ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the quality status of rubella virus IgM diagnostic kits by national supervising sampling.</p><p><b>METHODS</b>Using legal inspection combining with exploratory study, the positive and negative coincidence rate, detection limit and repeatability of kits were verified.</p><p><b>RESULTS</b>The results showed that 15 of 16 batches of kits were qualified using legal inspection, and the passing rate was 93.8%. The unqualified item was negative coincidence rate. In exploratory study, only 11 batches (68.8%) complied with industry standard. The unqualified items were negative coincidence rate, detection limit and repeatability.</p><p><b>CONCLUSION</b>At present, rubella virus IgM diagnostic Kits have some quality problems in the market. It is recommended that we adopt industry standard and national reference panel in the registration inspection for the future, which will prompt enterprises to improve quality.</p>
Subject(s)
Humans , Antibodies, Viral , Immunoglobulin M , Quality Control , Reagent Kits, Diagnostic , Reference Standards , Rubella , Diagnosis , Rubella virusABSTRACT
Objective To develop an efficient recombinant expression system of alanine aminotransferase ( ALT ) in order to build the foundation for the preparation of feedstock related ALT reference materials.Methods A new human ALT gene was synthesized by optimizing the codons of the nucleic acid sequence encoding human ALT using bioinformatic tools, and then it was cloned into pRSF-Duet expression vector.The recombinant plasmid pRSF-Duet-ALT was transformed into E.coli BL21 and the target protein expression was induced by 2 mmol/L isopropyl β-D-1-thiogalactopyranoside ( IPTG ) .The expression condition for soluble protein was optimized by changing the inducer concentration, shaking speed and induction temperature. The soluble protein was purified by nickel ion affinity chromatography and dextran molecular sieve chromatography, and identified by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis( SDS-PAGE) and Western blot analysis.The activity and stability of recombinant proteins in serum matrix under different storage conditions were detected.Results The usage frequency in E.coli of ALT codons was more than 10%after codon optimization.The expressions of soluble proteins were increased by optimizing the induced expression conditions, including a final concentration of 2 mmol/L of IPTG, and continued incubation with shaking at 150 rpm for 8 h at 25℃.The purified protein was identified as ALT by SDS-PAGE and Western blot with ALT activities of up to 80 000 U/L.Recombinant ALT could be stable for 2-8 d at 2-8 ℃or 25 ℃with a relative standard deviation of less than 5%.Conclusions An efficient recombinant expression system of ALT was developed successfully by codons optimization.The obtained recombinant protein could achieve the requirements of reference material feedstock.
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Objective To verify the industry standard for total IgE quantitative labelling immunoassay kit .Methods Different methods of kits were used and were verified in accordance with industry standard .Results The appearance ,limit of blank ,linearity , accuracy ,precision and stability could meet requirements ,while specificity of individual kits was just partly qualified .Conclusion The establishing of the industry standard for total IgE quantitative labelling immunoassay kit was reasonable ,which could help to standardized the experimental methods and technical requirements and promoted the improvement of quality ,and offer the basis for the admistration .
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This paper briefly introduces the working procedure of in vitro diagnostic products (IVD) industrial standards, and elaborates the importance of professional standards for production and supervision. Based on the analysis of working progress during the past 10 years, some problems and countermeasures on project setting, participation, standard material, personnel training, work cycle are put forward, which are helpful for the future development of the IVD.
Subject(s)
Humans , Diagnostic Techniques and Procedures , Reference Standards , Reference StandardsABSTRACT
Objective To prepare the HPV genotyping control materials. Methods Three hundred cervical smears with different HPV genotypes were collected and detected by surface plasmon resonance and sequencing. The primers for specific genotype were designed according to GenBank. The recombinant plasmid was constructed through PCR amplification, ligation and transformation. Thirty recombinant plasmids were identified through PCR amplification, sequencing, and the sequences were compared using BLAST. Results The collected HPV infectious samples contained 30 different genotypes including HPV 6,16,18 and so on.The fragment sequences of PCR amplification were concordant with the designed. The fragment sizes of the other types ranged from 1 500 to 2 000 bp except HPV CP8304. And the 30 recombinant plasmids identified by PCR were concordant with the target. The identity of BLAST was 99%. In the fragment of 1500 bp in length, 11 bases were inconsistent with the reference sequence. Conclusions Genotyping control materials were developed successfully. The human papillomavirns genotyping control materials covered all the most common types and included 14 types with high-risk, 3 types with medium-risk and 13 types with low-risk.
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Two hybridomas (TD_1, TD_2)secreting monoclonal anti-idiotypic antibodies (anti-ld) againstanti-HBs were established by fusing the myeloma cells, IR983F, with the spleen cells of LOU/Crat which had been immunized with mouse monoclonal anti-HBs to the a determinates and syn-genic rat polyclonal anti-HBs. TD_2(Ab_2)showed the ability to inhibit the binding of horse anti-HBs with HBsAg and was also neutralized by anti-HBs from various origins. Ab_2 can induce an-ti-anti-ld (Ab_3)when injected it into LOU/C rats introperitoneally. Ab_3 can react specifically withHBsAg. These data indicate that TD_2 monoclonal anti-ld against anti-HBs are the anti-ld bearingthe internal image of HBsAg epitope that could mimic HBsAg and possess the immunogenicity ofHBsAg.