Ochratoxin a (OTA) contamination detection by enzyme-linked immunosorbent assay (ELISA)

Indonesia is a country with a tropical climate with high temperatures, rainfall, and humidity, which are conducive to the growth of fungi in various agricultural commodities, food and feed. Ochratoxin A (OTA) is the result of secondary metabolites from the toxigenic fungi of the genus Aspergillus and Penicillium. OTA is carcinogenic, nephrotoxic, neurotoxic, and it can cause health problems. OTA can be detected by an enzyme-linked immunosorbent assay (ELISA). The ELISA method has more advantages than other methods, because it can detect samples quickly, easily, economically, specifically, and sensitively. The purpose of this study was to detect the presence of ochratoxin A (OTA) contamination in samples by ELISA. The results of this study showed that OTA contamination in 50 samples were 92%, with an average value of 6.18 ppb. The levels of OTA contamination in samples were below the regulation.


Introduction
Indonesia is a country with a tropical climate with high temperatures, rainfall, and humidity, which are conducive to the growth of fungi in various agricultural commodities, food and feed [1,2] Agricultural commodities that are damaged and have a high water content are easily contaminated by fungi such as Aspergillus flavus and A. parasiticus, which can produce mycotoxin as secondary metabolites such as aflatoxin, A. ochraceus, and Penicillium, which can produce ochratoxin, Fusarium moniliforme, which produces fumonisin toxin, and Fusarium graminareum, which produces the toxins zearalenone and deoxynivalenol [3].Mycotoxin contamination may be impacted by micronutrient bioavailability variables, pests, and insect damage brought on by climatic changes.Climate, in addition to these causes, serves as a key agroecosystem commanding force of fungal colonization and mycotoxin excretion [4].Thus, it is anticipated that climate change will have a significant impact on both the global environment and the sustainability of the food and feed production system [5].
Ochratoxin is a group of mycotoxins that are harmful to animal and human health.Based on the level of toxicity, ochratoxin types consist of A, B, and C, which have differences in their chemical structures.Ochratoxin A (OTA) is the most abundant and toxic of all ochratoxins [6,7] OTA is categorized as a group 2B substance by the International Agency for Research on Cancer (IARC) [8].Furthermore, OTA is nephrotoxic, teratotoxic, hepatotoxic, genotoxic, neurotoxic, and it can cause health problems.The formation of DNA adducts, the inhibition of protein synthesis, the disruption of 1253 (2023) 012100 IOP Publishing doi:10.1088/1755-1315/1253/1/012100 2 cellular energy production, the start of oxidative stress, the induction of apoptosis, effects on mitosis, the induction of cell cycle arrest, and interference with cytokine pathways are all reported to be OTA's genotoxic effects.The primary causative agent of Balkan Endemic Nephropathy (BEN), a chronic, progressive condition that results in kidney failure [9].The presence of OTA in food and feed products has raised concerns and been identified as a potential global threat to human and animal health [10]; [11,12].
Considering the risks associated with OTA for animal and human health, detection and quantification are crucial.Food and feed quality control includes extraction and analysis using thin chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS), and immunochemical methods.One of the immunochemical methods is the enzyme-linked immunosorbent assay (ELISA), which is based on the specific reaction between antigen and antibody and is faster, more accurate, and more inexpensive [13].The purpose of this study was to detect the presence of ochratoxin A (OTA) contamination in samples by ELISA.

Methods
Samples of feed ingredients (corn, bran) and feed were collected from several areas in West Java.Materials needed in the study included methanol, distilled water, 96-hole microplates that already contained antibodies (coated antibody plates), uncoated microplates, ochratoxin standard, conjugate solution, substrate solution, and reaction stop solution.

Sample preparation
A total of 20 grams of sample was weighed and then added to 100 ml of 70% methanol, shaken for 3 minutes, and filtered using Whatman ® 1 paper.

ELISA analysis for OTA detection
All materials to be tested should be adjusted to room temperature.An uncoated microplate was prepared, and then 200 µL of conjugate were added to each well, added 100 µL standards or samples to the conjugate.Once mixed well, transfer 100 µL content to the antibody-coated plate, then incubate for 10 minutes.It was then washed three times with distilled water and dried before being placed in 100 µL of substrate and incubated for 5 minutes.After that, 100 µL of stop solution were added.Then the optical density (OD) was read with an ELISA reader at a wavelength of 450 nm.The readings and concentrations of aflatoxin in the samples were calculated using the equation of the line generated by the standard curve (concentration vs. % inhibition).

Results and discussion
Sample detection by ELISA is a fairly effective method for detecting OTA contamination.ELISA is an immunochemical method based on a specific reaction between antigen and antibody that has high sensitivity and specificity, using enzymes as indicators.The advantages of the ELISA method are sensitivity, fast and easy application, low cost, and efficiency.Every time you are going to do a test on a sample, it is better to do a linearity or calibration test against the standard by making a standardized concentration level and then calculating the regression value.A good regression value is close to 1 [14].The determination of linearity is to determine the ability of a method to provide a linear response from measurements of standard concentration series; this is necessary for quantitative analysis.Linearity is achieved by making a line equation at several concentrations of standard OTA levels.The results of the linear regression analysis show the value of the coefficient of determination (R2) > 0.9 (Figure 1).This shows a strong linear relationship because there is a linear relationship between concentration and % OTA inhibition.The results of this study are in line with the results of other studies, namely the linearity of OTA by ELISA, which also shows the coefficient of determination (R2) > 0.9 [15,16].The results of the analysis of OTA contamination levels in samples of corn, bran, and feed (Table 1) using the ELISA method showed that the level of OTA contamination was 92% (46 samples) of 50 total samples.This shows that most of the samples were contaminated with OTA, with an average contamination of 6.18 ppb (Table 1.).However, the concentration of OTA in 50 samples of feed and feed ingredients was still below the regulations set by the European Commission (EU) with a maximum residue limit (MRL) of 100 ppb [17].

d = not detected OTA = ochratoxin A LOD (limit of detection) OTA = 1.9 ppb
A study in Pakistan conducted to evaluate the occurrence of ochratoxin A (OTA) by ELISA reported that 10 feed samples and 19 feed ingredient samples had OTA concentrations above the EU maximum regulatory limits [18].Another study in Turkey detected OTA in feed and feed ingredients using ELISA and reported 71.91% of samples were positive for OTA contamination, with the highest concentrations of up to 305.6 ppb in dairy cow feed, 222.40 ppb in cattle fattening feed, 96.30 ppb in chicken feed, and 157.20 ppb in feed ingredients [7].
Based on eco physiological studies, the level of OTA contamination can be affected by temperature, water availability and photoperiod.One of the primary elements in the agro-ecosystem that affects fungal colonization and OTA generation in crops has always been climate.The environment in which crops will be cultivated is predicted to change significantly over the next 10 to 25 years, with the atmospheric CO2 concentration likely to double or triple along with bouts of excessive rainfall or times of catastrophic drought.These data give preliminary support for the result 1253 (2023) 012100 IOP Publishing doi:10.1088/1755-1315/1253/1/0121004 that a potential rise in temperature may lead to a decrease in both A. carbonarius spoiling and OTA production [5].
Livestock that consume contaminated feed containing OTA absorb it into the bloodstream, where it can accumulate in the organs responsible for its detoxification and elimination.Therefore, it is very important to ensure the safety of milk, eggs, and meat before human consumption [19].OTA contamination can be prevented by implementing good management practices starting from the process of planting, harvesting, and storing agricultural products so as to produce quality agricultural, feed, food products and avoid contamination with hygienic practices [20,21].

Conclusions
The level of OTA contamination detected by ELISA in the samples was up to 92% with an average contamination value of 6.18 ppb, but the concentration of OTA in the samples analyzed was still below the European Commission regulatory limits.There is a threat that OTA will accumulate if livestock continue to consume feed contaminated with OTA.It may result residues in animal products, which can endanger to animals and humans health.

Figure 1 .
Figure 1.Linearity of OTA by ELISA

Table 1 .
OTA occurrence and concentration level in samples in West Java.