A modified immuno-polymerase chain reaction for the detection of β-glucuronidase from Escherichia coli

Chang, Tsung Chain; Huang, Su

doi:10.1016/s0022-1759(97)00125-7

Cited by 25 publications

(13 citation statements)

References 15 publications

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“…Although immuno-PCR is commonly used for the detection of Escherichia coli (8 ) and other bacteria (13,14 ), we demonstrate that a modified immuno-PCR method can also detect S. aureus in patient specimens. The Pyrogallol Red-molybdate (PRM) protein dye-binding assay is used for urinary protein determination (1-3 ).…”

mentioning

confidence: 87%

Detection of Staphylococcus aureus by a Sensitive Immuno-PCR Assay

Huang¹,

Chang

2004

Clinical Chemistry

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Staphylococcus aureus causes a wide variety of diseases in humans, the clinical courses of which range from boils and furuncles to more serious diseases such as septicemia and pneumonia (1 ). Although a significant cause of community-acquired infection, most life-threatening cases of S. aureus disease are hospital-acquired and are associated, in many cases, with indwelling vascular devices or catheters (2 ). The standard method of diagnosing S. aureus is to culture an isolate from a blood agar plate and then use a latex test to identify S. aureus.A specific product of S. aureus is protein A (3 ). Protein A is a cell-wall constituent of S. aureus and is mainly covalently linked to the peptidoglycan structure (4 ); however, ϳ8 -30% of the protein is secreted into the medium during the exponential growth phase (5 ). This property has been used to develop a latex agglutination test and an ELISA to identify and detect S. aureus. We describe here a sensitive immuno-PCR assay to detect S. aureus protein A. After optimization of the reaction conditions and the use of flexible plates with 96 V-bottomed wells, which were compatible with both the ELISA washer and the thermal cycler for PCR, we automated both detection methods.Anti-protein A antisera were created by immunizing rabbits with protein A purified from preparative polyacrylamide gels. The IgG fraction of the antisera was purified by DEAE ion-exchange chromatography (6 ). The protocol for biotinylating the antibody is described elsewhere (7 ). The avidin-biotinylated DNA complex was prepared fresh before use by mixing avidin and the biotinylated DNA at a molar ratio of 1:4. Avidin (2 g/L) was first diluted with bovine serum albumin (BSA) diluent to 2 g/L, and then 10 L was mixed with 2 L of the biotinylated DNA (120 mg/L). The mixture was incubated at room temperature for 10 min and then further diluted with BSA diluent before use.The protocol for immuno-PCR was a modified version of the modified protocol of Chang and Huang (8 ) (Fig. 1A). Briefly, microtiter plates were coated with 50 L/ well of anti-protein A IgG (10 mg/L in phosphate-buffered saline) for 1 h at 37°C, washed five times, and blocked with 75 L of the BSA diluent for 1 h at 37°C. The plates were washed five times, and 50 L of 10-fold serial diluted antigen (protein A; 10 Ϫ9 -10 Ϫ18 g/mL) was added to each well. After incubation for 1 h at 37°C, the plates were washed five times, and 50 L of the biotinylated anti-protein A (1 g/L) was added to each well. The plates were incubated at 37°C for 1 h and then washed 12 times; 50 L of the avidin-biotinylated DNA complex (1:10 000 dilution) was then added to each well. The plates were again incubated at 37°C for 1 h and washed 12 times before the PCR step. The negative control was performed in the same way, except that the BSA diluent was substituted for the antigen solution. The buffer used throughout for plate washing was phosphate-buffered saline containing 0.5 mL/L Tween 20, whereas the BSA diluent was used for the dilution of antigen, biotinylated an...

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confidence: 87%

Detection of Staphylococcus aureus by a Sensitive Immuno-PCR Assay

Huang¹,

Chang

2004

Clinical Chemistry

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“…For example, non-specific interactions of the Ab to molecules other than the target Ag may result in false positives using the IPCR method. Several studies have now shown that the most sensitive detection system for an indirect IPCR assay was obtained by using the same polyclonal Ab for both capture and detection of Ag, indicating a reduced probability of nonspecific (intra-species) Ab interactions (Chang and Huang, 1997;Sugawara et al, 2000;Niemeyer et al, 1997). 2.…”

Section: Ipcrmentioning

confidence: 99%

“…These predictions should be empirically tested to arrive at the actual estimation of amplification potential of the method. Although there are reports of single-molecule detection using IPCR (Case et al, 1999;Chang and Huang, 1997). Most IPCR applications in the literature cite at least 1000 molecules of Ag are necessary for a significant interpretable signal.…”

Section: Ipcrmentioning

confidence: 99%

Applications of real-time immuno-polymerase chain reaction (rt-IPCR) for the rapid diagnoses of viral antigens and pathologic proteins

Barletta

2006

Molecular Aspects of Medicine

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“…A sensitive method for in vitro labeling of MoAbs using the photobiotin was introduced in our single determinant Immuno-PCR method. [13][14][15][16] A solution of the appropriate MoAb in PBS in a polypropylene tube was mixed with photobiotin in aqueous solution (the ratio for molarity of antibodies and photobiotin is 50). The tube was placed in an ice water bath 10 cm below a sunlamp and illuminated for 15 minutes.…”

Section: Preparation Of Biotinylated Moabsmentioning

confidence: 99%

“…13,14 Subsequently, this method has been used in other areas with convincing results. [15][16][17][18][19][20][21][22] In the current study we report the application of the single determinant Immuno-PCR system to detect circulating TAA in the sera of patients with various carcinomas.…”

mentioning

confidence: 99%