The aim of the present study was to establish a quantitative method for the measurement of serum human augmenter of liver regeneration (hALR) using competitive inhibition that is applicable in the clinic. A monoclonal antibody to hALR was used as the primary antibody and the pure hALR protein was used as a standard for competition with Eu3+-labeled hALR (Eu3+-hALR) to plot a standard curve. Serum samples from 90 patients with various liver diseases due to hepatitis B virus (HBV) infection were used for a competitive reaction with Eu3+-hALR. A regression analysis of the results was performed using the standard curve to calculate the serum concentration of hALR. The minimum detectable value using direct competitive measurement established by Eu3+-hALR was 1 ng/ml, with a positive linear correlation within the range of 200 ng/ml. In the sera of the 90 patients, the hALR level in the severe hepatitis group was the highest, followed by that in the acute hepatitis group. The serum hALR levels in the cirrhosis and chronic hepatitis groups were significantly higher compared with those in the normal control groups (P<0.01). The direct competitive measurement method of serum hALR established in the present study has high sensitivity, specificity, stability and reliability, meets clinical requirements and may be used as potential index in clinical tests.
Augmenter of liver regeneration (ALR) is a non-specific hepatocyte growth-promoting factor with heat stability, identified by Hagiya
Recombinant plasmid pQE30-hALR was constructed in the Institute for Viral Hepatitis (Chongqing Medical University, Chongqing, China), as previously described (
The serum samples were collected from patients in the outpatient or inpatient ward of the Department of Infectious Diseases, the Second Affiliated Hospital, Chongqing Medical University between September 2005 and April 2006. The diagnosis met with the Viral Hepatitis Prevention & Treatment Strategy released by the Chinese Society of Hepatology and Society of Infectious Diseases (2005)(
i) Pronucleus expression of hALR: following identification, the recombinant plasmid pQE30-hALR was transformed into
Anti-hALR hybridoma (AAMA) cells were established in our laboratory (
Direct antigen competition was used to coat the 96-well ELISA plate with an optimal working concentration of anti-hALR (monoclonal antibody 100 μl/well, 1:800) at 4°C overnight. BSA/PBST was added (3%, 200 μl/well) and the well was blocked at 37°C for 3 h. Standard purified hALR protein (50 μl, 2 μg/ml) diluted with PBS at 1:10, 1:50, 1:100, 1:500, 1:1,000 and 1:5,000 was mixed with 50 μl Eu3+-hALR in the same reaction well for competitive inhibition reaction in the plate shaker and incubated at 37°C for 1 h. Three parallel wells were used for each concentration. Fluorescence enhancing solution (100 μl) was added to each well, the plates were incubated at 37°C for 5 min and the resulting samples were analyzed by TRFIA to create a standard curve. The serum samples from the patients with various liver diseases were used to replace the standard protein and competitively react with Eu3+-hALR in the blank, negative antibody control and negative quality control serum wells. A regression analysis of the results was performed using the standard curve to calculate the hALR concentration in the sera of the 90 patients with various liver diseases. Calf serum was used as a medium to prepare serial concentrations of hALR at 5, 10, 20 and 40 ng/ml to be detected with the direct competitive assay, and then the coefficient of recovery and the variation coefficient were calculated.
The data are expressed as mean ± SD. SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) was used to compare intra-group differences using a Student’s t-test. P<0.05 was considered to indicate a statistically significant result.
The recombinant plasmid pQE30-hALR showed high expression in the host bacteria and the molecular weight of the product was approximately 15 kDa (
The harvested anti-hALR monoclonal antibody ascites were measured by ELISA with an optimal working concentration of 1:800 (
A standard curve for competitive inhibition of Eu3+-hALR and hALR was constructed. The fluorescence values of standard hALR and Eu3+-ALR at various concentrations were measured by TRF. A standard curve was constructed using matched software (Sym-Bio Life Science, Zhejiang, China), with the concentration of standard hALR on the x-axis and the ratio of fluorescence value in reaction wells/measured maximal fluorescence value on the y-axis, as shown in
As shown in
Using calf serum as a medium, the coefficient of recovery was detected.
ALR is a recently discovered growth factor that promotes liver regeneration and, similar to other cellular factors such as IGF and EGF, plays an important role in the regeneration of hepatocytes. We aim to understand the hALR concentration in the sera of patients with hepatitis and cirrhosis, and further investigate its interaction with these diseases.
In previous studies (
The results indicated that the serum level of hALR in patients with viral hepatitis was higher than that in normal serum. In particular, severe hepatitis had the highest hALR level, which was 15-fold that of normal serum and 6 to 7-fold that of common hepatitis; the hALR level in acute hepatitis was 3-fold that of normal serum, and cirrhosis and chronic hepatitis had 3-fold higher serum hALR levels than normal serum. The hALR levels determined in the present study were higher than those reported in a previous study (
We noted that the serum hALR levels increased quickly in severe hepatitis caused by hepatitis B virus (HBV) infection. The reason for this increase is due to hepatocytes undergoing marked necrosis and releasing large amounts of hALR into the blood accompanied by increased secretion from other organs, including the kidney (
Based on the present results, the competitive inhibition method has sufficient accuracy, specificity and sensitivity to meet the requirements of clinical application. Through continuous optimization and widespread practice, this method may be used as a clinical diagnostic index for liver diseases and other diseases associated with hALR (such as urinary system diseases) (
This study was supported by the National Natural Science Foundation of China (No.s 30271178 and 30570826).
Purification of human augmenter of liver regeneration (hALR). Lanes 1 and 2: induction of SG(pQE30-hALR); lane 3: protein marker; lanes 4 and 5: purification of SG(pQE30-hALR) 15 and 5 mg/ml, respectively.
Identification of purified human ugmenter of liver regeneration (hALR) by capillary electrophoresis (CE).
Reactivity of anti-hALR monoclonal antibody. Lane 1: hALR expressed in SG13009; lane 2: human serum albumin. hALR, human augmenter of liver regeneration.
The standard curve of human augmenter of liver regeneration (hALR).
OD450 of hALR combined with mouse ascites in different concentrations.
Concentration of mouse ascites diluted with PBS (v/v) | |||||
---|---|---|---|---|---|
| |||||
Mouse ascites | 1:200 | 1:400 | 1:800 | 1:1000 | 1:2000 |
SP2/0 ascites | 0.214±0.08 | 0.184±0.05 | 0.155±0.05 | 0.164±0.08 | 0.138±0.06 |
AAMA ascites | 0.985±0.15 |
0.939±0.07 |
0.896±0.04 |
0.785±0.01 |
0.671±0.06 |
Compared with corresponding concentration of SP2/0 ascites,
P/N>2.1,
P/N maximum value.
ELISA, enzyme-linked immunosorbent assay.
Serum hALR levels in 90 patients with various liver diseases.
Clinical group | No. of cases | hALR concentration (ng/ml) |
---|---|---|
Normal control | 10 | 3.77±1.55 |
Acute hepatitis | 5 | 10.14±3.26 |
Chronic hepatitis | 30 | 8.44±2.78 |
Cirrhosis | 30 | 10.11±4.32 |
Severe hepatitis | 15 | 57.34±18.96 |
P<0.01 vs normal control.
hALR, human augmenter of liver regeneration.
Recovery percentages of the direct competitive reaction.
Addition hALR (ng/ml) | Repeated wells (n) | Detected concentration hALR (ng/ml) | Recovery percentage (%) | Variation coefficient (%) |
---|---|---|---|---|
5.0 | 3 | 4.16 | 83.2 | 7.12 |
10.0 | 3 | 8.52 | 85.2 | 4.84 |
20.0 | 3 | 18.73 | 93.7 | 7.35 |
40.0 | 3 | 33.54 | 83.9 | 9.86 |