| BackgroundC reactive protein (c-reactive protein,CRP) in acute infection was found in blood. It has been widely used in clinical auxiliary diagnosis of inflammation, because it can reflect the dynamic changes of infectious diseases. Related detection methods and detection products constantly emerging, but the current quantitative detection of CRP products are needed a device to complete, not suitable for the scene, community hospital and rural grassroots medical institutions. Therefore, a rapid colloidal gold immunochromatographic assay which couldn’t need to use any equipment has important clinical value and social significance.ObjectiveTo development a double antibody sandwich colloidal gold immune chromatography assay for semi-quantitative of C reactive protein.Methods1. The determination of CRP color point:according to the clinical diagnosis standard, determine the three fixed value points of the color card.2. The preparation of CRP internal quality control products:using low values CRP serum as matrix serum to dilute high values of serum CRP, in addition to add rheumatoid factor, three acyl glycerol interference material such as preparation of a series of indoor quality control, and use the roche reagent for review.3. The CRP test preparation and process optimization:using sodium citrate reduction of different particle size of colloidal gold, and using functional experiments to determine the optimal particle size of colloidal gold, optimum pH, the best coating protein and labelled protein; Using orthogonal experiment to determine the sample pad processing liquid composition and content; the dilution ratio of different samples were researched by the dilution multiple experiments of the whole blood, plasma and serum. Based on the basic experiment, and then we magnified 100 times of the basic reaction volume to verificate the stability of our technology.4. The test paper performance evaluation:using laboratory quality control products to study the dipsticks sensitivity, specificity, precision, batch difference, hooked (HOOK) effect, stability, and test environmental conditions.5. Clinical laboratory samples:200 clinical samples were detected by our technology to compared with control kit to confirm the consistency.Results1. To determine the CRP color points:10 μg/mL,50 μg/mL, 100μg/mL.2. Preparation CRP internal quality control products:including rheumatoid factor (150 IU/mL), three acyl glycerin (16 mmol/L) and contains no interference factors of CRP concentration 5 μg/mL, respectively,10 μg/mL,50 μg/mL,100 μg/mL, a total of 12 products. HOOK effect samples (1046 mg/L) and low-value samples (0.57 mg/L CRP), one copy each.3. Preliminary established colorimetry to detect CRP dipsticks preparation method, we choosed the colloidal gold with 1.6 and added 0.05% sodium azide to store. Adding 0.02 mol K2CO320 μL, labelled antibody 6 μg to per milliliter colloidal gold. Coating protein was 1.2 mg/mL. The dilution multiple experiments showed that whole blood and serum were diluted 200 times, plasma sample was diluted 120 times, and then adding 75 μL diluted samples to test. Strip preparation amplification experiments showed that the technology was stable.4. Strip performance test showed that 5 μg/mL CRP shows no color, and color was shown when 10 μg/mL. Using P1, P2, P3 to experiment 3 times, there was no difference in the color. And it consistents with the color stripe. Rheumatoid factor (150 IU/mL), three acyl glycerin (16 mmol/L) did not have interference to the results. The result of 1046 μg /mL CRP was not have HOOK.5. Using our method to detect 200 clinical samples, then compared to Wan Fu products. The results showed that the consistent rate of whole blood, serum, plasma, respectively:below 50 μg/mL were 100%; 50 μg/Ml,100 μg/mL were 93.8%,99% and 93.8%; More than 100 μg/mL were 92.6%,99%,96%.ConclusionWe developped a double antibody sandwich colloidal gold immune chromatography assay which could semi-quantitative C reactive protein and couldn’t need to use any equipment. |
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