immuno-PCR Research Articles

Diagnosis of genitourinary tuberculosis: detection of mycobacterial lipoarabinomannan and MPT-64 biomarkers within urine extracellular vesicles by nano-based immuno-PCR assay

We detected a cocktail of Mycobacterium tuberculosis lipoarabinomannan (LAM) and MPT-64 biomarkers within urine extracellular vesicles (EVs) of genitourinary TB (GUTB) patients by nano-based immuno-PCR (I-PCR) assay, i.e., magnetic bead-coupled gold nanoparticle-based I-PCR (MB-AuNP-I-PCR) and compared the results with I-PCR and Magneto-ELISA. The size (s) of urine EVs ranged between 52.6 and 220.4 nm as analyzed by transmission electron microscopy (TEM) and nanoparticle tracking analysis. Functionalized AuNPs (coupled with detection antibodies/oligonucleotides) were characterized by UV–vis spectroscopy, TEM, ELISA, PCR, Atomic Force Microscopy and Fourier Transform Infrared spectroscopy, while conjugation of capture antibodies with MBs was validated by UV–vis spectroscopy and Magneto-ELISA. Our MB-AuNP-I-PCR exhibited sensitivities of 85% and 87.2% in clinically suspected (n = 40) and total (n = 47) GUTB cases, respectively, with 97.1% specificity in non-TB controls (n = 35). These results were further authenticated by the quantitative SYBR Green MB-AuNP-real-time I-PCR (MB-AuNP-RT-I-PCR). Concurrently, I-PCR and Magneto-ELISA showed sensitivities of 68.1% and 61.7%, respectively in total GUTB cases, which were significantly lower (p < 0.05–0.01) than MB-AuNP-I-PCR. Markedly, a wide range (400 fg/mL–11 ng/mL) of LAM+MPT-64 was quantified within urine EVs of GUTB cases by SYBR Green MB-AuNP-RT-I-PCR, which can assess the disease dynamics. This study will certainly improve the current algorithms used in GUTB diagnostics.

Diagnosis of tuberculosis by nanoparticle-based immuno-PCR assay based on mycobacterial MPT64 and CFP-10 detection.

Aim: To improve the diagnostic accuracy of immuno-PCR (I-PCR) in tuberculosis (TB) patients by using functionalized gold nanoparticles (AuNPs) coupled with detection antibodies and oligonucleotides, and magnetic beads (MBs) conjugated with capture antibodies in the liquid phase. Materials & methods: MB-coupled AuNP-based I-PCR (MB-AuNP-I-PCR) assay was designed to detect a cocktail of Mycobacterium tuberculosis MPT64 and CFP-10 proteins in bodily fluids of TB patients. Results: The sensitivities of 89.3 (n=94) and 78.1% (n=73) were observed in pulmonary TB and extrapulmonary TB patients, respectively, with specificities of 97.9-98.3%. Notably, the sensitivities attained by MB-AuNP-I-PCR in smear-negative pulmonary TB and extrapulmonary TB patients were significantly higher (p < 0.05-0.001) than Magneto-ELISA and GeneXpert assay. Conclusion: The improved technology, as well as enhanced diagnostic accuracy of MB-AuNP-I-PCR, may lead todevelopment of an attractive diagnostic kit.

Transmembrane protease serine 5: a novel Schwann cell plasma marker for CMT1A.

ObjectiveDevelopment of biomarkers for Charcot‐Marie‐Tooth (CMT) disease is critical for implementing effective clinical trials. The most common form of CMT, type 1A, is caused by a genomic duplication surrounding the PMP22 gene. A recent report (Neurology 2018;90:e518–3524) showed elevation of neurofilament light (NfL) in plasma of CMT1A disease patients, which correlated with disease severity. However, no plasma/serum biomarker has been identified that is specific to Schwann cells, the most directly affected cells in CMT1A.MethodsWe used the Olink immuno PCR platform to profile CMT1A patient (n = 47, 2 cohorts) and normal control plasma (n = 41, two cohorts) on five different Olink panels to screen 398 unique proteins.ResultsThe TMPRSS5 protein (Transmembrane protease serine 5) was elevated 2.07‐fold (P = <0.0001) in two independent cohorts of CMT1A samples relative to controls. TMPRSS5 is most highly expressed in Schwann cells of peripheral nerve. Consistent with early myelination deficits in CMT1A, TMPRSS5 was not significantly correlated with disease score (CMTES‐R, CMTNS‐R), nerve conduction velocities (Ulnar CMAP, Ulnar MNCV), or with age. TMPRSS5 was not significantly elevated in smaller sample sets from patients with CMT2A, CMT2E, CMT1B, or CMT1X. The Olink immuno PCR assays confirmed elevated levels of NfL (average 1.58‐fold, P < 0.0001), which correlated with CMT1A patient disease score.InterpretationThese data identify the first Schwann cell‐specific protein that is elevated in plasma of CMT1A patients, and may provide a disease marker and a potentially treatment‐responsive biomarker with good disease specificity for clinical trials.

Phosphorylated abacavir analogue (ABC-1) has ameliorative action against Newcastle disease virus induced pathogenesis in chicken.

Newcastle disease virus (NDV) causes huge economic loss to the poultry industry due to high mortality and morbidity. The present study aimed to assess the protective role of novel phosphorylated analogue ABC‐1 in vivo in NDV‐infected chickens through the inhibition of fusion protein. Both NDV‐induced oxidative damage and protective role of novel phosphorylated ABC‐1 were evaluated in vital organs such as the liver and lung of chickens. Enzyme linked immunosorbent assay (ELISA) results showed that protein oxidation and nitration levels were significantly raised in NDV‐infected tissues compared to healthy controls, whereas these levels were reduced significantly (P < 0.05) in birds treated with phosphorylated compounds compared to the NDV‐infected group alone. Additional investigation with double immunofluorescence showed that the large amount of immuno colocalization and Western blot analysis also confirmed this observation through its band pattern in NDV‐infected birds compared to healthy birds, whereas these alterations were reduced in treatment with novel phosphorylated ABC‐1. The expression of fusion glycoprotein was studied by immuno colocalization, PCR, and flow cytometry, and results demonstrated that the novel phosphorylated analogues reduced the expression of fusion glycoprotein. These results put forth that novel phosphorylated ABC‐1 protects chickens from NDV‐induced pathogenesis, protein oxidation/nitration, and exerts potent antiviral activity.

Laboratory diagnosis and research progress of Lyme disease

Lyme disease is a natural zoonotic disease transmitted by the bite of tick. The clinical manifestations of Lyme disease are complex and varied, and it is easy to be misdiagnosed. Based on the review of the background knowledge of Lyme disease, this paper reviews the current situation of its diagnosis and the research progress of detection. Key words: Borrelia borrelia; Serologic testing; Polymerase chain reaction; Immuno PCR; Metabonomics

Method of Immuno-PCR in diagnostics of bacterial and viral infections

Immuno-PCR (I-PCR) combines the capabilities of two modern diagnostic methods of enzyme immunoassay (ELISA) and polymerase chain reaction (PCR), combination these methods causes a 100-10000 fold increase in sensitivity compared to a similar ELISA. The review considers the main variants of I-PCR, gives examples of possible use of the method for early and retrospective diagnosis of various diseases.

A novel immuno-PCR method using cDNA display

Immuno-PCR (IPCR) provides sensitive and versatile detection of a variety of antigens by conjugating a PCR-amplifiable DNA reporter to a specific antibody or an aptamer. Several methodologies have been developed to prepare appropriate DNA-antibody conjugates, but in most cases, it remains difficult to label polypeptides with high site-specificity and fixed stoichiometry. To address this issue, we first demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA via puromycin at the single molecule level. Several other in vitro display technologies (e.g., ribosome display, mRNA display) have similar simple nucleic acid-peptide linkage. However, they should be unsuitable for diagnostic applications because of their lability against heat and RNase. The newly developed system here, termed cDNA display mediated immuno-PCR (cD-IPCR), proved to work in direct- and sandwich-type detection of target proteins. Detection of a target in serum was also possible, using a VHH (variable domain of the heavy chain of a heavy chain antibody) antibody as a binding molecule. Although further improvement on sensitivity and quantitativity is necessary before the method becomes useful, we believe this work demonstrated a potential of cD-IPCR as an alternative novel format of IPCR.

A sandwich duplex immuno PCR for rapid and sensitive identification of Clostridium perfringens alpha and enterotoxin

The prevalence and lethality associated with C. perfringens alpha (CPA) and enterotoxin (CPE) toxaemia necessitate the need for rapid and definitive detection systems to initiate management measures. In the present study, a sandwich duplex immuno-capture PCR (SD-IPCR) was developed by employing IgY antibodies against a bivalent protein r-Cpae derived from CPA and CPE for antigen capture and reporter antibodies against truncated CPA or CPE conjugated to oligomers of distinguishable size for antigen revealing and signal amplification. The avian immunoglobulin's (IgY) were devoid of reactivity with S. aureus protein A (SpA), a commensal that often co-exists with C. perfringens. The assay was specific, had a detection limit (LOD) of 1 pg/ml for both CPA and CPE in PBS and improved the LOD by 104 folds compared to an analogous sandwich ELISA with same set of antibodies. In spiking studies, a ten-fold reduction in LOD was observed in case of intestinal tissue samples (10 pg/ml) however, no change in LOD was observed when SD-IPCR was applied on to faecal, serum or muscle tissue samples. Of the 136 natural samples examined, the SD-IPCR could detect CPA and CPE in 29.4% and 35.3% samples, while the sandwich ELISAs could detect the same in 25.7% and 25% samples respectively owing to the relatively lesser sensitivity. The LOD and specificity of the SD-IPCR demonstrates its applicability as an efficient and rapid platform for direct detection CPA and CPE from diverse samples matrices in clinical microbiological and meat testing laboratories.

CDNA Display Mediated Immuno-PCR (cD-IPCR): A Novel PCR-based Antigen Detection Method.

Immuno-PCR (IPCR) is a powerful method in antigen detection where a PCR-amplifiable DNA reporter is conjugated to a specific antibody or an aptamer for the target molecule. In the development and application of IPCR, successful conjugation of a protein (an antibody) with a reporter DNA becomes challenging. To address this issue, we recently demonstrated the feasibility of IPCR based on cDNA display, a 1:1 covalent complex of a polypeptide and its encoding cDNA at the single molecule level. The cDNA display molecule for IPCR is generated first by transcribing the DNA that encodes the detection antibody into an mRNA by in vitro transcription. A puromycin DNA linker is then ligated to the mRNA and then in vitro translation and reverse-transcription are performed to generate the cDNA display molecule. The molecule is then directly used in antigen detection and subsequent qPCR. This method can be applied to detect various antigens in biological samples, if sequences of their single-domain antibodies (VHHs) or peptide aptamers are known.

Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay.

PurposeImmuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen.MethodsGNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR.ResultsTransmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6.ConclusionUnlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis.

Development of immunomagnetic droplet-based digital immuno-PCR for the quantification of prostate specific antigen

Immuno-PCR (IPCR) is an immunoassay that employs DNA as the signal generator and utilizes both the versatility of antigen–antibody interactions and the exponential amplification power of PCR.

Starry Heart Valve: A Story Behind CAT Scratch

SESSION TITLE: Bacterial Infections 1 SESSION TYPE: Affiliate Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: Bartonella species are zoonotic bacterial pathogens in humans. B.quintana and B.henselae are the most common agents causing human infections. B.henselae infection is associated with exposure to cats and their fleas; endocarditis may occur in patients with known valvular disease. CASE PRESENTATION: A 62-year-old female, with no prior history of heart disease, presented with 10 days of bilateral leg swelling, malaise and exertional dyspnea. She lived with her 4 cats. Examination was notable for bilateral pitting edema extending to her waist with visible scratch marks on her abdomen and lower extremities. Laboratory data revealed new pancytopenia and an elevated serum creatinine (2.1 mg/dl). She was started on clindamycin for cellulitis. Blood cultures were negative. Transthoracic echocardiogram, obtained to evaluate the severe edema, showed an aortic valve vegetation measuring 2.7 cm x 1.3 cm with aortic insufficiency. The lesion was confirmed with a transesophageal study. The patient was deemed to have culture negative endocarditis; antibiotics were changed to vancomycin, ceftriaxone and doxycycline. B.henselae and B.quintana IgG titers were positive with the titer of > 1:1024. She underwent an aortic valve replacement. PCR of the heart valve tissue was positive for Bartonella. The bacterial colonies were strongly positive by Warthin Starry stain and demonstrated strong reactivity with a Bartonella immunostain. Vancomycin and ceftriaxone were discontinued; she was transitioned to doxycycline 100 mg twice daily with rifampin 300 mg twice daily for six weeks followed by doxycycline monotherapy for 6 months. DISCUSSION: Patients with Bartonella endocarditis generally present with subacute, nonspecific symptoms including fever, fatigue, weakness, and weight loss, like our patient. Our patient’s exposure to her four cats, with frequent scratches on her thighs and lower abdomen, and a culture-negative valuvular vegetation, raised the suspicion of Bartonella endocarditis. There are several methods to confirm the presence of Bartonella infection including tissue cell culture, Warthin-Starry staining of the resected valve, immuno histologic methods, serologic testing, and PCR amplification using valve specimens. If Bartonella is proven, treatment with 6 weeks of doxycycline along with 2 weeks of gentamycin and rifampin is recommended. However, there are no set guidelines defining optimal duration of therapy. Anecdotal evidences support extended doxycycline therapy for 3-6 months to prevent potential dissemination. CONCLUSIONS: Clinicians should suspect Bartonella endocarditis in patients with culture-negative endocarditis who have had extensive contact to cats. Reference #1: Chomel B et al: Fatal Case of Endocarditis Associated with Bartonella henselae Type I Infection in a Domestic Cat. Journal Of Clinical Microbiology; Nov. 2003, p. 5337-5339 Reference #2: Drancourt M et al: Bartonella (rochalimaea) quintana endocarditis in three homeless men. N Engl J Med 1995;332: 419-23 DISCLOSURE: The following authors have nothing to disclose: Thushara Paul, Siddhant Datta No Product/Research Disclosure Information

Immuno-PCR with digital readout

Immuno-PCR (IPCR) combines the versatile ELISA antigen detection with ultrasensitive PCR signal amplification, thereby enabling the highly sensitive detection of a broad range of targets with a typically very large dynamic detection range. The quantification of the antigen is usually achieved by real-time PCR, which provides a correlation between the target concentration and amplified DNA marker. We here report on the implementation of digital droplet PCR as a means for direct quantification of DNA copies to enable the highly sensitive detection of protein biomarkers. To this end, two alternative approaches, based on either magnetic microbead-based IPCR or a microplate-release IPCR were tested. The latter format worked well and revealed an extraordinary high robustness and sensitivity. While rtIPCR already fulfills typical immunoassay acceptance criteria, ddIPCR enables improved accuracy and precision of the assay because signal response and analyte concentrations are directly correlated. The utility of the novel ddIPCR technology is demonstrated at the example of two cytokines, interleukin 2 and interleukin 6 (IL2, IL6, respectively), with an overall average CV% of 5.0 (IL2) and 7.4 (IL6).

Development of a novel immuno-PCR for detection of avian leukosis virus

Avian leukosis virus (ALV) is an important pathogen for various neoplasms, including lymphoid, myeloid, and erythroid neoplasms, and it causes significant economic loss in the poultry industry. Several efficient methods for the detection of ALV have been reported. However, these previously developed approaches are based on either PCR or immunoassays. Here, we used a proximity ligation technique and combined PCR with the immunoassay to develop a novel immuno-PCR (Im-PCR) approach for the detection of ALV. Our data showed that the Im-PCR had high specificity and sensitivity to ALV. The Im-PCR method selectively reacted to ALV but not to the other avian viruses tested. The limit of detection of Im-PCR could reach 0.5 TCID50. Moreover, the results of Im-PCR were in agreement with results from commercial ELISA when the clinical cloaca samples were used for ALV detection. The present results demonstrate that the novel Im-PCR method can be efficiently applied to detect ALV in a clinical setting. Our data also highlight that Im-PCR may have promising applications in the diagnosis of pathogens.

Feasibility of immuno-PCR technology platforms as an ultrasensitive tool for the detection of anti-drug antibodies.

During the early clinical development of a receptor-IgG1 fusion protein (Drug X), a risk based strategy was utilized to evaluate immunogenicity from Phase I through Proof of Concept clinical studies. Three different technology platforms, enzyme-linked immunosorbant assay, electrochemiluminescent assay and newly emerging immuno-PCR were utilized for evaluation of immunogenic response. An ELISA with adequate sensitivity but significant drug interference was replaced by electrochemiluminescent method with improved drug tolerance; however, an inverse correlation was observed between the administered dose and the incidence of anti-drug antibodies. Further evaluation of an immuno-PCR showed reduced drug interference enabling the detection of anti-drug antibodies in the presence of excess amount of Drug X. Immuno PCR assay proved to be a feasible platform for anti-drug antibody characterization.

Immuno-PCR Assay for Sensitive Detection of Proteins in Real Time.

The immuno-PCR (IPCR) assay combines the versatility and robustness of immunoassays with the exponential signal amplification power of the polymerase chain reaction (PCR). Typically, IPCR allows a 10-1,000-fold increase in sensitivity over the analogous enzyme-linked immunosorbent assay (ELISA). This is achieved by replacing the signal-producing antibody-enzyme conjugate of an ELISA with an antibody-DNA conjugate that serves as a marker for PCR amplification. The amplification power of the PCR allows for the detection of even single molecules of nucleic acid templates, making it well suited for a broad range of applications. Here, we describe the application of an IPCR assay for detection of trace amount of antigens using ricin as an example.

Anthrax Outbreak Among Cattle and its Detection by Extractable Antigen 1 (EA1) Based Sandwich ELISA and Immuno PCR

Anthrax, a fatal zoonotic disease caused by spore forming bacteria Bacillus anthracis is predominantly witnessed among herbivorous animals that are highly sus- ceptible to the pathogen. Typically B. anthracis spores existing in soil, initiate the infection during grazing and animal to animal spread has been rarely reported. A sus- pected outbreak of anthrax was observed among cattle in Gundlupet taluk neighboring Bandipur national park and tiger reserve during January 2013. Blood samples collected from domestic cattle in Gundlupet region were examined by EA1 sandwich ELISA and immunocapture PCR. Of the 64 samples examined, 12 recovered during the course of outbreak were found positive by the diagnostic methods and further resulted in recovery of B. anthracis isolates that were confirmed by standard methods indicating prevalence of the pathogen among diseased cattle. The outbreak could be contained by the administration of oxytetracycline and live spore vaccine. The isolates recovered from the cattle were susceptible to the major antibiotics penicillin, ampi- cillin, ciprofloxacin, nalidixic acid, tetracycline, erythro- mycin and chloramphenicol whereas resistance could be seen for the antibiotic neomycin (25 %). The ambiguity for initial symptomatic identification due to the absence of bleeding from natural orifices could be overcome by the EA1 immunoassay kits employed in this study.

A sensitive immunosorbent bio-barcode assay based on real-time immuno-PCR for detecting 3,4,3',4'-tetrachlorobiphenyl

A functionalized gold-nanoparticle bio-barcode assay, based on real-time immuno-PCR (IPCR), was designed for the determination of 3,4,3',4'-tetrachlorobiphenyl (PCB77). 15nm gold nanoparticles were synthesized, and modified with thiol-capped DNA and goat anti-rabbit IgG. The nanoparticle probes were used to replace antibody-DNA conjugate in the IPCR, and were fixed on the PCR tube wall via the immune reaction. Real-time PCR was performed to quantify the DNA signal directly. Under optimized conditions, the new method was used to detect PCB77 with a linearity range from 5pgL(-1) to 10ngL(-1), and the limit of detection (LOD) was 1.72pgL(-1). Real samples of Larimichthys polyactis, collected from the East China Sea, were analyzed. Recovery was from 82% to 112%, and the coefficient of variation (CV) was acceptable. The results were compared with GC-ECD, revealing that the method would be acceptable for providing rapid, semi-quantitative, and reliable test results for making environmental decisions.

TaqManR Proximity ligation technology for the detection of heterodimeric adhesion receptors on lymphocytes

TaqManR proximity ligation technology (TaqManR PLA) is an innovative advancement of immuno PCR. It allows a fast and quantitative detection of vicinal proteins or protein–protein interactions from cell lysates by combining antibody–antigen binding with a real-time PCR detection. We tested if TaqManR PLA also was applicable to investigate and relatively quantitate adhesion receptor heterodimers such as the alpha-4/beta-1 integrin on the surface of intact cells.Both, alpha-4, beta-1 and the alpha-4/beta-1 heterodimer were detected on the surface of lymphocytes by TaqManR PLA. Results were specific, reproducible and comparable to flow cytometric data. However, preciseness of reactions varied dependent on the antibody pairs used. Co-detection of proximate identical subunits suggested clusters of alpha-4 and/or beta-1 on the cell surface which we confirmed by microscopy.We conclude that real-time PCR-based TaqManR PLA is of limited applicability to investigate heterodimeric receptor molecules such as the alpha-4/beta-1 integrin. Determination of an abundance ratio of alpha-4/beta-1 in relation to total alpha-4 or beta-1 was not possible and real-time detection did not allow conclusions on the surface distribution of molecules. The related in situ PLA developed for microscopy allows visualizing proximate protein interactions and might be an interesting alternative for research into receptor heterodimers and their surface distribution on immune cells.

Detection of hepatitis B virus core antigen by phage display mediated TaqMan real-time immuno-PCR

The core antigen (HBcAg) of hepatitis B virus (HBV) is one of the markers for the identification of the viral infection. The main purpose of this study was to develop a TaqMan real-time detection assay based on the concept of phage display mediated immuno-PCR (PD-IPCR) for the detection of HBcAg. PD-IPCR combines the advantages of immuno-PCR (IPCR) and phage display technology. IPCR integrates the versatility of enzyme-linked immunosorbent assay (ELISA) with the sensitivity and signal generation power of PCR. Whereas, phage display technology exploits the physical association between the displayed peptide and the encoding DNA within the same phage particle. In this study, a constrained peptide displayed on the surface of an M13 recombinant bacteriophage that interacts tightly with HBcAg was applied as a diagnostic reagent in IPCR. The phage displayed peptide and its encoding DNA can be used to replace monoclonal antibody (mAb) and chemically bound DNA, respectively. This method is able to detect as low as 10ng of HBcAg with 10(8)pfu/ml of the recombinant phage which is about 10,000 times more sensitive than the phage-ELISA. The PD-IPCR provides an alternative means for the detection of HBcAg in human serum samples.