Establishment And Application Of Magnetic Beads Enrichment Method Of The Low-level HBsAg

BackgroundHepatitis B virus (HBV) infection is a serious harm to human health. Hepatitis Bsurface antigen (HBsAg) is an important serological markers to diagnosis and evaluatehepatitis B. In HBV "window period", after the acute phase and recovery, self-limitedinfection carriers, there will be low-level HBsAg. Patients with low-level HBsAg may bedamaging liver and persistent virus replication. If leaked, it may affect the patients treated,or cause HBV spread through the surgery, blood transfusion and other ways. DetectingLow-level HBsAg effectively have important clinical and epidemiological significance.At present, the higher sensitivity method to detecte low-level HBsAg ischemiluminescence immunoassay(CLIA). It’s sensitivity reached0.05IU/ml. But due tothe need for the large-scale instrument, the hospital is hard to popularization. Testing costsis high and not for most patients bear. So it had some limitations. Enzyme-LinkedImmunosorbent Assay (ELISA)is simple, convenient, fast, but the sensitivity is not high,the sensitivity of domestic kit is0.5IU/ml. It often appear weak positive or false negativeresult to low–level HBsAg. How to more effective and economic increase sensitivity ofELISA test is important. This topic will establish a method of specimen enrichment based on magnetic beadsas medium (magnetic beads enrichment method). Analyzing the characteristics of magneticbeads enrichment methods, to elevate the sensitivity of ELISA detection of low-levelHBsAg. The results will clarify the adsorption protein mechanism of magnetic beads andmake magnetic beads extract more protein. Looking for reasonable experimentalconditions of adsorption of pure and serum HBsAg, provide a reference to magneticbeads’ application to adsorption and separation of other proteins in clinical. The resultswill clarify the role of magnetic beads enrichment HBsAg, and provide an effective way toimprove clinical sensitivity of ELISA detection of low-level HBsAg, and make magneticbeads enrichment method be used in clinical practice.Objective1. To determine magnetic beads enrichment method by study the performance of thecarboxy-bead adsorption of pure and serum HBsAg. And analyze the characteristics ofmagnetic beads enrichment method by compare with other protein enrichmentmethods (high-speed centrifugal, polyethylene glycol (peg) dialysis, polyacrylamidegel particle adsorption, vacuum dry).2. To determine the lowest detection limit that magnetic enrichment method can achieve,and investigate the significance of magnetic beads’ function to improve detectionsensitivity and low-level HBsAg positive rate. Investigate the application of magneticbeads enrichment mathod in other tests by magnetic beads concentration positiveHBVDNA and HBeAg serum samples.Methods1. The amount of protein before, after magnetic beads adsorbing plasma-derived HBsAgwas determined by BCA kits, the adsorption ability of HBsAg was judged. Theabsorbance values before and after magnetic beads adsorbing serum HBsAg were determined by ELISA kits. Established the optimal experimental conditions ofmagnetic beads concentration of serum HBsAg by change conditions of the amount ofbeads, serum pH value, adsorption temperature, adsorption time, PBS pH value, eluenttype, eluent concentration,and so on.2. After concentration methods of magnetic beads enrichment method, high-speedcentrifugal, polyethylene glycol (PEG) dialysis, polyacrylamide gel particle adsorption,vacuum dry deal with the serum samples, compared the ELISA test results before andafter concentration. Analysised the advantages and feasibility of magnetic beadsenrichment method.3. Calibrated the concentration of clinical HBsAg-positive serum, then judged theminimum ELISA detection limit of HBsAg after magnetic beads concentration. Aftermagnetic beads concentrated HBsAg-negative, HBsAg-positive dilution serum,low-level HBsAg, judged whether there would be false positive, be affected by othermicro-organisms, whether to improve low-level HBsAg positive rate.4. After electrophoresising the eluate of magnetic beads had absorpted HBsAg, HBeAg,and HBcAb positive serum, judged the specificity of adsorption. ELISA test andreal-time quantitative PCR were used to detect HBeAg and HBVDNA after adsorption,judged whether magnetic beads also concentrated HBeAg and HBVDNA.Results1. Adsorption HBsAg capacity of megnetic beads was affected by HBsAg concentration,temperature, amount of beads, incubation time. The adsorbed HBsAg could be elutedby10mMNaOH, and the elution rate was71.02%and the retained biological activitywas88.15%. Adsorption serum HBsAg capacity of megnetic beads was effected bythe bead dosage, temperature, adsorption time, pH of serum and PBS, type andconcentration of eluent, and so on.2. After concentrated by magnetic beads enrichment method, high-speed centrifugal,polyethylene glycol (peg) dialysis, polyacrylamide gel particle adsorption, vacuum dry, the S/CO values were higher than before concentration(P<0.01). Any twoconcentration methods had significant difference (P <0.01), the level of enrichmentwas in descending order as follows: vacuum dry, magnetic beads enrichment method,high-speed centrifugation, polyethylene glycol dialysis, polyacrylamide gel particleadsorption.3. After magnetic beads concentration, the minimum detection limit of ELISA was0.1IU/ml, the sensitivity was higher than before concentration(the minimum detectionlimit was0.1IU/ml), the S/CO value was in good correlation to HBsAg concentration(r=0.998). Not increased the nonspecific detection of HBsAg (P>0.05). It was nosignificant statistical difference between the S/CO value before and afterconcentration HBsAg-positive serum each diluted by HBsAg-negative serum,HCV-positive serum and syphilis-positive serum(P>0.05). The positive detection rateof low-level HBsAg was82.1%, it was significant statistical difference between beforeand after concentration (χ~2=17.765, P <0.01).4. the electrophoresis band of elution was more complex, in addition to the consistentbands of pure HBsAg, as well as other bands. After concentration, the S/CO value ofHBsAg and the concentration of HBVDNA were higher than before.Conclusion1. Successfully established magnetic beads enrichment method. it was simple, fast, lowcost, etc, but susceptible to be affected by bead dosage, temperature, adsorption time,pH and other factors.2. Megnetic beads enrichment method could improve the sensitivity of ELISA detectionof HBsAg, it could be used clinically to improve the detection rate of low-levelHBsAg, it was important to monitor low-level HBsAg.3. Megnetic beads enrichment method compared with other enrichment methods, had asimple, convenient and rapid benefit, it was suitable to promote the use of clinicaland blood bank. 4. The enrichment protein principle of megnetic beads was electronic exchange, it wasnon-specific, could be used in further testing of other projects of hepatitis B or otherclinical proteins.