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After adding the sample, a red vertical line is visible in the reaction window, but the background is not clear enough. What should I do?

This may occur when the sample flows across the membrane, but it does not affect the test result; readings taken within the specified time frame are valid as long as the background remains clear. If, at the designated reading time, the red vertical line has not disappeared and the background is blurred, the sample may have been hemolyzed—please use a fresh specimen to repeat the test.

What diluent should be used to dilute the serum?

What should be the interpretation when bands appear at both ends of the T‑line region, while the middle remains a clear, unbroken line?

When interpreting the test line (T), the criterion is whether the bands at both ends are clearly visible: a clear band at both ends indicates a positive result. This is due to the edge effect during chromatography, whereby the migration rate in the center is faster than at the edges; the central region is more easily washed away, causing the antigen–antibody complexes to concentrate at the edges, which appear sharper than the central area.

What are the requirements for the serum to be tested when using the colloidal gold immunochromatographic assay?

Venous blood is collected without anticoagulant; the resulting serum, obtained either by natural settling or centrifugation, should be at least 200 µL. Fresh samples are recommended for timely analysis. If circumstances necessitate delaying testing, ensure that thawed or refrigerated serum samples are allowed to equilibrate fully to room temperature before aliquoting. For samples stored under refrigeration or freezing, follow these guidelines: serum can be kept at 2–8°C for up to one week; if analysis is delayed beyond this period, store at –20°C or lower. Avoid repeated freeze–thaw cycles, as they may reduce antibody titers; it is advisable to aliquot samples in small portions for storage. Hemolyzed, lipemic, or icteric sera are unsuitable, as their high viscosity and dark color can slow chromatographic processes, obscure background readings, and interfere with binding reactions and result interpretation.

What conditions are suitable for storing colloidal gold immunoassay test kits?

The colloidal gold assay is performed at room temperature; refrigerated serum samples and test reagents must be allowed to equilibrate fully to room temperature before use. A key advantage of the colloidal gold immunochromatographic assay is its simplicity and speed. To minimize waiting time for temperature equilibration, it is recommended to store the reagents in a cool, well‑ventilated environment at room temperature and to process samples immediately upon receipt, avoiding refrigeration of both samples and reagents. If a chromatography card is opened after being refrigerated without sufficient room‑temperature equilibration, it may absorb moisture, compromising assay performance. To prevent moisture uptake after opening, any opened test strip should be used promptly. Therefore, colloidal gold test strips should be stored sealed under dry, light‑protected conditions at room temperature and never frozen; they are intended for immediate use.

What is the principle behind using colloidal gold chromatography to detect autoantibodies?

Colloidal gold immunochromatographic assay (GICA) involves immobilizing highly purified antigens—such as CCP, M2, Sm, U1RNP, SSA, SSB, Scl‑70, Jo‑1, and P0—along with IgG in the form of distinct bands on the test line (T line) and the control line (C line) of a nitrocellulose membrane (NC membrane). A colloidal gold‑labeled reagent is adsorbed onto the conjugate pad. When the sample to be tested is applied to the sample pad at one end of the test strip, it migrates forward by capillary action, dissolving the colloidal gold‑labeled reagent on the conjugate pad and allowing it to bind to proteins in the sample. As the sample reaches the test line (T line), the colloidal gold‑labeled autoantibodies in the sample specifically bind to the immobilized antigens on the T line, becoming trapped and accumulating at the test zone, producing a visible coloration that can be readily interpreted. Meanwhile, any unbound labeled reagent continues to migrate past the test zone, separates from the conjugate, and is captured in the control zone, forming the control line.
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