Gold label false positive, can it be remedied?
Whether false positive results occur occasionally or repeatedly, a systematic approach to the diagnosis of such problems is also required to implement the most appropriate remedial measures. The most obvious reasons are the first to be noticed, mainly the reaction of the gold label with the capture antibody, specific charge attraction, hydrophobic interactions and gold-sulfur binding forces.
Attention should then be paid to non-specific cross-reactions in antibodies, specific sample properties and transmembrane flow properties. Use controls to quickly find the source of a problem.
Below is a description of the system's diagnostic methods and a reference to the possible causes listed previously. When using any systematic diagnostic method, only one parameter can be changed when using controls. In this way, a process of elimination can be adopted. The following questions may be helpful:
1. Is it due to charge? Changes in the pH of the detection system (pH 5-11) indicate the presence of positive charges and binding of the colloidal gold particles to the capture antibody.
2. Is it due to hydrophobic force? This may occur in the solid phase, in the capture antibody zone or in the gold-labeled zone. Changing the active agent concentration in the system can provide clues as to whether the hydrophobic interaction is a major factor.
3. Is it the reason why Jin-SH is attracted? It most likely occurs with the cysteine and arginine groups in the capture antibody band and sample immersion band. Close inspection of these two areas, as described in the following sections, can reveal the problem.
A systematic approach to solving the false positive problem:
For most testing methods used in rapid testing test strips, generally only one test strip (or "half strip") is used. There is no need to choose completely dry test strips. During the detection process, the nitrocellulose membrane is placed directly in the micropores containing the gold standard solution, sample and chemicals. The chemicals are normally placed in the drying box. In this way, several tests can be performed quickly and easily, and without the need for fully assembled units and drying ovens. However, if many problems arise due to the drying step, then complete assembly must be performed before inspection.
These questions about the system approach can be grouped into the following five categories related to testing devices and components.
Is the problem related to the gold standard solution?
This problem can be easily solved by changing the gold conjugate solution - for example, replacing the original gold conjugate solution with BSA-gold at the same concentration and the same pH value. If the problem still persists, the most likely cause is the charge effect. If false signals no longer occur after changing the gold conjugate solution, then the most likely cause of the problem is a problem with the original gold conjugate solution or a problem with the labeled antibody. Other controls here are similar gold conjugates and gold conjugates containing monoclonal and polyclonal antibodies. There are also many gold conjugate solutions on the market that can be used for this type of assay.
Below is an example of a false positive signal due to a gold conjugate. The application is a test strip for detecting infectious diseases in clinical samples (serum). False positive results were seen in all non-positive samples. When BSA-gold gold conjugate solution was used, the false positive signal disappeared. After using another non-specific gold conjugate, the false positive signal also disappeared. However, when the non-clinical control sample was replaced, the false positive signal still existed when using the specific gold conjugate solution in PBS at pH 7.2, but there was no false positive signal when using other gold conjugate 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 has nothing to do with the sample or the capture antibody, but with the high sensitivity of the gold conjugate to the capture antibody mat. These gold labels most likely contain bare gold particles, which may bind to capture antibodies. False positive signals can be eliminated by using new gold conjugate solutions and careful preparation of the gold conjugate.
Is it because of capturing antibodies?
The controls used in this case are similar capture antibodies or other types of antibodies. However, before using these controls, stripping the BSA capture protein can indicate whether the problem is occurring elsewhere. It is also possible that the false positive signal is not due to the antibody itself, but to some protective agent in the antibody. In this case, dialysis in a suitable buffer (e.g. 10 mmol PO4) can improve the situation. If the false positive signal disappears using other antibodies, then it is obvious that the specific capture antibody caused the false positive signal, and the action to be taken is to replace the antibody or remove it. For example, in actual operation, rabbit monoclonal antibodies sometimes produce this kind of situation. This is mainly due to the effect of hydrophobic force and requires special methods to deal with it.
Pregnancy tests that detect ßhCG in urine found false positive signals for all samples, regardless of whether the samples themselves were positive or non-positive. Changing the pH value did not weaken the signal, nor did changing the gold standard solution. Even using the PBS buffer control sample did not eliminate the signal.
The problem is most likely due to capture antibodies. All signals were eliminated after stripping off the capture antibody band and replacing it with BSA. After dialyzing the initial capture antibody in 10 mmol of PO4, the final results were consistent with the actual conditions of the sample. Based on the above situation, we can conclude that these antibodies were once suspended in a buffer containing SH components (such as thimerosal), and we need to eliminate or avoid these factors.
Is it a membrane problem?
The most important process in the development of any test strip is selecting the appropriate membrane. Some membranes may be more suitable than others for specific types of detection or for specific samples. Developers should have a variety of membranes from all manufacturers and in a variety of pore sizes so that quick comparisons can be made. For example, the hydrophobicity of the membrane was evident during the drying process, so it was clear that this batch of membranes would produce random false positive signals.
The selection of membranes for use in rapid detection technologies should take into account not only the required flow rate and protein binding properties, but also the homogeneity of the membrane during preparation. The controls that need to be used here are membranes with different pore sizes and different sources, as well as different areas of the same batch of membranes.
The initial test was developed so that it would not produce false positive or false negative results during the laboratory design phase. Only after all tests are passed and the testing technology is determined can it be transferred to the production design stage.
When scaling up to thousands of devices for assembling test strips, we should be recording many randomly generated false positives in each production batch anyway. Adjusting the pH, using another gold conjugate, and checking the capture antibody twice did not immediately reveal the cause of the false positive. The test was re-tested in the small-scale laboratory design stage. Since the false positive results occurred when there was no C-band, it can be explained that the cause of the false positive results was due to the nitrocellulose membrane.
For laboratory design studies, only small batches of membranes were used. The results of such a small amount do not indicate whether they are suitable for large-scale production. Large batches of membranes have been found to have distinct hydrophobic regions (i.e., areas not normally wetted) that can cause nonspecific binding of colloidal gold particles to capture antibodies.
Is it because of chemicals?
During the assembly process of the rapid test strip, the gold conjugate solution and solid support (membrane, conjugate pad or sample pad) may be pre-treated with chemical reagents. Added chemicals may include salts, active agents, proteins, sugars and polymers. Some chemicals can increase the chance of false positives.
During the development of test strips for the study of animal proteins in serum, some false positive results occurred. During the laboratory design phase, before applying the test strip, the test is initiated by adding preparation buffer to the serum sample and gold conjugate solution, and then dipping the test strip into the microwells. Replacing the gold standard solution has no effect. Changing the acidity of the buffer did not affect the results. Changing the capture antibody did not eliminate the random occurrence of false positive results.
In the case of non-positive serum samples, false positive results are only observed after a few minutes of buffer has been added to the serum sample prior to maceration with the serum sample. The buffer contains 5% strong active agent. After reducing the concentration of the active agent to 1% and then allowing the serum and colloidal gold particles to mix with the buffer for several hours, no false positive results were observed. This shows that excess active agent will strongly affect the colloidal gold particles, remove the antibodies on the surface of the particles and produce bare gold particles, thereby allowing them to interact with the capture antibodies.
Is it because of the sample?
Due to factors such as changes in acidity, sample contamination, or urinary tract content, as mentioned above, samples can cause unpredictable false positive signals. In order to determine whether a sample is the cause, many different samples need to be tested, including similar samples and samples from different sources. In addition to the amount of biological or organic matter in the sample, the problem may also lie in the buffer used. If this is the case, another buffer needs to be used as a control. The simplest way to adjust is to change the acidity of the sample. This method can quickly determine whether the problem is caused by the effect of charge attraction in the sample. It may also be necessary to filter the sample, either independently of the detection procedure or as part of the detection procedure. In production and development, rapid detection technology for hormones in urine is used to detect a large number of different urine samples. Its specificity can reach 97%, and its sensitivity can reach 98%. However, several false positive results were discovered from stored samples during later testing phases.
After measuring the acidity of these samples, it was found that the acidity increased, but the influence of acidity can be ruled out after testing with control buffer samples with equal acidity levels. No false positive results were produced when urine samples were filtered. Microscopic observation then revealed that bacterial contamination of the sample was the main cause of false positives. In this way, we can conclude that an increase in bacterial content increases the hydrophobicity of the sample, which causes nonspecific effects of colloidal gold particles and capture antibodies. Fresh samples do not cause similar problems.
