Should you pay attention to several problems in ELISA? - Huaqiang Electronic Network

In recent years, the continuous occurrence of mad cow disease and clenbuterol has been shocked. It has been found that feed under certain conditions not only poses a serious threat to animal and human health, but also causes unexpected economic losses and huge political influence. As a source of food safety, feed safety has become a hot issue that has attracted wide attention from all levels, from international organizations and government departments to civilians. Many countries and non-governmental organizations in the world have actively increased the ranks of feed and food safety control. Various countries and regions have also stepped up efforts to strengthen relevant organizations and formulate a series of laws and regulations. For example, the United States and Japan have revised the Feed Safety Law, the European Union has established the European Food Administration, and will implement new feed and food management regulations. China has also promulgated the Regulations on the Administration of Feed and Feed Additives and implemented feed safety projects. However, even the best policies and measures are inseparable from the technical support of feed testing. The test results are not only the scientific basis for formulating policies and standards such as MRL, but also an important basis for taking action and resolving trade disputes. In recent years, the most effective and most influential implementation is the step-by-step detection method for the enforcement analysis of illegal drugs and veterinary drug residues in feed food safety testing. Mainly for screening, confirmation and quantitative methods.


The screening method is the first step in performing the assay for high-throughput analysis of large numbers of samples. The purpose is to detect the presence or absence of a certain or a certain type of hazard. It is simple and fast, does not require special venues, does not require large instruments, and is generally less expensive. The screening method is based on a number of principles, but the most widely used is the enzyme-linked immunosorbent assay (ELISA). For example, the detection of illicit drugs such as clenbuterol, ractopamine, diazepam, furazolidone, and hexacetin in the feed, residual detection of clenbuterol in urine, clenbuterol in pig liver, and chloramphenicol in milk The various kits are all enzyme-linked immunosorbent assay (ELISA).

Since 2000, the Autonomous Region Veterinary Drug Feed Inspection Institute has used enzyme-linked immunosorbent assay to detect clenbuterol in feed. In 2004, it began to undertake large-scale feed safety testing projects issued by the Ministry of Agriculture and the autonomous region, mainly for feed, drinking water and urine. Detection of clenbuterol hydrochloride, ractopamine, diazepam, furazolidone, and hexacetin. According to the requirements of the Ministry of Agriculture, the enzyme-linked immunosorbent assay was used for screening. The positive ones were confirmed and quantified by high performance liquid chromatography and GC/MS. I have discussed the problems and precautions in the preliminary screening test using the enzyme-linked immunosorbent assay in the past few years for peer reference.

1, the choice of elisa kit http://

At present, there are many kinds of kits for enzyme-linked immunoassay, which are imported from abroad and developed and produced by ourselves. How to select a kit that meets the detection requirements and is inexpensive in many brands is a problem. According to the use of these kits, I found in the actual test that the following indicators are generally listed in the kit instructions: minimum detection limit; recovery rate; cross-reaction rate; standard curve map and data, the standard curve consists of at least 5 concentrations; The IC50 measured value and the number of deformation curve points are comparative specifications and can be selected.

2. Determination of the cut-off

The threshold value is first determined when actually using the enzyme-linked immunosorbent assay. By comparing the test results with the critical values, it can be determined that the sample is negative or suspicious. For banned drugs, the critical value of the test method is the limit of quantitation, which is the lowest limit of quantitation that can be achieved with existing accepted instrumental tests. For drugs with the highest residue limit (MRL), the cut-off value of the test method was 20 samples of the MRL concentration added and the mean of the three measurements was subtracted by 1.64 standard deviations. If the test result is less than the critical value is negative, greater than or equal to the critical value or the standard is suspicious, it needs to be confirmed by confirmatory method.

3. The detection limit can be added to each of the upper and lower concentrations of MRL and MRL.

When using the enzyme-linked immunosorbent assay in each laboratory, the detection limits of the respective laboratories should be checked. The limit of detection was the mean of 20 blank samples plus 3 standard deviations. If the detection limit obtained after the standard operation is greater than the critical value, the background interference of the kit is large, and the sensitivity of the kit cannot meet the detection requirements. The kit manufacturer should be contacted or discarded without using another kit.

4, enzyme-linked immunoassay method verification

When using the enzyme-linked immunosorbent assay to detect samples, the method should be verified. Mainly determine the following:

(1) Determination of the standard curve, the standard curve consists of at least 5 concentrations, and the number of measurements is not less than 5 times to determine the working concentration range and the IC50 range. Generally, the critical value is near IC50, and the detection sensitivity is high.

(2) Determination of sample detection limit.

(3) Sample addition recovery experiment. For the banned drugs, a limit of quantitation and a limit of 2 times the limit can be added. For drugs with the highest residue limit (MRL), one concentration above and below the MRL and MRL can be added.

Accuracy: If the recovery rate is greater than 40%, it meets the requirements of the corresponding testing standards. Precision: The intra-assay coefficient of variation has a coefficient of variation of less than or equal to 25% between each batch of parallel samples and less than or equal to 30% for banned drugs. The inter-assay coefficient of variation has a coefficient of variation of less than or equal to 30% for all samples and less than or equal to 40% for banned drugs.

To meet the above accuracy and precision requirements, it is indicated that the enzyme-linked immunosorbent assay is feasible and can be used to detect drug residues.

5, other

(1) For a kit that can be used for multi-residue detection, a drug having a low cross-reaction rate is positively added to measure a cut-off.

(2) The following tests should be included in the actual test: at least two standard curves, two negative controls, two positive cut-off controls, and two parallel samples to be tested. After the test results come out, the shape of the standard curve (according to the test requirement is S-shaped), whether the IC50 is close to the critical value and the recovery rate of the positive sample, and the parallel state of the sample can determine whether the test is reliable.