ABSTRACT
To establish a novel colloidal gold immunochromatography assay (GICA) for rapid, sensitive and accurate detection of Haemophilus influenzae infection by using the outer membrane protein P6 as detection target. First, the linear antigen epitope located in the extracellular domain of the P6 protein (GenBank accession number: AGH02799) was predicted by bioinformatics analysis. The region (62-75 aa of the protein) with strong antigen specificity was chosen and synthesized. Two rabbits were then immunized by the polypeptides (14 aa) for production of polyclonal antibodies. Then, the recombinant P6 proteins were also obtained to produce polyclonal antibodies. Finally, based on the two antibodies, a novel colloidal GICA for detection of Haemophilus influenzae infection was established and the specificity, sensitivity, repeatability and stability of this method were evaluated. At the same time, the method was tested in clinical simulation, and the plate culture method was used to verify its accuracy. The test strip for Haemophilus influenzae infection was successfully prepared. The detection limit of the test strip was as low as 1×105 CFU/mL and the whole process can be completed within 15 minutes. The strip specifically recognized Haemophilus influenzae and did not react with nine of other common respiratory pathogens such as Streptococcus pneumoniae, Moraxella catarrhalis, Mycoplasma pneumonia, and Legionella pneumophila. And the strips could be stored at 25 °C for at least 6 months without losing sensitivity or specificity. The coincidence rate between the results of 200 clinical samples and the plate culture method was 90.5%. Haemophilus influenzae protein P6, which possessed a high degree of surface antigen accessibility and antigencity, could be used as a marker for Haemophilus influenzae detection. The immunochromatographic colloidal gold test strip which bears the features of rapidity, convenience and sensitivity provides a unique tool for the on-site surveillance and diagnosis of Haemophilus influenzae infection in clinical test.
Subject(s)
Animals , Humans , Rabbits , Chromatography, Affinity , Diagnostic Tests, Routine , Reference Standards , Gold Colloid , Chemistry , Haemophilus Infections , Diagnosis , Haemophilus influenzae , Limit of Detection , Sensitivity and SpecificityABSTRACT
In this study,we estimated the application value of detecting Mycobacterium tuberculosis (MTB) specific IgG/IgM antibodies for tuberculosis diagnosis with colloidal gold immunochromatography assay (GICA).We collected 332 effective serum samples and their background information,including 260 patients with tuberculosis and 72 healthy individuals.The means of GICA was used to detect MTB specific IgG/IgM antibodies.Results were compared with the clinical diagnosis and the results of bacteriological tests.The SPSS 22.0 software was used to analyze the results,and when P＜0.05 the difference was statistically significant.The sensitivity and specificity of GICA were 41.15％ and 91.67％,and the sensitivity of the bacterial positive and negative patients were 51.38％ and 33.77％,respectively.The positive rate of IgG/IgM antibodies detection with GICA (41.15％) was much higher than that of bacteria with acid-fast stain of sputum smear (18.84％) and sputum bacteria cultivation (36.15 ％) (P ＜ 0.05) respectively.The positive rate of the combination of tuberculosis antibody detection,sputum bacterial culture and sputum smear was 61.54％,higher than the result of single method or combination of two methods.The detection of specific antibodies against MTB in serum with GICA is sensitive,specific,rapid and convenient,which can be used in clinical screening.Meanwhile,there are still certain limitations of this method,and the sensitivity and specificity need to be improved.Therefore,the GICA can be used as an auxiliary diagnosis combined with sputum bacteriology,imaging test and clinical features rather than diagnose tuberculosis alone.
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Objective To investigate the clincial value of ELISA combined with colloidal gold immunochromatography in the detection of anti-cyclic citrullinated peptide (CCP) antibody in patients with rheumatoid arthritis (RA).Methods From October 2014 to October 2016,120 patients with RA who admitted to Fuyang People 's Hospital were selected as RA group,and 120 healthy subjects were selected as the control group.Serum samples of patients were collected and tested for anti-CCP antibody by ELISA and ELISA combined with colloidal gold immunochromatography.Results ELISA method was lower than ELISA combined with colloidal gold immunochromatography test (x2 =3.943,P ＜ 0.05).The sensitivity,specificity,positive predictive value,negative predictive value of ELISA combined with colloidal gold immunochromatography assay were 93.33％,91.67％,84.85％,96.49％,respectively,which were higher than single ELISA method (all P ＜ 0.05).Conclusion ELISA combined with colloidal gold imnmunochromatography assay for detection of serum anti-C.CP antibody in patients with RA is effective and can obtain high diagnostic sensitivity and specificity,and it is worthy of promotion.
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Objective To use the colloidal gold immunochromatography (GICA ) method to conduct the methodological prelimi-nary evaluation on pepsinogen Ⅰ (PGI) reagent kit .Methods The method provide by the Preliminary Evaluation of Clinical Quan-titative Experimental Methods :Approval Guide Second Edition (EP10-A2) formulated according to the Clinical Laboratory Stand-ards Institute (CLSI) was used to continuously detect the low ,moderate and high concentrations of serum for 5 d ,Then the related data were collected for analyzing the dispersion degree ,linearity ,offset ,precision and so on .Results The PGⅠ quality control ser-um (concentration 25 ,50 ,100 μg/L) was detected at low ,medium and high concentration levels ,the linear regression equation ob-tained by analysis was Y=0 .9939X+0 .7433 ,correlation coefficient (R2 )=0 .9992 ;the offsets were 0 .37 ,0 .77 ,0 .78 μg/L re-spectively ,total imprecision was 3 .04% ,1 .17% and 1 .08% respectively .Conclusion The GICA related technical indicators of PGⅠreagent kit reach the standards of EP10-A2 document ,the detection results are accurate with high sensitivity and good stability , and conform to the requirements of clinical applications .
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Objective To explore the value of urine retinol-binding protein 4 (RBP4)detected by colloidal gold immunochromatography assay in evaluating the renal dysfunction state in kidney transplant patients. Methods A total of 141 kidney transplant patients followed-up by our urology center were included in this study. The RBP4 levels in the urine of the participants were measured by both enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatography assay. The consistency of the two methods was evaluated by kappa test. The patients were divided into three groups according to the results of colloidal gold immunochromatography assay, in which dark red, light red and colorless representing the low, medium and high levels of RBP4, respectively. The relationship between patient clinical manifestations and RBP4 results was analyzed. Results The two methods of ELISA and colloidal gold immunochromatography assay showed high consistency in detecting RBP4 concentration in the urine of kidney transplant patients (kappa=0.813, 95%CI: 0.763-0.933). According to the results of colloidal gold immunochromatography assay, the low, medium and high levels RBP4 groups had significantly different fasting glucose (P=0.028), serum creatinine (P=0.021), blood urea nitrogen (P=0.012), albumin (P=0.014), hemoglobin (P=0.026) and proteinuria (P=0.015). Conclusion Colloidal gold immunochromatography assay detecting urine retinol binding protein 4 can achieve semi-quantitative results via reaction color of the strip. It is reliable, sensitive and easy to perform, making it easy for the early diagnosis of renal tubular injury after kidney transplantation during follow-up.
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Objective To investigate the sensitivity and specificity of colloidal gold immunochromatography assay in rapid detection of respiratory syncytial virus (RSV)antigen.Methods A total of 197 nasal-pharyngeal swabs (NPs)or nasal-pharyngeal aspirates (NPAs ) obtained from patients were tested by RSV assay kits, i. e., colloidal gold immunochromatography assay.The results determined by reverse-transcription polymerase chain reaction (RT-PCR)were taken as reference.Compared to the results of RT-PCR,the sensitivity of the kit was different when testing different types of specimens or specimensobtained from patients of different ages.Results A total of 95 (48.2%)samples were positive for RSV tested by RT-PCR.The sensitivity and specificity of colloidal gold immunochromatography assay were 34.7% and 100%, respectively compared with RT-PCR results.The positive rate of the assay was higher in testing NPAs than NPs (36.2% vs 8.6%,P < 0.01 ), which was the same in the sensitivity (22.1% vs 12.6%). The positive rate of colloidal gold year old,RT-PCR method should be used for RSV detection in clinical practice to avoid missed diagnosis.
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Objective To evaluate the clinical applicability of Gold Lebel,ELISA and LiCA,through detecting HBsAg by all the three methods.Methods A total of 1 000 sapmles were selected,and each sample was detected HBsAg by using three methods. Each positive sampale was detected repeatedly to identify false negative and false positive.Results In all the three methods,the most sensitive and specific method was LiCA,next was ELISA,and the last was Gold Lebel.But Gold Lebel method was more sim-ple and rapid for preliminary screening for emergency or preoperative patients with its sensibility of 1 ng/mL.ELISA had too many operating steps to bring false negative and false positive,while its high sensitivity and specificity and low price might be accepted by commonage and fit for mass simples.LiCA′s especially high stability,accuracy,easily operating could better reflex HBV replication and therapy effects of antiviral drugs in spite of its slight cost.Conclusion The suitable HBsAg detection method should be chosen according to clinical demands.
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Objective:To develop a sensitive,rapid,simple gold immunochromatography assay for human cardiac troponin I(cTnI).Methods:The cTnI was used to immunize Balb/c mice by route method.The spenocytes were fused with SP2/0 myeloma cell lines.Six hybridoma cell lines,secreting stably McAbs to cTnI antigen have been obtained and screened.The 3F10 was coupled with colloidal golds,the 3A7 biotinylated.Streptavidin was coupled with the nitrocellose strip to prepare an test apparatu.According to red line in nitrocellose,thus can analysis the results.Results:The sensitivity of cTnI measured by this apparatus was 0.4 ng/ml.The 30 patients with acute myocardial infarction(AMI)were detected for cTnI by this apparatus and by the product of PBM Co USA as standard.The coincidence rate of both two assays was 93.6%. Conclusion:This method is a sensitive,specific simple and rapid assay.