Establishment and Application of a Method for the Determination of Ganoderic Acid A

A method for the quantitative determination of ganoderic acid A was constructed using the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA), and this method was used to determine the ganoderic A contents of Ganoderma lucidum samples in the market. .e conjugate of ganoderic acid A and bovine serum albumin was used for four rounds of immunization on test rabbits to obtain rabbit antiganoderic acid A antibody IgG. .e enzyme-labeled plate was coated with the conjugate of ganoderic acid A and ovalbumin. .e first stage reaction in the indirect competitive ELISA was that the conjugate of ganoderic acid A in the sample competed with the conjugate coated on the enzyme-labeled plate to bind rabbit antibodies. .e second stage reaction was the combination of goat anti-rabbit IgG–horseradish peroxidase and rabbit antiganoderic acid A antibody IgG..e results of the determination of ganoderic acidA standard by this method showed that the coefficient of variation of repeated wells in the group was <5%, the detection limit of ganoderic acid Awas 0.6 μg/L, and ganoderic acid A had a substantial dose-response relationship in the content range of 0.9–72.9 μg/L (R � 0.994). .is method was used to measure the ganoderic A content of 12 varieties of G. lucidum in the market and showed the obvious differences in the ganoderic acid A contents of the different varieties. .is method is simple, fast, and of great importance to the quality control of Ganoderma products.


Introduction
Ganoderma is rich in a variety of active ingredients, and its pharmacological effects are different. Triterpene is one of the main active components of Ganoderma lucidum. Triterpene has liver-protective [1], antitumor [2,3], immunitystrengthening [4,5], and antioxidation functions [6,7], and it lowers blood lipid and blood sugar [8,9]. According to investigation and analysis, the triterpenoid contents of different types of G. lucidum vary greatly, and the triterpenoid contents between wild and cultivated G. lucidum differ greatly [10][11][12]. Ganoderic acid A accounts for the largest proportion of G. lucidum triterpenes and is one of the Ganoderma triterpenoid markers [13]. Ganoderic acid A plays a major role in improving immunity [14]. erefore, the development of a fast and simple method with a low detection limit for the determination of ganoderic acid A is of great importance for the quality control of Ganoderma products. At present, the detection methods of ganoderic acid A include visible light colorimetry [15], UV spectrophotometry [16,17], high-performance liquid chromatography [18][19][20][21], and chromatography-mass spectrometry [22,23]. e main drawbacks of these methods are the high cost of the highly specialized instrument, difficulty in popularizing the method, and long detection time. erefore, providing a simple and rapid detection method of ganoderic acid A is an urgent problem that needs to be solved. e purpose of this research was to use the principle of enzyme-linked immunosorbent assay (ELISA) to construct a method for the quantitative determination of ganoderic acid A and determine and analyze the ganoderic acid A contents of different G. lucidum varieties in the market. ELISA includes antigen coating, sample loading reaction, and substrate color development. e method used in this experiment was indirect competitive ELISA. e sample loading reaction involved two stages. e first stage is the competition between the ganoderic acid A in the sample and the conjugate coated on the enzyme-labeled plate to bind rabbit antiganoderic acid A antibody IgG, and the second stage is the combination of goat anti-rabbit IgGhorseradish peroxidase (HRP) and rabbit antiganoderic acid A antibody IgG.

Preparation of the Conjugate of Ganoderic Acid A and
Bovine Serum Albumin. Bovine serum albumin (500 mg) was dissolved in 8 mL of 2% sodium bicarbonate-tetrahydrofuran (THF) solution (1 :1). Ganoderic acid A (100 mg), 1-hydroxybenzotriazole (140 mg), N,N-diisopropylcarbodiimide (140 mg), and 1 mL of THF were stirred at 0-5°C for 1 h to form an activated ester. e 20% of the total mass of the activated ester solution was added to the bovine serum albumin solution and reacted at room temperature for 2-4 h. e solution was freeze-dried for 48 h to obtain a powder. e powder was dissolved in 50 mL of purified water and filtered with a 0.45 μm filter membrane to remove insoluble materials. e filtrate was collected and permeated with a permeable membrane with a molecular weight cutoff of 3 kD for 10 h. e purified water was changed every 2 h. e solution was freeze-dried for 72 h after permeation treatment to obtain a solid powder, which is the conjugate of ganoderic acid A and bovine serum albumin.

Preparation of the Conjugate of Ganoderic Acid A and
Chicken Egg Albumin. Bovine serum albumin was replaced with chicken egg albumin in 2.2.1.

Preparation of Rabbit Antiganoderic Acid A Immune
Serum and Purification of Antibody IgG. Two healthy Japanese white rabbits were selected, and the conjugate of ganoderic acid A and bovine serum albumin was used as the immunogen. Four rounds of immunization were carried out, and the dose of the conjugate was 1 mg per round for each rabbit. e first round of vaccination conjugate was mixed with Freund's incomplete adjuvant. A subcutaneous multipoint injection was used. e second, third, and fourth rounds of immunization were performed after 14 days. e latter three rounds of inoculation did not need an adjuvant and were injected by ear vein at an interval of 7 days. At 7 days after the fourth round of vaccination, the antiganoderic acid A immune serum was separated by cardiac blood sampling, Purified to IgG, by A protein affinity chromatography column, and concentrated to original volume 4°C save.

Determination of the Effect of Ganoderic Acid A Antibody.
e titer of rabbit antiganoderic acid A antibody IgG was determined by indirect competitive ELISA. e conjugate of ganoderic acid A and bovine serum albumin was dissolved in carbonate buffer at pH 9.6 and coated on an enzyme-labeled plate at a concentration of 20 μg/mL. After standing for 24 h at 4°C, the coating buffer was discarded and washed with a phosphate-buffered solution with Tween (PBST) three times. e rabbit antiganoderic acid A antibody IgG solution (100 μL) diluted 1 : 5 times with the sample diluent was added in the small holes in rows 1 and 2, and the rabbit antiganoderic acid A antibody IgG solution (100 μL) absorbed by bovine serum albumin and diluted 1 : 5 times with the sample diluent was added to the small holes in rows 3 and 4 (treatment method: 10% bovine serum albumin solution was prepared with sample diluent, 4 mL of the bovine serum albumin solution was added to 1 mL of rabbit antiganoderic acid A antibody IgG solution, and let stand at 4°C for 48 h so that the treated solution was equivalent to the original antibody solution diluted by 1 : 5 times). In the last column of the four rows of holes, 100 μL of negative serum with 1 : 100 dilution was added as the control. e solution was shaken in a microplate constant temperature shaker at 200 r/min for 30 min at 37°C. After 30 min, the reaction solution was discarded and washed with PBST three times. e HRP-IgG solution (100 μL) diluted with 1 : 1000 sample diluent was added to the holes and shaken in the microplate constant temperature shaker at 200 r/min for 30 min at 37°C. After 30 min, the reaction solution was discarded and washed with PBST three times. TMB substrates A and B were mixed as the substrate working solution.
e substrate working solution (100 μL) was added to each well and shaken in the microplate constant temperature shaker at 200 r/min for 15 min at 37°C. en, 50 μL of stop solution was added to each well. After 3 min, a microplate reader was used to measure the absorbance of each test well at a wavelength of 450 nm.

Selection and Verification of the Coating Antigen.
Indirect competitive ELISA was used to verify the effectiveness of the five coating antigens that competed with ganoderic acid A. Ganoderic acid A, the conjugate of ganoderic acid A and chicken egg albumin, the conjugate of ganoderic acid A and bovine serum albumin, chicken egg albumin, and bovine serum albumin were selected as the coating antigens. Each antigen was applied on one row of holes on the enzyme-labeled plate. e coating and washing process is as described in 2.2.4. Ganoderic acid A standard product was diluted with sample diluent to a concentration of 1 μg/mL. en, 50 μL of this product was added to columns A-D in each row, and 50 μL of sample diluent was added to columns E-H as the control. e rabbit antiganoderic acid A antibody IgG solution (50 μL) diluted with sample diluent to 1 : 200 was added to each row and placed in the microplate thermostatic oscillator at 200 r/min for 30 min at 37°C, and the subsequent steps were as described in 2.2.4.

Establishment of Indirect Competitive ELISA for Ganoderic Acid A and Formulation of Regression Curve.
e process of coating the ELISA plate and coating and washing the conjugate of ganoderic acid A and chicken egg albumin are as described in 2.2.4. Ganoderic acid A standard was dissolved with sample diluent and divided into 10 dose groups (0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, 218.7, and 656.3 μg/L). Ganoderic acid A standard solution (50 μL) was added to the corresponding wells of the enzyme-labeled plate, and three wells were used for each concentration. e rabbit antiganoderic acid A antibody IgG solution (50 μL) diluted with sample diluent 1 : 200 was added to each well and placed in the microplate thermostatic oscillator at 200 r/min for 30 min at 37°C, and the subsequent steps followed that in 2.2.4. e coefficient of variation (C·V) of the absorbance values of repeated wells in the group was calculated using the formula: C·V � (standard deviation/average value) × 100%. e relative absorbance value of each dose group was calculated as follows: relative absorbance value � A/ A 0 × 100%, where A represents the average absorbance value of a certain concentration of ganoderic acid A reaction well, and A 0 represents the average absorbance value of the 0 μg/L ganoderic acid A reaction well. e relative absorbance value of each dose group of ganoderic acid A was analyzed by ANOVA to compare the significance of the differences between the groups. e regression curve and regression equation of the relationship between the relative absorbance value and the concentration of ganoderic acid A were obtained by curve fitting.

Verification of the Specificity of Ganoderic Acid A by
Indirect Competitive ELISA. Twelve test substances of the same triterpenoids as ganoderic acid A, namely, ganoderic acid B, ganoderic acid Y, lucidenic acid A, ginsenoside Rb1, betulinic acid, jujuboside B, soy saponin Ab, glycyrrhizic acid, saikosaponin D, Anemarrhena saponin, aescin, and notoginsenoside R1, were selected. After proper dilution, the determination was conducted according to the method in 2.2.6. If the test substance had certain cross-reactivity and a dose-response relationship existed, the regression curve and regression equation of the relationship between the relative absorbance value and content of each test substance were prepared. According to the regression equation, the 50% inhibition concentration of the test substance (50% inhibition concentration is the sample concentration that corresponds to 50% of the relative absorbance value on the regression curve) was calculated, and the cross-reaction rate with ganoderic acid A was calculated by the formula: cross-reaction rate � (50% inhibitory concentration of ganoderic acid A/50% inhibitory concentration of the tested substance) × 100%. If the tested substance had no obvious cross-reactivity and cannot reach the 50% inhibition rate at the maximum detection concentration, the cross-reaction rate was estimated to be less than a certain value according to the calculation formula.

G. lucidum Sample Processing Method and Determination of Ganoderic Acid A Recovery
Rate. Dried G. lucidum was pulverized and passed through a 20-mesh sieve. A 2.5 g sample was placed in a 100 mL flask with a stopper, added with 25 mL of absolute ethanol, stoppered and shaken for 1 min, soaked for 24 h, then stoppered and shaken for 1 min, and filtered with filter paper. An appropriate amount of sample solution was collected, gradually diluted five times with the sample diluent, and subjected to an indirect competitive ELISA method as established in 2.2.6. Ganoderic acid A standard was determined to draw the regression curve. According to the relative absorbance value of the sample, the content of ganoderic acid A that corresponded to the sample solution was checked on the standard curve. e ganoderic acid A content of the dried G. lucidum was expressed by X in microgram per kilogram according to formula (1). After obtaining the ganoderic acid A content of the dried G. lucidum, standard ganoderic acid A was added at 0.5, 1.0, 1.5, and 2 times the content to the G. lucidum samples and then determined using the same method. e recovery rate of the sample addition was obtained according to the formula: recovery rate of sample addition � (measured value of spiked sample − measured value of sample)/spiked amount × 100%.
where ρ is the content of ganoderic acid A in the sample extract found from the standard curve, in microgram per liter; V is the volume of sample extraction solution, in liter; N is the dilution factor of the sample; M is the mass of the sample, in kilograms.

Results and Analysis of Ganoderic Acid A Antibody Titer
Determination. e average absorbance value of the repeat wells was calculated after indirect competitive ELISA. e result was considered positive if the average absorbance value of the test group or the average luminosity value of the negative control group ≥2.1.
e judgment results under each dilution multiple of the treatment group and untreated group are shown in Table 1. In this test, the maximum dilution factor at which a positive result appears was determined as the efficacy value of the test antibody. From Table 1, the efficacy value of the antibody in the group after the absorption of bovine serum albumin was 1 : 3125, and the efficacy value of the antibody in the group that had not been absorbed by bovine serum albumin was 1 : 78125. e results showed that the inoculation of the conjugate of ganoderic acid A and bovine serum albumin produced a considerable amount of antibody IgG in the test rabbits, of which a large amount of antibody IgG bound to bovine serum albumin, and only a small part of antibody IgG bound to ganoderic acid A.

Results and Analysis of the Choice of Coating Antigen.
After the absorbance values of the five coating antigens were measured by indirect competitive ELISA, the competitiveness presentation rate of each coating antigen was calculated according to the formula: competitiveness presentation rate � (the average absorbance value of the control group-− the average absorbance value of the competition group)/ the average absorbance value of the control group ×100%. e effectiveness of the coating antigen was analyzed by the competitiveness presentation rate. e results are shown in Table 2. Only the conjugate of ganoderic acid A and chicken egg albumin achieved a high presentation rate. e reasons for the lower rate of the other coating antigens are different: e reason for the low presentation rate of Ganoderma acid A coating is that it can not bind to the enzyme label effectively; the reason for the low presentation rate of chicken egg white albumin coating is that the anti-Ganoderma acid A antibody can not bind to it; the reason for the low presentation rate of bovine serum albumin, Ganoderma acid A, and bovine serum albumin is that a large number of antibovine serum albumin antibodies interfere with the action of Ganoderma acid A antibody in the reaction, which further verifies the analysis in 3.1, that the antibody obtained in Step 2.2.3 contains a large amount of IgG bound to bovine serum albumin. e experiment at this stage confirmed the feasibility of the conjugate of ganoderic acid A and chicken egg albumin as a coating antigen. e conjugate of ganoderic acid A and chicken egg albumin can effectively interact with the rabbit antiganoderic acid A IgG in 2.2.3 without binding to other nonspecific IgGs.

Establishment of Indirect Competitive ELISA for Ganoderic Acid A and Formulation of Regression Curve.
e results of the indirect competitive ELISA of the 10 dose groups of ganoderic acid A showed that the absorbance value showed a gradual decline as the concentration of    ganoderic acid A increased. e C·V of the absorbance value of repeated wells in each group was calculated as <5%. One-way ANOVA was performed on the absorbance value of each dose group. e results showed substantial differences in the absorbance values between the adjacent dose groups 0.3, 0.9, 2.7, 8.1, 24.3, 72.9, and 218.7 μg/L, whereas the differences in the absorbance values between the dose groups 0, 0.1, and 0.3 μg/L and between the dose groups 218.7 and 656.1 μg/L were unremarkable. Further refinement of the group found that the absorbance of ganoderic acid A at the concentration of 0.6 g/L was significantly different from that of 0 g/L, and the absorbance of Ganoderic acid A at the concentration of 121.5 g/L was significantly different from that of 656.1 g/L. us, the data of dose groups 0.9, 2.7, 8.1, 24.3, and 72.9 μg/L were used for curve fitting. e regression curve of the relationship between the relative absorbance value and the concentration of ganoderic acid A was prepared by taking the relative absorbance value as the abscissa and the concentration of ganoderic acid A as the ordinate. e results are shown in Figure 1. Figure 1 shows that the concentration of ganoderic acid A and relative absorbance had the best correlation degree under the power function relationship, and the value of R 2 reached 0.994.

Results and Analysis of the Specificity of Indirect ELISA for Ganoderic Acid A.
e results of the cross-reaction of ganoderic acid A with the 12 kinds of triterpenoid test substances are shown in Table 3. Among them, only ganoderic acid B showed certain cross-reactivity and doseresponse relationship, but the cross-reaction rate with ganoderic acid A was still less than 0.1%. Based on the solubility of the test substance in the sample reaction solution, the maximum reaction concentration of the other 11 test substances was 10 mg/L, which still did not reach the 50% inhibition rate. Under the same conditions, the 50% inhibitory concentration of ganoderic acid A was 3.058 μg/L; thus, the estimated cross-reaction rate of these test substances was less than 0.03%. Specificity analysis showed that the method has good specificity for the determination of ganoderic acid A.

Results and Analysis of Ganoderic Acid A Recovery Rate in Ganoderma Samples.
e ganoderic acid A content of dried G. lucidum was 0.28 × 10 6 μg/kg, and the corresponding doses of 0.5, 1.0, 1.5, and 2 times of its content were 0.14 × 10 6 , 0.28 × 10 6 , 0.42 × 10 6 , and 0.56 × 10 6 μg/ kg, respectively. e corresponding doses of ganoderic acid A standard was added to the filtered G. lucidum sample, and the determination results are shown in Table 4. Table 4 shows that the range of standard added recovery was 98.3% ± 6.2%, which indicates that the sample processing method had a high recovery effect and can be applied in the determination of samples.

Determination and Analysis of Ganoderic Acid A in
Different Kinds of G. lucidum. Table 5 shows the determination results of the content of ganoderic acid A in the dried G. lucidum from the family Ganodermataceae. e table shows that the contents of ganoderic acid A in the four kinds of G. lucidum were very different. Single-factor ANOVA showed that the content of ganoderic acid A in G. lucidum was significantly different from the other three types (P < 0.001); the ganoderic acid A content of G. applanatum was significantly different from that of G. leucocontextum and G. atrum (0.01 < P < 0.05); the ganoderic acid A content of G. atrum was not significantly different from that of G. leucocontextum (P > 0.05). Table 6 shows the determination results of the ganoderic acid A contents of dried G. lucidum analogues to the family Polyporaceae. ese dried analogues, which are commonly called G. lucidum, contained a large amount of ganoderic acid A, which even exceeded those in G. leucocontextum and G. atrum. A comparison of the results in Tables 6 and 5 showed that G. lucidum had the highest ganoderic acid A content among all the tested samples. According to one-way ANOVA, the results were significantly different from other varieties (P < 0.01).

Conclusions
is study used synthetic immunogen to obtain antiganoderic acid A antibodies in the immune serum of experimental rabbits. An indirect competitive ELISA with good specificity and sensitivity for the detection of ganoderic acid A was constructed using synthetic coating antigen and antiganoderic acid A antibody, compared with the high-performance liquid chromatography adopted by Hongling Dong, Duanping Lu, and others [24,25], compared with the liquid chromatography-tandem mass spectrometry adopted by Liangqin Liu and Yongli Liu [26,27], and compared with the enzyme-linked immunosorbent assay method constructed by Sakamoto Seiichi, Gorawit Yusakul, and others [28][29][30]. In comparison, this method has a lower detection limit. e established detection method was used to determine the ganoderic acid A contents of G. lucidum samples. e results showed that ganoderic acid A contents of different varieties were substantially different, and the content of ganoderic acid A was the highest in G. lucidum. e method is simple and fast for the determination of ganoderic acid A and is of great importance for the quality control of Ganoderma products.

Data Availability
All data generated or used during the study are included in the submitted article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
e first two authors contributed equally to this manuscript.