Quantitative hepatitis B core antibody and quantitative hepatitis B surface antigen:Novel viral biomarkers for chronic hepatitis B management

The management of hepatitis B virus(HBV)infection now involves regular and appropriate monitoring of viral activity,disease progression,and treatment response.Traditional HBV infection biomarkers are limited in their ability to predict clinical outcomes or therapeutic effectiveness.Quantitation of HBV core antibodies(qAnti-HBc)is a novel non-invasive biomarker that may help with a variety of diagnostic issues.It was shown to correlate strongly with infection stages,hepatic inflammation and fibrosis,chronic infection exacerbations,and the presence of occult infection.Furthermore,qAnti-HBc levels were shown to be predictive of spontaneous or treatment-induced HBeAg and HBsAg seroclearance,relapse after medication termination,re-infection following liver transplantation,and viral reactivation in the presence of immunosuppression.qAnti-HBc,on the other hand,cannot be relied on as a single diagnostic test to address all problems,and its diagnostic and prognostic potential may be greatly increased when paired with qHBsAg.Commercial qAnti-HBc diagnostic kits are currently not widely available.Because many methodologies are only semi-quantitative,comparing data from various studies and defining universal cut-off values remains difficult.This review focuses on the clinical utility of qAnti-HBc and qHBsAg in chronic hepatitis B management.

TCR-mimic antibody-drug conjugates targeting intracellular tumor-specific mutant antigen KRAS G12V mutation

Limited clinical application of antibody-drug conjugates(ADCs)targeting tumor associated antigens(TAAs)is usually caused by on-target off-tumor side effect.Tumor-specific mutant antigens(TSMAs)only expressed in tumor cells which are ideal targets for ADCs.In addition,intracellular somatic mutant proteins can be presented on the cell surface by human leukocyte antigen class I(HLA I)molecules forming tumor-specific peptide/HLA I complexes.KRAS G12 V mutation frequently occurred in varied cancer and was verified as a promising target for cancer therapy.In this study,we generated two TCR-mimic antibodydrug conjugates(TCRm-ADCs),2E8-MMAE and 2 A5-MMAE,targeting KRAS G12 V/HLAA*0201 complex,which mediated specific antitumor activity in vitro and in vivo without obvious toxicity.Our findings are the first time validate the strategy of TCRm-ADCs targeting intracellular TSMAs,which improves the safety of antibody-based drugs and provides novel strategy for precision medicine in cancer therapy.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Analysis of sperm membrane antigens relevant to antisperm antibody using Western blot

To identify the sperm membrane antigens associated with antisperm antibody. Methods: The antisperm antibody in serum was tested by ELISA. Antisperm antibody positive sera from 18 infertile men and 15 infertile women were used. The molecular weight (MW) of sperm membrane antigens associated with the antisperm antibody was analyzed with antisperm antibody positive serum using Western blot. Results: Eight kinds of MW of sperm membrane antigens were identified. The ratio of identification on the 78 KD(60.7 %), 60KD (71.4 %), 51 KD (14.9 %) and 23 KD (14.29 %) sperm antigen was higher than others. Conclusion: Sperm membrane antigens with MW of 78 KD, 60 KD, 51 KD and 23 KD were associated with antisperm antibody and immunological infertility. (Chin J Andro12002; 16: 345)

GENE ENGINEERING EB VIRUS MEMBRANE ANTIGEN IN DETECTION OF MA-IgA ANTIBODY(COMPARISON WITH VCA-IgA AND EA-IgA ANTIBODIES)

With gene engineering EB virus membrane antigen as the diagnostic antigen, indirect immunofluo-rescence (IF) assay was used to detect IgA antibody against EB virus membrane antigen (MA-IgA) in sera from 202 nasopharyngeal carcinoma (NPC) patients and 315 controls (normal and patients with other tumors). MA-IgA antibody was positive in 96.8% of the pretreatment NPC patients with a GMT of 1:36.3. MA-IgA detection by this method was more sensitive than EA-IgA detection by IE. In contrast, patients with tumors other than NPC were negative for MA-IgA antibody. 9.1% of VCA-IgA positive persons were MA-IgA positive with a GMT of less than 1:5. No MA-IgA positive was found in VCA-IgA negatives. The results indicated that this method was relatively specific. In the treatment group, the positive rate and GMT of MA-IgA antibody declined with increase in survival time and the decline was faster than VCA-IgA. When recurrence or distant metastasis developed, similar to VCA-IgA and EA-IgA antibodies, the positive rate and GMT of MA-IgA antibody increased to its pretreatment level. Therefore, MA-IgA detection might be valuable in the early diagnosis and monitor of NPC.

CHARACTERIZATION OF MONOCLONAL ANTIBODY CL58 AGAINST CARCINOEMBRYONIC ANTIGEN(CEA)AND STUDY OF ITS BIODISTRIBUTION

Objective: To study the preparation and characterization of monoclonal antibody (McAb) against carcinoembryonic antigen (CEA). Methods: CEA antigen was extracted from metastasized liver of patients with colorectal cancer and used for the preparation of McAb against CEA by hybridoma technique. Immunoreactivity of McAb to CEA antigen was evaluated using ELISA. Mouse ascites was purified by two steps, high performance liquid chromatography (HPLC) using protein A and high performance hydroxylapatite (HPHT). Normal adult tissues and tumor specimen were used for immunohistochemical evaluation of the McAb. Isotope 99mTc labeled CEA McAb was used for biodistribution in tumor-bearing mouse. Results: Purified CEA antigen was a glycoprotein of 180 kD. Anti-CEA McAb affinity constant was 7.4×109/M. The McAb showed positive staining in 54–88% of colorectal cancer, gastric cancer and lung cancer, while negative for normal tissues. 24 hours after injection of 99mTc labeled McAb, tumor ID%/g was higher than 15% and tumor/blood, tumor/kidney and tumor/liver were 1.82, 1.51 and 2.92 respectively. T/NT ratios of other viscera were over 3.0. Conclusion: Purified CEA antigen had very good immunogenicity. The anti-CEA McAb was highly specific. 99mTc labeled McAb was stabled both in vivo and in vitro. In vivo distribution result was satisfactory. McAb CL58 may be useful for RII and RIGS.

HLA antigens and anti-sperm antibody production in Iranian vasectomized men

Dear Editor： Anti-sperm antibodies （ASAs） are composed of numerous antibodies interacting with multiple sperm antigens that play a role in fertility. In males, ASAs cause ＇immune infertility＇ by decreasing sperm counts and normal forms, as well as reducing sperm motility and viability, markedly reducing the likelihood of natural conception. The development of ASA in the male depends on the release of sequestered antigens on germ cells following the disruption of the blood-testis barrier.

Methodological aspects of anti-human leukocyte antigen antibody analysis in solid organ transplantation

Donor human leukocyte antigen(HLA)-specific antibodies(DSA) play an important role in solid organ transplantation. Preexisting IgG isotype DSA are considered a risk factor for antibody mediated rejection, graft failure or graft loss. The post-transplant development of DSA depends on multiple factors including immunogenicity of mismatched antigens, HLA class Ⅱ typing of the recipient, cytokine gene polymorphisms, and cellular immunoregulatory mechanisms. De novo developed antibodies require special attention because not all DSA have equal clinical significance. Therefore, it is important for transplant clinicians and transplant immunologists to accurately characterize DSA. In this review, the contemporary immunological techniques for detection and characterization of anti-HLA antibodies and their pitfalls are described.

Antigen Detection in Canine Blastomycosis: Comparison of Different Antibody-Antigen Combinations in Two Competitive ELISAs

This present study was designed to evaluate four different Blastomyces dermatitidis antibody-antigen combinations (B5896 and T-58 antibodies and B5896 and WI-R antigens) for the detection of antigen in 36 urine specimens from dogs with blastomycosis using a standard indirect ELISA (STD) and a biotin-streptavidin ELISA (B-SA). The antigen detection sensitivity values ranged from 81% (B-SA: T-58 Ab + WI-R Ag) to 100% (STD and B-SA: B5896 Ab + WI-R Ag;B5896 Ab + B5896 Ag) with the antibody-antigen combinations in the two assays. Optimal detection was evidenced when the B5896 Ab was allowed to react with the urine specimens for 30 min at 37?C and then placed in the B-SA ELISA plates containing the B5896 Ag. The greatest absorbance value obtained with this antibody-antigen com-bination was 0.903 (range of 0.596 - 0.903) as compared to the control value of 1.246. The difference between the control absorbance and the test absorbance values was 0.343 which was considerably greater than the control-test values with the other combinations. This study thus showed that the results obtained in antigen detection assays are dependent upon the antibody used to react with the urine specimens as well as the antigen used in the enzyme immunoassay.

Comparative antibody study for antigen detection in urine specimens for diagnosis of blastomycosis using a competitive enzyme-linked immunosorbent assay

Diagnosis of blastomycosis is often done using a combination of clinical signs and cytologic or histopathologic identification of the organism, Blastomyces dermatitidis, from infected tissues. However, these methods are time consuming, invasive, and still lead to misdiagnosis. A competitive enzyme-linked immunosorbent assay (ELISA) can be used for detection of B. dermatitidis antigens, which are present in urine specimens of infected patients. The current study evaluates the use of various antibodies for detection of antigen in dog urine specimens, to provide a better diagnosis of blastomycosis in the future. Our results show that different antibodies against B. dermatitidis produce various sensitivities for antigen detection. The most realistic antibodies for immunodiagnostic tests would be antibodies that can be obtained in larger quantities, i.e. vaccination using a yeast lysate in a laboratory setting. We found that these antibodies produce a comparable and reliable result to that of antibodies obtained from an infected patient.

Prospective study of SEVA TB peroxidase assay for cocktail antigen and antibody in the diagnosis of Tuberculosis in suspected patients attending a tertiary care hospital located in rural area

Objective:To evaluate inhouse developed SEVA TB peroxidase enzyme immunoassay using cocktail of mycobacterial excretory-secretory antigens(ES-31,ES-43 & EST-6) for antibody detection and their affinity purified antibodies for antigen detection in tuberculosis suspected patients.Methods:Inhouse developed SEVA TB peroxidase enzyme immunoassay was evaluated prospectively in 73 suspected pulmonary and 46 extra-pulmonary tuberculosis patients during November 2008~March 2009 in a tertiary hospital located in rural area.Results:Assay on prospective analysis showed 100% correlation of pulmonary tuberculosis(PTB) and extrapulmonary tuberculosis(EPTB) acid fast bacilli positivity and antitubercular treatment in 11 cases.Thirty nine PTB and 12 EPTB cases showed negative for EUSA test and were also not given antitubercular therapy.However 30 PTB and 27 EPTB cases showing ELISA positivity were neither acid fast bacilli positive nor antitubercular therapy treated.These cases may possibly have dormant infection and need further diagnosis.In EPTB cases ELISA was observed to be more useful than AFB smear test.Conclusions:This inhouse developed user-friendly peroxidase ELISA can be used as an adjunct test of smear microscopy or culture techniques for routine screening of patients suspected of PTB or EPTB.

Comparison of Antibody Detection with Yeast Lysate Antigens Prepared from Blastomyces dermatitidis Dog Isolates from Wisconsin and Tennessee

Blastomyces dermatitidis, the causative agent of blastomycosis, a potentially lethal dimorphic fungal disease of humans and animals has been difficult to diagnose in the clinical laboratory. We are attempting to develop and improve immunodiagnostic assays by producing novel yeast lysate reagents for the detection of antibodies in blastomycosis. The objective of this study was to use lysate antigens prepared from four B. dermatitidis antigens isolated from dogs infected with blastomycosis from two different endemic areas (Wisconsin and Tennessee) testing for the detection of antibodies in serum specimens from immunized rabbits and infected dogs using the indirect ELISA. In the dog sera, absorbance values ranged from 0.774 to 1.350, while the rabbit sera values ranged from 0.533 to 1.191. Antigen T-58 appeared to lack any geographical specificity in antibody detection, which could prove useful in future immunodiagnostic detection of blastomycosis infections.

Comparison of antibody detection with <i>Blastomycesdermatitidis</i>yeast lysate antigens in serum specimens from immunized rabbits and infected dogs

This present study was designed to evaluate B. dermatitidis antigens, prepared from two isolates (B5896, 597), when the yeast cells were allowed to lyse in distilled water for one day or seven days. The indirect enzyme-linked immunosorbent assay (ELISA) was used to determine the ability of the lysate reagents to detect antibodies in 30 rabbit and 30 dog serum specimens. Mean absorbance values with B5896 lysate antigen ranged from 1.637 (day 1) to 1.461 (day 7) and absorbance values with 597 antigen ranged from 1.579(day 1) to 1.396 (day 7) with the serum specimens from immunized rabbits. Serum specimens from infected dogs yielded absorbance values ranging from 1.672 (day 1) to 1.763 (day 7) with the B5896 and values ranging from 1.909 (day 1) to 1.224 (day 7) with the 597. Optimal reactivity was obtained with the day 1 lysate using both lysate antigens against the rabbit sera and with the 597 antigen against the dog sera. Slightly greater reactivity was evidenced with the day 7 B5896 antigen when the dog sera was tested. Comparative studies are continuing in order to produce an optimal anti-genic preparation for antibody detection in blastomycosis.

Comparison of Two Enzyme Immunoassays and Four Lysate Antigens for the Detection of Antibody in Canine Blastomycosis

Blastomycosis, the systemic fungal disease of humans and animals caused by Blastomyces dermatitidis and the cryptic species Blastomyces gilchristii, is often misdiagnosed as a bacterial or viral pulmonary disease. Therefore, the development of improved immunodiagnostic assays for this disease has been the primary focus of research in our laboratory. The present study was designed to evaluate four Blastomyces yeast-phase lysate antigenic preparations (human, 597, Eagle River, WI;dog, ERC-2, WI;Human, B5927, Mountain Iron, MN;soil, 85, Georgia, ATCC 56920) for their ability to detect antibody in 48 serum specimens from dogs with diagnosed blastomycosis using an indirect ELISA (STD) compared to a biotin-streptavidin ELISA (B-SA). All four lysate antigens were able to detect antibodies in the specimens with mean absorbance values ranging from 0.930 (B5927) to 1.142 (ERC-2) with the STD ELSA and from 1.395 (B5927) to 1.775 (85) with the B-SA ELISA. The results indicated that both ELISA methods could be utilized for antibody detection, but the B-SA ELISA exhibited greater sensitivity than the STD ELISA with all four of the lysates.

DETECTION OF EB VIRUS EARLY ANTIGEN IN NASOPHARYNGEAL CARCINOMA (NPC) USING A NEW MONOCLONAL ANTIBODY--BAE-5

A mouse monoclonal antibody against EB virus EA-R named BAE- 5 was prepared applying hybridoma technique by use of n-butyric add and croton oil induced Raji cells as immunogen.And then, 34 NPCs and 29 mm-NPC neoplasms were detected by the BAE-5 using APAAP immunohistochemical staining method.The results Indicated that all of the 34 NPCs and 9 of the 29 non-NPC neoplasms could reacted with the BAE-5 in spite of the columnar epithelium reserve cells showing positivity also. It is reasonable to come to the conclusion that most of the NPC cells can not only harbour EBV DNA but some of them be able to express EA-R. Some non-NPC neoplasms being demonstrated BAE-5 posttlvlty may be due to the presence of EBV EA- R antigenic epitope or polypeptides simulating the structure of EA-R. The authors think that these findings have theoretical significance for understanding the role of EBV- encoded poiypeptides, especially EA complex, In the development and progression of NPC as well as some non- NPC neoplasms harbouring EBV-DNA.

Detection of Antibody in Dogs with Blastomycosis Using <i>Blastomyces dermatitidis</i>Yeast Phase Lysate Antigens

The objective of our study was to compare two B. dermatitidis yeast phase lysate antigens [ERC-2, dog Wisconsin;85, soil Georgia, ATCC 56,920] for detecting antibody in 38 serum specimens [pre-treatment, 30-day, and 60-day post treatment] from dogs with diagnosed blastomycosis. The mean absorbance values obtained with the two antigens (N = 38) were ERC-2 = 2.359 and 85 = 2.189. The mean absorbance values when the sera were divided into the three treatment groups were as follows pre-treatment: Isolate ERC-2 had an absorbance value of 2.418;Isolate 85 had an absorbance value of 2.688, 30-day post treatment: ERC-2 had an absorbance value of 2.452;85 had an absorbance value of 2.303 and 60-day post treatment: ERC-2 had an absorbance value of 2.150;85 had an absorbance value of 2.073 with the mean absorbance values of all treatment groups were ERC-2: 2.229 and 85: 2.141. This study indicates the potential for further evaluations of the two lysate antigens with regard to antibody detection in dog sera with the ERC-2 reagent slightly more reactive than the 85 lysate antigen.

Evaluation of rabbit antibody response against 8 and 16 kDa recombinant subunits of antigen B from Echinococcus granulosus

Objective:To immunize rabbits with 12 and 16 kDa recombinant subunits of antigen B from Echinococcus granulosus(E.granulosus) and measuring polyclonal antibody and humoral immune response using ELISA and gel diffusion.Methods:Two mentioned antigens were cloned and expressed in expression vector and purified by affinity chromatography.Four young rabbits were selected and challenged intradermallt with yielded recombinant antigens.Rabbits’ sera were collected post infection and were tested using ELISA and gel diffusion for polyclonal antibody detection 10 days after last injection.Results:The specific antibody against the recombinant peptides was efficiently produced within 4 weeks post infection.Conclusions: Produced recombinants proteins could induce the immune response of the rabbits successfully. This process might improve the clarification of diagnosis and vaccination as regards hydatidosis.

Anti-PD1 antibody and not anti-LAG-3 antibody improves the antitumor effect of photodynamic therapy for treating metastatic breast cancer

Photodynamic therapy(PDT)has limited effects in treating metastatic breast cancer.Immune checkpoints can deplete the function of immune cells;however,the expression of immune checkpoints after PDT is unclear.This study investigates whether the limited e±cacy of PDT is due to upregulated immune checkpoints and tries to combine the PDT and immune checkpoint inhibitor to observe the e±cacy.A metastatic breast cancer model was treated by PDT mediated by hematoporphyrin derivatives(HpD-PDT).The anti-tumor effect of HpD-PDT was observed,as well as CD4þT,CD8þT and calreticulin(CRT)by immunohistochemistry and immunofluorescence.Immune checkpoints on T cells were analyzed byflow cytometry after HpD-PDT.When combining PDT with immune checkpoint inhibitors,the antitumor effect and immune effect were assessed.For HpD-PDT at 100 mW/cm2 and 40,60 and 80 J/cm2,primary tumors were suppressed and CD4þT,CD8þT and CRT were elevated;however,distant tumors couldn't be inhibited and survival could not be prolonged.Immune checkpoints on T cells,especially PD1 and LAG-3 after HpD-PDT,were upregulated,which may explain the reason for the limited HpD-PDT effect.After PDT combined with anti-PD1 antibody,but not with anti-LAG-3 antibody,both the primary and distant tumors were signi-cantly inhibited and the survival time was prolonged,additionally,CD4þT,CD8þT,IFN-þCD4þT and TNF-þCD4þT cells were signi-cantly increased compared with HpD-PDT.HpD-PDT could not combat metastatic breast cancer.PD1 and LAG-3 were upregulated after HpD-PDT.Anti-PD1 antibody,but not anti-LAG-3 antibody,could augment the antitumor effect of HpD-PDT for treating metastatic breast cancer.

Prostate cancer with elevated free prostate-specific antigen density:A case report

BACKGROUND Prostate cancer is the second most common cancer among men worldwide,and prostate-specific antigen(PSA)is often used in clinical practice to screen for prostate cancer.Normal total PSA(tPSA)level initially excludes prostate cancer.Here,we report a case of prostate cancer with elevated free PSA density(fPSAD).CASE SUMMARY A patient diagnosed with benign prostatic hyperplasia underwent prostatectomy,and the postoperative pathological results showed acinar adenocarcinoma of the prostate.The patient is currently undergoing endocrine chemotherapy.CONCLUSION We provide a clinical reference for diagnosis and treatment of patients with normal tPSA but elevated fPSAD.