Development of a Novel Urine Alzheimer-Associated Neuronal Thread Protein ELISA Kit and Its Potential Use in the Diagnosis of Alzheimer's Disease

Abstract

Background: Alzheimer's disease (AD) is an age-related chronic degenerative disease that damages the nervous system. A noninvasive and simple method for early detection of AD is extremely important for the diagnosis and prognosis of AD. Thus, we aimed to develop an enzyme-linked immunosorbent assay (ELISA) kit to detect urine Alzheimer-associated neuronal thread protein (AD7C-NTP), and to evaluate its clinical value for the diagnosis of AD.

Methods: Immunogenic AD7C-NTP peptide fragments were synthesized by the solid-phase method and used for immunizing mice or rabbits to generate anti-AD7C-NTP antibodies. The urine AD7C-NTP ELISA kit was then established; the generated mouse anti-AD7C-NTP antibody was used as a capture antibody, the biotin-labeled rabbit anti-AD7C-NTP antibody was used as a detection antibody, and avidin labeled by horseradish peroxidase was used as a substrate. The first morning urine specimens of 121 AD patients and 118 age-matched controls were collected, and the urine AD7C-NTP levels were detected by the above ELISA kit.

Results: Mouse and rabbit anti-AD7C-NTP antibody ELISA titer was found to be 1:8,000 and 1:32,000, respectively. A single band with a relative molecular mass of 41 kDa was found in human brain specimens by Western blot assay, which was identified as AD7C-NTP antibody. The urine AD7C-NTP concentration of the AD patients was higher than that of the age-matched controls, the sensitivity was 89.3% and the specificity was 84.7%.

Conclusions: Our study demonstrated that our newly developed urine AD7C-NTP ELISA kit has suggested potential for diagnosing AD in a Chinese population, suggesting it may be a useful diagnostic kit for detecting early AD.

Keywords: Alzheimer disease; Alzheimer-associated neuronal thread protein; enzyme-linked immunosorbent assay; urine.

Figure 1

Immunohistochemical staining of AD7C‐NTP in the mouse frontal lobe and hippocampus. The expression of AD7C‐NTP in the mouse frontal lobe (A) and hippocampus (B) was detected by immunohistochemistry using mouse and rabbit anti‐AD7C‐NTP antibodies, respectively. The AD7C‐NTP immunopositive cells are stained in brown color. Scale bar = 50 mm.

Figure 2

AD7C‐NTP is expressed in the temporal lobe and hippocampus of AD patients. The protein expression of AD7C‐NTP in the temporal lobe (A) and hippocampus (B) of AD patients was measured by Western blot assay using specific mouse anti‐AD7C‐NTP antibody. 1:6,000, 1:6,000 dilution of anti‐AD7C‐NTP antibody; 1:3,000, 1:3,000 dilution of anti‐AD7C‐NTP antibody; M, prestained protein standard.

Figure 3

Stability test of the coated ELISA plate. The reduction of anti‐AD7C‐NTP antibody activity of coated plate following between the two groups, 37°C for 4 days and 4°C for 8 months, were compared.

Figure 4

The concentration of urine AD7C‐NTP. (A) The standard curve of urine AD7C‐NTP ELISA detection. (B) The area under the curve of urine AD7C‐NTP ELISA detection. When the best critical point of the ROC curve of urine AD7C‐NTP is 1.5 ng/ml, the sum of sensitivity and specificity reaches the maximal value; that is, at a sensitivity of 89.3% and specificity of 84.7%, the area under the curve is 0.926 and the diagnostic value is the highest.