the polyclonal antibody against human angiotensinogen is located on the NH2 terminus of human angiotensinogen (34-124 aa) and that the epitope for the monoclonal antibody against human angiotensinogen is on the COOH terminus of human angiotensinogen (394-485 aa).


Figure 1
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  		Fig. 1. A: epitope mapping. GST, glutathione S-transferase; hAGT, human angiotensinogen; Poly and Mono, poly- and monoclonal antibodies. B: absorbance values. Polyclonal antibody-coated ELISA plates were incubated at 37°C for 1 (open bars), 8 (gray bars), or 15 (solid bars) days. C: a corresponding standard curve.

 
Plate stability. To address their stability, we incubated the polyclonal antibody-coated ELISA plates at 37°C for 1, 8, or 15 days. Then different concentrations of human angiotensinogen standards were applied to the plates and processed, and the absorbance values were obtained. Each concentration of human angiotensinogen standards (n = 6) exhibited similar absorbance values beyond 15 days (Fig. 1B). These data clearly indicate that the polyclonal antibody-coated ELISA plates are stable ≥15 days after antibody coating.

Standard curves. The corresponding standard curve exhibited a high linearity (0.31–20 ng/ml), with absorbance values ranging from 0.03 to 2.34 (n = 6 at each concentration; Fig. 1C). The correlation coefficient was >0.99.

Detection limit. The lowest sensitivity for this system (0.31 ng/ml) was determined using the guidelines of the National Committee for Clinical Laboratory Standards Evaluation Protocols.

Plasma and urinary angiotensinogen concentrations. Angiotensinogen concentrations in plasma obtained from 10 healthy volunteers ranged from 28 to 71 µg/ml (Fig. 2A) . Angiotensinogen concentrations in urine obtained from seven healthy volunteers ranged from 7.1 to 35 ng/ml (Fig. 2B). Urinary creatinine concentrations of seven healthy volunteers were 0.93–2.2 mg/ml. The ratio of urinary angiotensinogen concentration to urinary creatinine concentration was 5.0–30 µg/g (Fig. 2C).


Figure 2
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  		Fig. 2. A: angiotensinogen concentrations in plasma obtained from 10 healthy volunteers. Samples were applied to the ELISA system in triplicate, and averaged values were obtained. B: angiotensinogen concentrations in urine obtained from 7 healthy volunteers. Samples were applied to the ELISA system in triplicate, and averaged values were obtained. C: ratio of urinary angiotensinogen concentration to urinary creatinine concentration.

 
Addition and recovery test. Different known concentrations of human angiotensinogen standards were added to a human plasma sample or a human urine sample, and the added angiotensinogen concentrations were calculated as the difference between angiotensinogen concentrations in samples with and without added angiotensinogen. Recovery rates were >82.9% for plasma and >87.6% for urine (Table 1).


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  		Table 1. Addition-and-recovery test

 
Sample stability. To address stability of samples, we performed two tests: a freeze-and-thaw test and an accelerated test. In a freeze-and-thaw test, one plasma sample was frozen and thawed 0, 1, 2, and 3 times, and plasma angiotensinogen concentrations were measured by the ELISA system. No significant differences were observed (Fig. 3A). In an accelerated test, one plasma sample was incubated at 37°C for 0, 1, 3, and 7 days, and plasma angiotensinogen concentrations were measured by the ELISA system. No differences were observed (Fig. 3B). These data clearly indicate that angiotensinogen in plasma samples is stable without preservatives or proteinase inhibitors, except EDTA.


Figure 3
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  		Fig. 3. A: freeze-and-thaw test. B: accelerated test.

 
Intra-assay test. Intra-assay tests were performed using three different concentrations of human angiotensinogen standards. Coefficients of variation among the wells ranged from 4.4 to 5.5% (Table 2).


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  		Table 2. Intra-assay test

 
Interassay test. Interassay tests were performed using three different concentrations of human angiotensinogen standards. Coefficients of variation among the plates ranged from 4.3 to 7.0% (Table 3).


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  		Table 3. Interassay test

 
Specificity. To confirm cross-reactivity with other proteins, several human proteins (20 µg/ml) were applied to the ELISA system and processed. Cross-reactivity for other human proteins in this ELISA system was negligible (Table 4). In addition, downstream products of angiotensinogen (20 µg/ml) were also applied to the ELISA system. The cross-reactivity for angiotensin peptides in this ELISA system was also negligible (Table 4).


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  		Table 4. Specificity

 
Effect of pH of test samples on sensitivity of ELISA. As described above, plasma samples were diluted 1:8,000 and urine samples 1:8 in PBS-based dilution buffer (pH 7.0); therefore, pH of diluted plasma and urine samples should be ~7.0. To address this issue, seven urine samples were prepared (pH 5.5–7.0, 6.3 ± 0.6). After the samples were diluted with the dilution buffer, pH values of the samples were integrated (pH 6.7–7.0, 6.8 ± 0.2). Moreover, diluted urine samples worked well with ELISA (15.9 ± 6.4 ng/ml). These data clearly indicate that the change of urine sample pH does not affect the sensitivity of ELISA.

Conclusion. We developed antibodies and a sensitive and specific quantification system for human angiotensinogen using a sandwich ELISA. This human angiotensinogen ELISA will be a useful tool to investigate the relationship between urinary angiotensinogen excretion rate and reactivity to antihypertensive drugs in hypertensive subjects.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants R01 DK-072408, P20 RR-017659, and R01 HL-026371, the Health Excellence Fund from the Louisiana Board of Regents, and Sankyo Co., Ltd.


   ACKNOWLEDGMENTS
 
The authors acknowledge the excellent technical assistance of My-Linh Rauv, Duy V. Tran, and Dale M. Seth (Tulane University).

Portions of this study were presented in abstract form at the 17th Scientific Sessions of the Inter-American Society of Hypertension, Miami Beach, FL, May 6–10, 2007.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Kobori, Tulane Univ. Health Sciences Center, 1430 Tulane Ave., #SL39, New Orleans, LA 70112-2699 (e-mail: hkobori{at}tulane.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. Katsurada and H. Kobori contributed equally to this work. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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