Double Antibody Sandwich ELISA Method Research Articles

Development of a double antibody sandwich ELISA method for the quantitative detection of serum C-reactive protein based on nanobody

In this study, we successfully developed a nanobody-based double antibody sandwich ELISA kit for the detection of clinical serum C-reactive protein (CRP) by using two novel CRP specific nanobodies.The developed method exhibited a linear detection range of approximately 6–200 ng/mL, with a detection limit of 1 ng/mL. Furthermore, the method demonstrated excellent specificity, as there was no cross-reactivity with interfering substances such as total bilirubin and hemoglobin and so on. To assess reproducibility, independent measurements of the samples were conducted under experimental conditions, resulting in intra- and inter-batch coefficients of variation below 10% and a recovery rate of 93%–102%. These results indicate robust reproducibility of the method.To evaluate the performance of the developed kit, we collected 90 clinical samples for correlation analysis with commercial kits. The results showed a high correlation coefficient value (R2) of 0.98, indicating accurate concordance between the developed and commercial kits.In conclusion, our study successfully developed a nanobody-based double antibody sandwich ELISA kit to detect clinical serum CRP. The utilization of nanobodies represents a significant advancement in the field of CRP immunoassay development. The developed kit demonstrates excellent performance characteristics and holds promise for clinical applications.

Development of a double-antibody sandwich enzyme-linked immunosorbent assay for rapid detection of VZV

BackgroundVaricella zoster virus (VZV) is the pathogen of varicella and herpes zoster, it is necessary to develop a rapid, sensitive and specific detection method for the prevention and control of related diseases. MethodsWe inserted the gB protein extracellular region gene (gB-ex, 1–2208 bp) of VZV into lentivirus vector, and then obtained the recombinant gB protein through mammalian expression system. BALB/c mice were immunized multiple times with purified gB protein as immunogen. Then four strains of high affinity monoclonal antibodies targeting gB protein were prepared by cell fusion technique. Monoclonal antibodies 5G4 and HRP-4E9 were selected as capture and detection antibodies respectively, and a double-antibody sandwich ELISA method was established for detection. ResultsThe detection limit of the DAS-ELISA was 156 PFU/mL, and there was no cross-reaction with Herpes simplex virus-1/Herpes simplex virus-2/Pseudorabies virus. The coefficients of variation of intra-assay and inter-assay repeatability were less than 5%. ConclusionsIn this study, a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was established for the detection of VZV. The assay has good sensitivity, specificity and repeatability, which provides strong technical support and product guarantee for the rapid clinical detection of VZV.

Development of a double-antibody sandwich ELISA targeting the receptor binding domain of TcdB toxin of ST11 type Clostridium difficile of porcine origin

Clostridium difficile is an important zoonotic intestinal pathogen, which is widely present in humans and a variety of animals. The ST11 type C. difficile is one of the most widespread and harmful subtypes in the world. As a large country in pig farming, China lacks efficient methods for detecting C. difficile of porcine origin, leaving hidden dangers for the prevention and control of C. difficile. The aim of this study was to develop a specific and sensitive double-antibody sandwich ELISA for the epidemiological investigation of ST11 type C. difficile of porcine origin. Firstly, a 97 kDa receptor binding domain (RBD) was expressed in a prokaryotic host and purified. A hybridoma cell line AE2D3 capable of stably secreting monoclonal antibody targeting the RBD was screened, and the antibody subtype was determined to be IgG2b (κ). Secondly, a double antibody sandwich ELISA method was developed, where the monoclonal antibody targeting the RBD was used as a detection antibody, and the rabbit polyclonal antibody was used as a capture antibody. The chessboard method was used to determine the matching concentration of the capture antibody and the detection antibody, the antigen coating conditions, the blocking conditions, the incubation conditions for detection antibody and samples to be tested, as well as the reaction conditions of HRP-conjugated and reaction conditions of TMB chromogenic solution. The negative cutoff OD450 was 0.152, and no cross-reaction with 13 strains of non-ST11 type C. difficile was found. The minimum detection concentration of RBD was 8.83 ng/mL. This specific and sensitive double-antibody sandwich ELISA provides a reliable serological detection method for epidemiological investigation of the ST11 type C. difficile in pig industry.

Development of Patient-Derived Human Monoclonal Antibodies Against Nucleocapsid Protein of Severe Acute Respiratory Syndrome Coronavirus 2 for Coronavirus Disease 2019 Diagnosis

The pandemic caused by emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a global public health threat. Illustrating human antibody responding to viral antigen could potentially provide valuable information for basic research and clinical diagnosis. The antibody can be used as a complement to the viral detection for the rapid diagnosis of infected patients. Compared with spike protein (SP), nucleocapsid protein (NP) is normally conserved and highly immunogenic in many coronavirus members. As a major antigen, NP is a potential target for the diagnosis of SARS-CoV-2 infection. Here, we constructed a combinatorial fragment of antigen-binding (Fab)antibody phage library based on peripheral blood-derived from five coronavirus disease 2019 (COVID-19) infected donors. From the library, 159 Fab antibodies were obtained and identified by panning with NP. Among them, 16 antibodies were evaluated for their binding properties and epitopes recognition. Among these 16 antibodies, two well-paired antibodies were finally screened out for SARS-CoV-2 diagnosis by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) method. Our works may provide a potential resource for the clinical diagnosis of SARS-CoV-2 infection.

Effects of Chinese Herbal Medicines on Lipid Metabolism and Immunity Function in Laying Hens

Background: Lipid is considered as one of the essential nutrients for both humans and animals. Lipid accumulation within the body may induce several health disorders. Disease is are caused easily in aged laying hens. It is necessary to improve lipid metabolism and enhance immunity in laying hens. Traditional Chinese veterinary medicine (TCVM) with no toxicity and low drug residues is becoming more and more popular. The current study aimed to study the effect of Chinese herbal medicines on lipid metabolism and immunity function in chicken to provide basis for clinical reference. Methods: In this laboratory investigation during 2018-2019, a total of 400 60-week-old hens were randomized into 10 groups to measure lipid metabolism by colorimetry and immunoglobulin by double antibody sandwich ELISA method. A total of 500 28-day-age chickens were randomized into 10 groups to measure immune organ index. The experiment lasted for 42 days. Result: Our investigations have showed that Jujube (ZJM) has significant effects on the count of Serum total cholesterol (TC), Triglyceride (TG), Low-density lipoprotein-c (LDL-C) and High-density lipoprotein-c (HDL-C) (p less tha 0.01, p less than 0.05),the count of Immunoglobulin-G (IgG) (p less than 0.05) and the immune organ indexes (p less than 0.05) in 30 mg/kg group than control group10.The present work will be a complementary contribution to the ability of ZJM on improving lipid metabolism and immunity function in chickens.

The effect of warming kidney and dredging collaterals on the clinical effect and urinary C5b-9 in patients with idiopathic membranous nephropathy

Objective To observe the effect of the method of warming kidney and dredging collaterals on the clinical effect and the content of urine C5b-9 in patients with idiopathic membranous nephropathy with spleen kidney yang deficiency and blood stasis. Methods A total of 60 idiopathic membranous nephropathy patients with spleen kidney yang deficiency and blood stasis type were randomly divided into the conventional western medicine treatment group (control group), Jingui-Shenqi pill and Taohong-Siwu decoction plus conventional western medicine treatment group (treatment group), 30 cases in each group. The Scr was detected by deproteinized alkaline picric acid method, and BUN was detected by rate method, and serum albumin (ALb) was detected by bromocresol green dye binding method, and 24 hours urinary protein was measured by pyrogallol red colorimetry, and the double antibody sandwich ELISA method was used for detection of urinary C5b-9. Results The Jingui-Shenqi pill combined with Taohong-Siwu decoction plus conventional western medicine treatment has obvious curative effect on patients. The total effective rate was 83.3 in the treatment group (25/30), and the control group was 60% (18/30). After treatment, the Alb (33.5 ± 7.95 g/L vs. 28.8 ± 6.10 g/L, t=2.569) in the treatment groupwas significantly higher than that in the conventional treatment group (P<0.01). While the 24 h urine protein (2.40 ± 0.92 g/24 h vs.3.60 ± 2.3 g/24 h, t=2.653), the contents of C5b-9 in urine (42.5 ± 17.50 ng/mg vs.71 ± 25.2 ng/mg, t=5.088) in the treatment group were significantly lower than those in the conventional treatment group (P<0.01). Conclusions The method of warming kidney and dredging collaterals can improve the clinical symptoms, improve serum albumin level, reduce the 24 hour urine protein and urinary C5b-9 content of idiopathic membranous nephropathy of spleen and kidney yang deficiency and blood stasis type. Key words: Glomerulonephritis, membranous; Syndrome of yang deficiency of spleen and kidney; Syndrome of static blood blocking collaterals; Tao Hong Si Wu Tang; Jingui-Shenqi pill; Urinary albumin excretion rate; Urine C5b-9

PARAS INTERLEUKIN-18 PENDERITA TUBERKULOSIS PARU DAN PERAWAT SEHAT BERISIKOTUBERKULOSIS

Tuberculosis is an infectious disease which is the second cause of death in the world. Indonesia belongs to the third ranks as themost prevalent tuberculosis country. However the eradication is programmed only to focus on finding the case and treatment of theactive tuberculosis patients. Health care workers are at risk to tuberculosis infection, but there is no examination yet for early detectionactivity of tuberculosis. In order to know the activity of tuberculosis, other examinations are needed such as IL-18 examination. Today, no research about IL-18 is performed yet in Indonesia; therefore this study is performed in order to know the difference of IL-18level in active tuberculosis patients and nurses at risk. This study is to know the difference between IL-18 plasma of active tuberculosispatients and nurses at risk by analysis. A cross sectional, observational analytical study of 8 nurses at risk of tuberculosis and 8 activetuberculosis patients, has been conducted from February up to April 2007, at the Dr. Soetomo General Hospital and Karang TembokHospital in Surabaya. The diagnosis of active tuberculosis patients was based on positive sputum bacteriological examination, positiveradiology examination and who never had received anti-tuberculosis drugs. Nurses at risk of tuberculosis consisted of those who had beenworking more than 2 years, and was examined by negative bacteriological and radiology examination, TB-dot and positive tuberculinskin test with a diameter – 10 mm. IL-18 examination was done by double antibody sandwich ELISA method (MBL/Medical &amp; BiologicalLaboratories Co.Ltd). IL-18 level in active tuberculosis patients was 491.4–1215.3 pg/ml (mean 794.6 pg/ml, SD 222.6), in nursesat risk of tuberculosis was 88.9–429.0 pg/ml (mean 256.2 pg/ml, SD 137.6). There was a significant difference of IL-18 level amongactive tuberculosis patients and nurses at risk of tuberculosis (p &lt; 0.001); the IL-18 level in active tuberculosis patients was significantlyhigher than in nurses at risk of tuberculosis.

Collembolans and soil nematodes as biological regulators of the plant pathogen Fusarium culmorum

A field study was conducted to investigate biocontrol and interaction effects of important members (Folsomia candida, Collembola, and Aphelenchoides saprophilus, Nematoda) of the soil food web on the plant pathogenic fungus Fusarium culmorum in wheat straw. The soil fauna was introduced in minicontainers in different numbers and combinations and exposed to either Fusarium-infected or non-infected wheat straw. Minicontainers were established in the topsoil of a winter wheat field after harvest. After 2 and 4 weeks, biomass of F. culmorum was detected in samples of soil and wheat straw using double antibody sandwich (DAS) ELISA method. Furthermore, individual density of collembolans and nematodes was determined. The content of Fusarium biomass was reduced significantly throughout all treatments after 2 weeks. After 4 weeks of minicontainer exposure, Fusarium biomass decreased significantly in treatments containing collembolans and nematodes in single culture compared to the control. The results demonstrate the potential of collembolans and nematodes as biological regulators. Furthermore, the introduced soil fauna contributes to a sustainable control of fungal plant pathogens in wheat straw, thus reducing the risk of plant diseases as an important ecosystem service for soil health.

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

ABSTRACTAn effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40 ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.

Serum Levels of Mannose-Binding Lectin in Atopic and Healthy Mongolian Adults

Objectives: Mannose-binding lectin (MBL) is a vital protein of an innate immune system synthesized by the liver. The serum level of MBL is associated with an increased susceptibility to infection with a high risk of allergic and autoimmune diseases. The aim of this study was to determine the profile of serum levels of MBL in atopic and healthy Mongolian subjects. Methods: Serum samples were collected from 219 healthy Mongolian adult blood donors and 216 atopic subjects. We analyzed the MBL level in each serum by using the double-antibody sandwich ELISA method. Results: The mean serum level of MBL was 3088.28 ± 669.8 ng/ ml in atopic subjects and 2165.07 ± 708.5 ng/ml in healthy groups. In males, the MBL mean serum level of atopic subjects was 3012.8 ± 783.16 ng/ml and 2073.33 ± 678.26 ng/ml in the healthy group of males. The MBL mean serum level of female atopic subjects was 3138.22 ± 580.6 ng/ml and 2263.61 ± 733.1 ng/ml in the healthy group of females. Low association and significant differences were observed between MBL levels of atopic and healthy subjects. Conclusion: The serum level of MBL in atopic subjects was comparatively higher than in subjects of the healthy group. Significant differences were observed between mean levels of MBL between the different age groups.

Development, identification and application of 33 monoclonal antibodies against cardiac troponin T

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs' epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche's was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche's.

Etiological agents distribution and epidemiology of viral diarrhea in children below 5 years old in He′nan province, 2008-2015

Objective To investigate the infectious status, etiological spectrum and epidemiological characteristics of rotavirus (group A/B/C), calicivirus (novovirus Ⅰ/Ⅱ, sapovirus), astrovirus and enteric adenovirus in diarrhea cases below 5 years old from 2008 to 2015 in Henan provinces. Methods Totally 2541 stool samples were collected from cases below 5 years old in four sentinel hospitals. All stool specimens were tested for group A rotavirus by double antibody sandwich ELISA method. G/P genotyping of group A rotavirus was determined by nested multiplex PCR. Viral RNA was extracted from all samples and rotavirus (group B/C), calicivirus, astrovirus and enteric adenovirus were detected by two-step reverse transcription-polymerase chain reation (RT-PCR)/PCR. Results One thousand four hundred and twenty-one out of 2 541 samples were positive with a total positive rate of 55.9%, among which, 102 were mixed infection. The isolation rate of rotavirus was 36.0% (914 samples) (group A: 785 cases, group B: 36 cases, group C: 93 cases), calicivirus was 12.1% (308 samples) (novovirus Ⅰ: 64 cases, novovirus Ⅱ: 193 cases, sapovirus: 51 cases), astrovirus was 5.9% (151 samples), enteric adenovirus was 1.9% (48 samples). The group A rotavirus gene type combinations were composed mainly of G9P[8], G2P[4], G3P[8], G1P[8] and most cases were identified from September to November and March to May. Novovirus Ⅱ was predominant in calicivirus and most cases were identifed between March and May. Rotavirus or calicivirus infection was mainly among children aged 4—12 months or 3—5 years, respectively. Clinical manifestations included fever, diarrhea, vomiting, dehydration. Gender and region distributions differed according to the types of pathogen. Conclusions Group A rotavirus and novovirus Ⅱ are the major viral pathogen in diarrhea cases younger than 5 years old in Henan province. Different viral infections exhibit extinct epidemiologic and clinical characteristics. Key words: Diarrhea virus; Infant; Pathogen spectrum; Genotype; Epidemiological feature

Effects of astragalus polysaccharide on intestinal immune function of rats with severe scald injury

Effects of astragalus polysaccharide on intestinal immune function of rats with severe scald injury

Clinical effects of soluble interleukin-6 receptor detection on autoimmune rheumatic disease patients.

Objective : This study aims to explore the effects of soluble interleukin-6 receptor (IL-6R) on the clinical diagnosis and treatment of autoimmune rheumatic disease (ARD) patients. Sixty-eight ARD patients enrolled in our hospital recently were selected, the IL-6R levels of whom were detected by double-antibody sandwich ELISA method and compared with the IL-6 levels of normal subjects. The expressions of IL-6R in joint tissues were detected by section staining. The mechanism regarding the effects of IL-6R was postulated by administering the patients with blocking agent. The blood IL-6R level of ARD patients was 2-3 times higher than the IL-6 level of normal subjects with significant difference. The detection results of C-reactive protein and erythrocyte sedimentation rate show that both the indicators were significantly decreased (P=0.0098 and 0.0097 respectively). IL-6R was associated with autoimmunity based on the considerable expression in tissue sections, which was also verified by the alleviated symptoms after blocking IL-6R expression. Detecting soluble IL-6R is able to determine the patient's conditions and treatment effects. Meanwhile, soluble IL-6R detection can also serve as the inflammatory responses of ARD, as well as the determination index for abnormal immune responses and generated antibodies number, which is crucial for early diagnosis.

Study of the risk factors in disease progression and concentrations of VEGF and VCAM-1 in hand foot and mouth disease combined with encephalitis

Objective To explore the risk factors in disease progression and the significance of vascular endothelial growth factor （ VEGF ） and vascular intercellular adhesion molecule-1 （ VCAM-1 ） in hand foot and mouth disease （ HFMD） combined with encephalitis .Methods Altogether 92 subjects with HFMD were enrolled in the study and were divided into four groups , including group A （ ordinary group with no complication ） , group B （ severe group with complication ） , group C （ critical group with complication ） , group D （ recovery group with complication ） .Concentrations of VEGF and VCAM-1 in serum and cerebro-spinal fluid were detected by double antibody sandwich ELISA method .Multiple factors logistic regression＆nbsp;analysis was performed to analyze main risk factors in disease progression for two combinations , one for ordi-nary group and severe group , the other for severe group and critical group .The results were analyzed by SPSS16.0 statistical software.Results The concentration of VEGF and VCAM-1 in serum and cerebrospi-nal fluid had statistically significant differences among the four groups , but there was no significant difference between group A and group D , and between group B and group C .In addition , the statistically significant factors for prediction of disease progression were duration of fever , limb shaking, cerebrospinal fluid WBC , cerebrospinal fluid protein and EV 71 IgM between ordinary group and severe group , and cerebrospinal fluid WBC, respiratory rate and heart rate between severe group and critical group .The multiple factors logistic regression analysis revealed that limb shaking , cerebrospinal fluid protein , VEGF and VCAM-1 in serum were the main risk factors for disease progression from ordinary to severe （P=0.071, 0.019, 0.020, 0.025 and OR=147.629, 26.572, 5.958, 6.345）.And increased heart rate indicated the progression from se-vere to critical with P value of 0.001 and OR value of 2.69.Conclusion （1） Compared with group A, VEGF and VCAM-1 in serum and cerebrospinal fluid were highly expressed in patients with HFMD combined with encephalitis .Therefore , VEGF and VCAM-1 could be used as diagnostic criteria for auxiliary diagnosis of encephalitis in patients with HFMD and reflect the severity and prognosis to a certain extent .（ 2 ） Risk factors like limb shaking , cerebrospinal fluid protein , VEGF and VCAM-1 in serum would be helpful to early diagnosis of severe patients .Increased heart rate would be a significant factor for identification of patients with critical disease , according to which a timely treatment would be provided to prevent from worse . Key words: Hand foot and mouth disease combined with encephalitis; Vascular endothelial growth factor; Vascular cell adhesion molecule-1; Disease progression; Risk factor

Effect of the Haoqinqingdan decoction on damp-heat syndrome in rats with influenza viral pneumonia

ObjectiveTo investigate the effect of Chinese medicine prescription-Haoqinqingdan decoction on damp-heat syndrome in rats with influenza viral pneumonia and its influence on the immune function. MethodsA total of 48 Wistar rats were randomly divided into the normal control group, the damp-heat syndrome model group, the Haoqinqingdan decoction group (high, medium and low dose group) and the ribavirin group. The body temperature and weight of rats in each group were recorded after modeling. After treatment for 6 d, the concentration of T lymphocyte subgroup (CD3+CD4+, CD3+CD8+) was determined by flow cytometry. The OD value of IFN-γ/IL-4 was detected by double-antibody sandwich ELISA method, and its concentration was acquired through conversion. ResultsAfter modeling, the temperature and weight of rats in each modeling group showed the increasing trend (P<0.01). From the second day of treatment, there was significant difference in the body mass between groups, and the rat weight of the control group was higher than in the modeling group (P<0.05 or 0.01). With the advances of treatment, only the temperature in the medium and high dose Haoqinqingdan decoction groups declined significantly (P<0.05). After treatment, the CD4+/CD8+ ratio of the damp-heat syndrome model group decreased more significantly compared with the control group. Elevated CD3+ CD8+ percentages and declined CD4+/CD8+ ratios can be observed in the low dose group and ribavirin group (P<0.05). Moreover, the CD3+ CD4+ percentage of ribavirin group was lower than in the control group (P<0.05). After treatment, the IFN-γ and IFN-γ/ IL-4 levels in the peripheral blood of rats in the damp-heat syndrome group were obviously higher than in the control group (P<0.05). ConclusionsCompared with ribavirin, the high dose Haoqinqingdan decoction can improve the ratio of T lymphocyte subgroup and Th1/Th2 cell balance more effectively.

Prevalence of Hepatitis B e Antigen in Chronic HBV Carriers in North-central Nigeria

Hepatitis B virus (HBV) is an important clinical problem due to its worldwide distribution and potential of adverse sequelae, including hepatocellular carcinoma (HCC). We studied the prevalence of hepatitis B virus e antigen (HBeAg) among individuals determined to be HBV surface antigen-positive (HBsAg+) and analyzed the gender/age category associated with more active HBV infection. A total of 572 HBsAg+ individuals, as determined by a double antibody sandwich ELISA method, participated in the study. They were tested for HbeAg, using a lateral flow chromatographic immunoassay. One hundred and ten individuals were found to be HBeAg-positive giving an overall prevalence of 19.2%. Of these 110 individuals, 20 (18.2%) were females, and 90 (81.8%) were males. Thus, the prevalence of HBeAg appears to be higher in males than in females (p < 0.05). Our data also revealed that the prevalence of HBeAg was higher in patients between the age-group of 0-10 years and 11-20 years and appeared to decrease with increase in age. Taken together, our data show that approximately 1/5 of HBV-infected individuals are HBeAg+, suggesting that the virus is actively replicating and infecting liver-cells thereby ensuring an HBV-transmission pool within the Nigerian population. We suggest strengthening of the childhood HBV vaccination programmes, massive intervention activities, and treatment programmes, especially among young people to reverse the possible devastating effect of HBV infection. The success of these efforts will depend on our resolution to make the elimination of HBV infection a top priority on the public-health agenda as we start the second decade of this new century.

Comparative pharmacokinetics of a tumour-targeting therapy candidate rh-IFNα2a–NGR with rh-IFNα2a administered intravenously in mice and rats

rh-IFNα2a-NGR is a promising anti-tumor candidate. The aim of present study was to compare pharmacokinetics of rh-IFNα2a-NGR with rh-IFNα2a. Pharmacokinetics and elimination were investigated after intravenous administration to mice and rats. Compared tumor and tissue distribution profiles between rh-IFNα2a-NGR and rh-IFNα2a were illustrated in the tumor transplanted mice of SP2/0 myeloma. Double antibody sandwich ELISA method was used to assess the level of both rh-IFNα2a-NGR and rh-IFNα2a in serum, tissue, bile and urine. After a single intravenous administration, the pharmacokinetic characters of rh-IFNα2a-NGR and rh-IFNα2a were described using a two-compartment model. No significant differences were observed between the two drugs in pharmacokinetic and elimination data. However, the concentration of rh-IFNα2a-NGR in tumor was 5.34 times and 1.52 times as high as that of rh-IFNα2a at 0.5 h (P < 0.01) and 1 h. In addition, immunohistochemical stain displayed rh-IFNα2a-NGR was predominantly located in tumor vascular tissues. rh-IFNα2a-NGR could be an agent for tumor vascular-targeting therapy and these findings provided references for further clinical study.

The Study of Eleutheroside on Immunological Function of Aging Rats

Objective: a study on effect of eleutheroside on the immunological function of aging model rats, exploring its immune mechanism of delaying aging.Method : Eleutheroside of different concentration after the effect of aging model rats ,calculating the cardiac index ,determination of T- lymphocyte proliferation faction by MTT colorimetric , determination the concentration of the IL-2, IL-6 and TNF-α by double antibody sandwich ELISA method. Result : Eleutheroside can improve the immune function of aged rats and have the centration-dependent .Conclusion: Eleutheroside can improve the immune function of the aged rats and delay the immune senescence.

Development and evaluation of an ELISA method for the measurement of kallikrein-related peptidase 5 (KLK5) in human serum

Kallikrein-related peptidases (KLKs) have been proposed as potential cancer biomarkers. Kallikrein-related peptidase 5 (KLK5) is a secreted trypsin-like protease of the KLKs. Until now, detection of KLK5 in both biological fluids and tissues has been described frequently due to the potential of being a new cancer biomarker. Our objective was to prepare KLK5 antibodies and establish an ELISA method for KLK5 to study the possible clinical application of KLK5 as a biomarker for malignancies. In this study, recombinant KLK5 protein was produced and purified using a prokaryotic expression system, and then used as immunogen to generate antibodies. High titers of specific antibodies were measured in serum of rabbits after the forth booster injection. And the titer of the antiserum reached 1:106. We have also generated monoclonal antibodies using hybridoma technology and the titer reached 1:105. The activity of KLK5 antibodies was characterized by Western blot and immunohistochemistry. To quantitatively examine KLK5 in serum samples, we established double antibody sandwich ELISA method using mouse mAb as capture and rabbit pAb as tracer antibody. We have detected KLK5 levels in ovarian cancer serum to ensure that our sandwich ELISA measurement to have high sensitivity and specificity. The ranges of linearity reached by the standard curves of the newly developed ELISA were 0.45 ng/mL to 125 ng/mL. The detection limit of the method, defined as the concentration of KLK5 can be distinguished, was 0.20 ng/mL. Median serum KLK5 levels were 3.77 ng/mL and 0.86 ng/mL in ovarian cancer patients and normal female, respectively (P ELISA assay for KLK5. Our preliminary findings prompt that KLK5 may be a new potential biomarker for the diagnosis and prognosis in patients with ovarian.