False positive signal

Characteristics of false positive results

A false positive result is the appearance of a colored line (red line for gold standard) on the detection band when there is no analyte in the sample. This line may appear in the early stages of detection or after a significant time delay. False signals may occur in many types of samples, or they may only occur in a specific type or source of sample.  

The basic reasons for false positives

During the specific binding of proteins to colloidal gold particles, the proteins are absorbed by three main forces on the surface of the colloidal gold.

1. Charge attraction: Colloidal gold particles carry a negative charge, which is due to the layer of negative charge carried by the reducing agent (often citrate) used to convert sodium chloroaurate into colloidal gold being adsorbed on the surface of the colloidal gold particles. Negative charges will attract proteins with positive charges and bring them close to the surface of the binding region, which may result in false positives. Proteins below their isoelectric point carry a positive charge and may therefore strongly attract the surface of colloidal gold particles. Especially for protein regions rich in lysine and arginine, they carry a large amount of positive charge below the isoelectric point of lysine (pH 10.4) and arginine (pH 12.5).  

2. Hydrophobic force: Once proteins are very close to each other (with a distance of less than 1nm), any hydrophobic region of the protein is likely to come into contact with and bind to the hydrophobic region on the surface of colloidal gold. Therefore, proteins rich in non-polar amino acids such as tryptophan, valine, leucine, isoleucine, or phenylalanine will strongly bind to the surface of colloidal gold.

3. Coordination bond binding force Coordination bond binding force is the strongest binding force among all attractive forces. Proteins containing a large amount of sulfur rich amino acids (due to the presence of cysteine and methionine) strongly bind to the surface of colloidal gold particles. This is due to the attraction between gold atoms (which have the ability to conduct band electrons) and sulfur atoms (which have valence electrons). When these three binding forces act on the gold labeled particles, they will generate false positive signals as described below and thus have a negative impact on the efficiency of the detection technology.

Problems caused by gold conjugate particles

For some reason, the surface of the gold labeled particles may be partially exposed due to the lack of protein coverage. During the drying of the gold standard solution or during the detection process, or if the gold standard is applied very little in the first step, these can all lead to the removal of antibodies. Regardless of the reason, exposed colloidal gold particles will be attracted by any positively charged protein, nitrocellulose, or nylon membrane. When colloidal gold particles pass through the capture line, the possibility of physical contact is very high due to the very small distance, which is particularly evident. Therefore, it is very important to effectively encapsulate colloidal gold particles and prevent protein detachment during storage, processing, or handling.

In addition to the adsorption of colloidal gold particles onto the capture line, the labeled antibody itself may be adsorbed onto the capture line under specific operating conditions. This may be attributed to charge or hydrophobic forces, or it may be due to the acidic or ionic environment during this process. Excessive gold standard particles can cause several problems. Firstly, it can increase the possibility of false positive signals on the capture line, or due to the large amount of gold standard solution passing through this line. Secondly, after the detection period, it will also increase the possibility of colloidal gold standard particles refluxing. Colloidal gold particles may also aggregate into clusters for various reasons, usually due to operational errors. If there are too many colloidal gold clusters, they may clog the membrane pores. If the clustering is caused by the hydrophobic interaction of colloidal gold, then colloidal gold particles will also adhere to the capturing antibody during the flow of the gold standard solution.

Regardless of the presence of the analyte, gold labeled antibodies may undergo non-specific immune reactions with capture antibodies. However, this method will make the labeled antibodies on the nitrocellulose membrane very close to the capture antibodies, thereby increasing the possibility of non-specific immune reactions. In addition, especially when using polyclonal antibodies, non-specific reactions may occur with other analytes in the sample, and the occurrence of such non-specific reactions mainly depends on the purity of the antibodies used.  

Issues related to solid support materials

The nitrocellulose membrane is very brittle and easily damaged upon contact. Therefore, it is very important to avoid any possible mechanical contact with the film during the tearing process. If the membrane is pressed onto the capture antibody band during the application of capture antibodies, it will increase the possibility of non-specific reactions of gold labeled particles during sample flow. There are two aspects that can cause residual gold particles to adhere to the capture antibody band - the slow flow rate of gold labeled particles in the membrane or the slow release rate of colloidal gold pads. If the pore size of the membrane is too small or there is not enough active substance on the detection band, and there is poor contact between the nitrocellulose membrane and the gold standard solution or absorption pad, then these situations will occur.

In addition, due to the hydrophobicity of nitrocellulose membrane, it can hinder the smooth flow of gold standard solution. Some samples may be very viscous (such as serum), which can also slow down the flow rate. When there is not enough sample or active substance in the detection system to move the gold standard along the detection band, colloidal gold particles will also adhere to the capture antibody band. If a lot of time is spent observing the detection results (usually more than 15 minutes), the membrane will dry out, and excess gold standard particles are likely to start flowing back from the absorption pad to the dried membrane. Due to the highly hydrophobic nature of the captured antibodies after drying, there is a high possibility of colloidal gold particles flowing from the absorbent pad towards the captured antibody bands after drying.

Chemical reagent issues

The closed production process, in a dry state, may make the membrane more hydrophobic, thus even causing more common background staining or false positive signals when capturing antibody bands. The use of excessive protein or active agents for sealing can generate high viscosity during sample flow.  

Some protective agents, whether in capturing antibodies, labeling antibodies, or samples, can produce false positive results. Thimerosal (a compound containing sulfur and mercury) and lysine (usually with a large positive charge at pH<10.4) are particularly important issues to pay attention to in detection.  

Remedial measures for false positive diagnosis

The most obvious reason is first noticed, mainly the reaction between the gold tag and the capture antibody; Specific charge attraction, hydrophobic interaction, and gold sulfur binding force. Then attention should be paid to non-specific cross reactivity in antibody, special sample characteristics, and transmembrane flow characteristics.

Is it due to electric charge? The change in pH value of the detection system (pH 5-11) indicates the presence of positive charges and the binding of colloidal gold particles to capture antibodies.  

Is it due to hydrophobic forces? This may occur in the solid phase, capturing the antibody region or gold label region. Changing the concentration of active agents in the system can provide insights into whether hydrophobic interactions are the main factor

Is it because of the attraction of gold SH? It is most likely to occur in the cysteine and arginine groups that capture antibody bands and sample immersion bands.

A systematic approach to solving false positive problems

Is the issue related to the gold standard solution? This problem can be easily solved by replacing the original gold standard solution with BSA gold at the same concentration and pH value. If the problem still persists, the most likely reason is the charge effect carried. If false signals are no longer generated after changing the gold standard solution, the most likely cause of the problem is either a problem with the original gold standard solution or a problem with the labeled antibody. The other controls here are similar gold standard solutions and gold standard solutions containing monoclonal antibodies and polyclonal antibodies.

Here is an example of a false positive signal generated due to a gold standard solution. The application is a detection strip for infectious diseases in clinical samples (serum). False positive results were observed in all non positive samples. After using BSA gold gold standard solution, the false positive signal disappeared. After using another non-specific gold standard solution, the false positive signal also disappeared. However, when non clinical control samples were replaced, PBS still showed false positive signals when using specific gold standard solutions at pH 7.2, while no false positive signals were observed when using other gold standard solutions. Changing the pH value of the buffer to 10 reduced the occurrence of false positive signals, but did not eliminate them. Therefore, we can conclude that the problem is not related to the sample or capture antibody, but to the highly sensitive gold standard solution for the capture antibody line. These gold tags are likely to contain naked gold particles, which may bind to capture antibodies.

Is it the reason for capturing antibodies? The control used in this case is similar capture antibodies or other types of antibodies. However, before using these controls, peeling off the BSA capture protein can indicate whether the problem occurred elsewhere. It is also possible that the false positive signal is not caused by the antibody itself, but by some protective agents in the antibody. In this case, dialysis in a suitable buffer solution (such as 10mmolPO) can improve the situation. If the false positive signal disappears after using other antibodies, it is obvious that the specific capture antibody caused the false positive signal. The measures taken are to replace the antibody or remove it. For example, in practical operation, rabbit monoclonal antibodies sometimes produce this situation, mainly due to the hydrophobic effect, which requires special methods to be used for treatment. (Sg whole blood excipient)

Is it a problem with the membrane? The most important process in the development of any detection strip is selecting the appropriate membrane. Some membranes may only be suitable for specific types of detection or specific samples compared to others. Developers should have various types of membranes from all manufacturers and membranes with different pore sizes in order to make quick comparisons. For example, the hydrophobicity of the membrane is evident during the drying process, so we can clearly know that this batch of membranes will produce random false positive signals. When choosing a membrane for rapid detection technology, not only should

Is it due to chemical drugs? During the assembly process of the rapid detection strip, the gold standard solution and solid support (membrane, binding pad, or sample pad) may have been pre treated with chemical reagents. The added chemical reagents can include salts, surfactants, proteins, sugars, and polymers. Some chemicals can increase the likelihood of false positives.  

Considering the required flow rate and protein binding properties, as well as the homogeneity of the membrane during the preparation process. The controls needed here are membranes with different pore sizes and sources, as well as different regions of membranes from the same batch.  

Is it due to the sample? Due to changes in factors such as acidity and sample contamination, as mentioned earlier, samples can cause unpredictable false positive signals. The simplest way to adjust is to change the acidity of the sample, which can quickly determine whether the problem is caused by the attraction of charges in the sample. It may also be necessary to filter the sample, and the filtering process can be independent of the detection program or used as part of the detection process.

Analysis of the causes of abnormal situations during testing

For false positives caused by individual cases, they may be due to the following reasons. For example:

1. Abnormal blood samples such as high blood lipids, high hemoglobin, and high chyle may cause non-specific adsorption, resulting in fine lines or false positives.

2. Insufficient centrifugation and prolonged sample storage can result in some cell parts being damaged into cell fragments. Hydrophobic bacterial fragments may cross react with captured antibodies and gold labeled particles, leading to false positives.

3. If the operation method is to directly add samples, the sample amount must be sufficient, otherwise it may lead to false positives; If the method of inserting the reagent strip into the sample is chosen, it cannot exceed the MAX line, otherwise it may lead to false positives.