Development and Evaluation of Simple Dot–Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food

The present study was aimed to develop and evaluate dot–blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot–blot format, recombinant SEA was directly coated on NCM dot–blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot–blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot–blot assays exhibited a sensitivity of ~48 ng ml^?1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot–blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot–blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.     

Development of a one-step immunochromatographic strip test using gold nanoparticles for the rapid detection of Salmonella typhi in human serum

An immunochromatographic strip test using gold nanoparticles was developed for the rapid detection of Salmonella typhi (S. typhi) in human serum. The strip test based on the principle of sandwich immunoassay by the specific binding of antigens from S. typhi O901 and antibody of S. typhi O901 on a nitrocellulose membrane. Antibody-gold nanoparticle conjugate was used as the label and was coated onto a glass fiber membrane, which was used as a conjugate pad. To create a test and control zone, antibody of S. typhi O901 and an anti-IgG were dotted on the nitrocellulose membrane, respectively. Positive samples were displayed as red dots at the test and control zones of the nitrocellulose membrane, while negative samples resulted in a red dot only in the control zone. The limit of detection (LOD) was found to be 1.14×10(5) cfu mL(-1), which could be visually detected by the naked eye within 15 min. This strip test provided a lower detection limit and analysis time than a dot blot immunoassay (8.88×10(6) cfu mL(-1) for LOD and 110 min for reaction time). In addition, our immunochromatographic strip test was employed to detect S. typhi in human serum effectively, with high accuracy. This strip test offers great promise for a rapid, simple and low-cost analysis of S. typhi.     

Dimethyl suberimidate as an effective crosslinker for antibody-enzyme conjugation.

Dimethyl suberimidate (DMS), a bifunctional reagent was used for the first time to crosslink the alpha-feto protein monoclonal antibodies (AFPMAb) to horse radish peroxidase (HRP). Three batches of conjugates were prepared, purified by Sephadex gel chromatography and evaluated for their immunological reactivity. The Rz values obtained for AFPMAb-HRP conjugate were 0.39 to 1.36. Under optimised conditions the ELISA results showed the optical density of 1.9. The iso-electric focusing for the conjugate revealed different degrees of crosslinking between antibodies and HRP. It was evident that isoperoxidase-C was involved in the crosslinking process. From the dot ELISA, as low as 25 pg of AFP in the test samples could be detected with AFPMab-HRP conjugate. The conjugate prepared by DMS was stable at 0 degrees C for more than 10 months.     

应用ELISA和ELFA定量检测牛奶中葡萄球菌肠毒素A方法的建立

探讨了Tecra SEs SET(ELISA法)和Vidas SET2(ELFA法)两种葡萄球菌肠毒素定性检测试剂盒用于定量检测牛奶中葡萄球菌肠毒素A(SEA)的可行性。根据GB/T27404-2008《实验室质量控制规范食品理化检测》的要求,对两种方法应用于定量检测时的检出限、校正曲线范围、相关系数和加标回收率指标进行了分析比较。实验结果显示,Tecra SEs SET对牛奶中SEA的检出限为0.79 ng/mL,校正曲线范围为0.79~10 ng/mL,相关系数r=0.997,SEA加标浓度为0.80、2.5和10 ng/mL时的回收率分别为110%、81%和100%。Vidas SET2的检出限为0.09 ng/mL,校正曲线范围为0.09~1.0 ng/mL,相关系数r=0.998,SEA加标浓度为0.1、0.25和1.0 ng/mL时的回收率分别为90%、95%和104%。上述结果结合对阳性样品的检测表明:这两种定性检测试剂盒能满足牛奶中SEA定量检测的要求。     

A modified rapid enzyme immunoassay for the detection of rabies and rabies-related viruses: RREID-lyssa.

This paper presents a modification of the previously described Rapid Rabies Enzyme Immuno-Diagnosis test (RREID) by using biotinylated antibodies, streptavidin conjugate and a mixture of monospecific polyclonal antibodies against several lyssaviruses. In the modified technique (RREID-lyssa), microplates were sensitized with a mixture of purified antibodies against ribonucleoprotein (RNP) from Pasteur virus (Lyssavirus serotype 1), European Bat Lyssavirus (EBL, unclassified) and Mokola virus (Lyssavirus serotype 3). Bound RNP was detected by the same antibodies labelled with biotin and peroxidase-strepavidin conjugate. These techniques were used for the detection of RNP of different Lyssavirus serotypes (rabies and rabies-related viruses). For lyssavirus specimens of serotype 1, the threshold of detection of RREID and RREID-lyssa were similar. However, a smaller amount of labelled antibodies was needed when biotinylated antibodies were used. For specimens infected by rabies-related strains (serotypes 2, 3, 4 and EBL), the threshold of detection of the RREID-lyssa was between two and 512 times lower than with the RREID. The sensitivity and the specificity of the RREID-lyssa for rabies virus (serotype 1) when tested on a small field trial (53 specimens) were found to be identical to the RREID. Consequently, RREID-lyssa can be a useful tool for diagnostic laboratories that receive specimens infected by rabies-related viruses.     

Binding of staphylococcal enterotoxin A to purified murine MHC class II molecules in supported lipid bilayers

The staphylococcal enterotoxins are a family of bacterial toxins that are thought to exert their pathogenic effects by the massive activation of T lymphocytes to produce lymphokines. Activation of T cells by these toxins is dependent on MHC class II+ APC. Recent studies from a number of laboratories have implicated MHC class II proteins as the APC surface receptor for a number of the staphylococcal enterotoxins. The present report shows that staphylococcal enterotoxin A, (SEA) binds to the purified murine MHC class II molecule I-Ed reconstituted in supported planar membranes, indicating that no other cell surface proteins are required for SEA binding. The Kd for SEA binding to I-Ed was determined to be 3.5 +/- 1.6 x 10(-6) M. Specific binding of SEA to I-Ad was also observed, but the interaction was of significantly lower affinity. Binding of SEA to purified I-Ed was blocked by antibodies against both the alpha- and the beta-chain of the I-Ed molecule, but not by antibodies specific for an unrelated MHC class II protein. Binding of SEA to I-Ad was blocked by an A beta d but not by an A alpha d-specific antibody. Planar membranes containing only lipid and purified I-Ed molecules were sufficient for activation of a V beta 1 expressing T hybrid by SEA. The T cells responded to as few as 180 toxin molecules per T cell.     

First External Quality Assessment of Molecular and Serological Detection of Rift Valley Fever in the Western Mediterranean Region

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis which affects humans and a wide range of domestic and wild ruminants. The large spread of RVF in Africa and its potential to emerge beyond its geographic range requires the development of surveillance strategies to promptly detect the disease outbreaks in order to implement efficient control measures, which could prevent the widespread of the virus to humans. The Animal Health Mediterranean Network (REMESA) linking some Northern African countries as Algeria, Egypt, Libya, Mauritania, Morocco, Tunisia with Southern European ones as France, Italy, Portugal and Spain aims at improving the animal health in the Western Mediterranean Region since 2009. In this context, a first assessment of the diagnostic capacities of the laboratories involved in the RVF surveillance was performed. The first proficiency testing (external quality assessment—EQA) for the detection of the viral genome and antibodies of RVF virus (RVFV) was carried out from October 2013 to February 2014. Ten laboratories participated from 6 different countries (4 from North Africa and 2 from Europe). Six laboratories participated in the ring trial for both viral RNA and antibodies detection methods, while four laboratories participated exclusively in the antibodies detection ring trial. For the EQA targeting the viral RNA detection methods 5 out of 6 laboratories reported 100% of correct results. One laboratory misidentified 2 positive samples as negative and 3 positive samples as doubtful indicating a need for corrective actions. For the EQA targeting IgG and IgM antibodies methods 9 out of the 10 laboratories reported 100% of correct results, whilst one laboratory reported all correct results except one false-positive. These two ring trials provide evidence that most of the participating laboratories are capable to detect RVF antibodies and viral RNA thus recognizing RVF infection in affected ruminants with the diagnostic methods currently available.     

Indirect immunofluorescence colony staining method for detecting bacterial pathogens of tomato.

An indirect immunofluorescence colony staining method was developed for the detection of important seed-borne bacterial pathogens of tomato. The method involves the use of specific antiserum for initial binding of target bacteria and visualization of positive colonies with a commercially available secondary antiserum conjugated with FITC and observed under a fluorescence microscope. The indirect method is especially suitable for laboratories, seed companies, and quarantine stations which have no facilities for conjugation of primary antiserum. It is more economical and overcomes the problems generally encountered with variable conjugate quality in new batches of conjugates prepared from the same stock of primary antiserum. The assay is easy to perform and results can be easily assessed by visual scoring or image analyser. Results are available in 4-5 days as compared to 30-45 days in traditional methods. The resulting bacterial culture can be tested by PCR or host infectivity and a culture can be stored for future reference. Used in combination with highly specific antibodies (commercially available monoclonal and recombinant antibodies) it can be used as a very sensitive detection tool and has application potential in localization studies as well. Choosing the right secondary conjugate is however necessary to get best results in the assay.     

A gold nanoparticles based immuno-bioprobe for detection of Vi capsular polysaccharide of Salmonella enterica serovar Typhi

A rapid and sensitive gold-nanobioprobe based immunoassay format has been presented for the detection of capsular Vi polysaccharide of Salmonella enterica serovar Typhi (surface antigen) using anti-Vi antibodies. The Vi antigen was extracted from serovar Typhi cells, under the optimised growth conditions for its over-expression. Anti-Vi antibodies were produced and conjugated with gold nanoparticles (GNPs) of definite size (~30 nm), which served as the nano-bioprobe in the detection system. A sandwich immunoassay was developed using nitrocellulose dot blot comb (8/12 wells) membranes immobilized with anti-Salmonella antibodies at the optimal concentration (43 ng spot(-1)). The Vi antigen in the clinical isolates, spiked samples and also in the standard strain (serovar Typhi Ty2) was detected by measuring the colour intensity of GNPs and correlating it with the concentration of serovar Typhi in samples. Using this developed immunoassay technique Vi positive serovar Typhi strains could be detected with a sensitivity of up to 10(2) cells mL(-1) in the clinical isolates as well as in the spiked samples. The developed immunoassay technique could be useful for the detection of typhoid fever and may be important from an epidemiological point of view.     

Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum

Monoclonal antibodies were raised against calmodulin purified from Dictyostelium discoideum. To increase its antigenicity, the calmodulin was conjugated to keyhole limpet hemocyanin; mice were immunized with the conjugate. Hybridomas producing antibodies against calmodulin were identified by screening culture supernatants with calmodulin coupled to bovine serum albumin. The specificity of antibodies from hybridoma culture supernatants was tested by Western blot of Dictyostelium cell lysates. For the purpose, methods were developed that permitted sensitive detection of calmodulin bound to membranes. The key elements of the blotting protocol were used of PVDF membrane, transfer conducted in phosphate buffer, and glutaraldehyde fixation after transfer. These methods permitted detection of as little as 0.1 ng of calmodulin spotted directly onto the membrane, or 10 ng transferred from an SDS polyacrylamide gel. Ten calmodulin-specific antibodies were identified; most of these reacted preferentially with the calcium-containing form of Dictyostelium calmodulin. Several of the monoclonal antibodies cross-reacted with calmodulin from bovine brain.     

Monoclonal antibodies labeled with colloidal gold for immunochromatographic express analysis of diphtheria toxin

A one-step immunochromatographic method for the detection of the diphtheria toxin in different water samples (phosphate buffer, milk, and human nasopharyngeal swab) was developed using a conjugate of monoclonal antibodies labeled with colloidal gold. The detection limit of diphtheria toxin was 10 ng/ml, the time of the analysis was 15 min. The use of silver to enhance the reaction sensitivity and scanning equipment to evaluate the immunochromatography results decreased the detection limit to 1.25 ng/ml.     

Fluorescence polarization immunoassay of ractopamine

A technique was developed for fluorescence polarization immunoassay (FPIA) of ractopamine, a toxic low molecular weight nonsteroidal growth regulator belonging to the most controlled contaminants of food products of animal origin. The assay is based on the competition between a sample containing ractopamine and ractopamine–fluorophore conjugate for binding to antibodies. The competition is monitored via changes in the degree of fluorescence polarization for plane-polarized excitation light, which differs for the free and antibody-bound forms of the conjugate. The optimal assay conditions were established, ensuring a high accuracy and minimal detection limit. The developed assay demonstrated a detection limit of 1 ng/mL and a range of detectable concentrations of 2.3–50 ng/mL, which met the requirements of sanitary control. The duration of the analysis was 10 min. The possible application of the developed FPIA was demonstrated with testing of turkey meat. The speed and simplicity of the proposed assay define its efficiency as a screening tool for safety of foods.     

Field trial of an immunoenzyme method using beta-lactamase as the monoclonal antibody marker for the capsular antigen of the plague microbe

Beta-lactamase (penicillinase) has been used as a marker of monoclonal antibodies in the enzyme immunoassay made with a view to the detection of Y. pestis capsular antigen and antibodies to it. The trial of the enzyme immunoassay with the use of the beta-lactamase conjugate in laboratories and under field conditions has revealed the advantage of this assay over hemagglutination tests commonly used for this purpose.     

Trans-diamminedichlorplatinum (II)-modified probes for detection of picogram quantities of DNA.

A novel simple nonradioactive method for detection of specific nucleotide sequences has been developed. This method consists of the hybridization of a target DNA with a DNA probe modified with trans-diamminedichlorplatinum(II) (trans-DDP) followed by detection of DNA/DNA hybrids with affinity-isolated anti-DNA-trans-DDP antibodies and poly-horseradish peroxidase-protein A conjugate. Major advantages of this approach are the low cost and the extreme simplicity of the labeling procedure, which involves only mixing of the reagents. The sensitivity of the proposed technique is sufficient to detect 0.8 pg of DNA in Southern blot hybridization and 25 fg in dot hybridization and permits colony screening.     

Development of a one-step strip for the detection of triazophos residues in environmental samples

Environmental and food safety issues now are recognized internationally, and pesticide residues play key roles as environment and food pollutants. It is crucial to develop methods for rapid determination of pesticide residues in environments and foods. A one-step strip based on nanocolloidal-gold-labeled monoclonal antibodies for detection of triazophos residue was developed. The nanocolloidal gold, with an average particle diameter of 25 nm (G25), was labeled to an antitriazophos monoclonal antibody. This conjugate was dispensed on the conjugate pad of a porous glass fiber. Ovalbumin hapten and goat anti-mouse IgG were dispensed on the nitrocellulose membrane and served as the test line (T-line) and control line (C-line), respectively. After conditions optimization, the one-step strip was finally developed for the residue determination of triazophos. The limit of detection (LOD) of the strip was 4 ng/mL for standard. The detection was not affected by the pH of the liquid sample but low total ion concentration will induce illegible C-line and T-line. The LOD for spiked samples of soil and water was 5 ng/mL, with run time of no more than 10 min.     

Immunoenzyme determination of the total level of ceruloplasmin in the serum from patients with hepatocerebral dystrophy]

An immunoenzymatic method for ceruloplasmin analysis (IEA) based on the use of horseradish peroxidase-labelled monospecific antibodies as markers has been developed. IEA can be used for direct measurements of ceruloplasmin in blood serum, as can be evidenced from the coincidence of calibration plots obtained after the use of potassium-phosphate buffer and ceruloplasmin-free sera. The procedure allows the determination of the total content of ceruloplasmin present in the blood sera of patients with hepatocerebral dystrophies both in the active and inactive forms. The minimum ceruloplasmin concentration detectable by this method is 5 x 10(-9) g/ml. The method was used to determine ceruloplasmin levels in the blood of patients with various grades of hepatocerebral dystrophy. Analysis of blood sera from 6 patients revealed that the ceruloplasmin concentrations determined by IEA were very close, whereas the oxidase activities of this protein differed more than 7-fold. The amount of enzymatically active ceruloplasmin as determined from the oxidase activity made up to 10-68% of the total ceruloplasmin content in the sera, depending of the severity of the pathology.     

Sensitive and rapid detection of 2,4-dicholorophenoxyacetic acid in water samples by using evanescent wave all-fiber immunosensor

Sensitive and rapid detection of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was achieved with a newly developed evanescent wave all-fiber immunosensor (EWAI). A reusable functional sensing surface of the immunosensor is prepared by covalent binding of 2,4-D-bovine serum albumin (2,4-D-BSA) conjugate to a self-assembled alkanethiol monolayer formed onto the fiber optic probe through heterobifunctional reagent. The quantification of free 2,4-D in samples was based on indirect competitive immunoreaction principle. Under optimum conditions, calibration curve obtained for 2,4-D had detection limits of 0.07 microg L(-1), the 50% inhibition concentration (IC(50)) was 3.93+/-0.03 microg L(-1) and the quantitative detection range was 0.22-69.5 microg L(-1). The antibodies binding on the sensor surface could be removed simply by the flow of a pepsin solution (pH 1.9), facilitating reuse of the same probe. The regeneration of the sensor surface allowed the performance of more than 100 assay cycles without significant loss of reactivity. The antibody showed negligible cross-reactivity against a few compounds structurally similar to 2,4-D. The immunosensor developed was successfully applied to the monitoring of 2,4-D in spiked water samples without significant effect of the matrix. The proposed portable immunosensor is promising for real-time on-site analysis of small molecules of environmental interest.     

Extremely sensitive and selective antibodies against the explosive 2,4,6-trinitrotoluene by rational design of a structurally optimized hapten

Antibodies are a promising tool for the fast and selective trace detection of explosives. Unfortunately, the production of high-quality antibodies is not trivial and often expensive. Therefore, excellent antibodies are a rare and limiting resource in fields such as biosensing, environmental analysis, diagnostics, cancer therapy, and proteomics. Here, we report the synthesis, bioconjugation, and application of the structurally optimized hapten 6-(2,4,6-trinitro)-phenylhexanoic acid to improve the selectivity and sensitivity of antibodies for the detection of one of the most important explosives, trinitrotoluene. With a conjugate of bovine serum albumin and a highly purified N-hydroxy-succinimide (NHS)-activated hapten, two rabbits were immunized to obtain polyclonal antibodies. The immunization process was monitored by enzyme-linked immunosorbent assay to gain information about the progress of antibody titer and affinity. Finally, the polyclonal antibodies reached an affinity constant of (5.1 ± 0.6) × 10(9) l/mol (rabbit R1) and (2.3 ± 0.2) × 10(9) l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC(50) value) of 0.1 ± 0.01 μg/l (R1) and 0.2 ± 0.02 μg/l (R2) and a working range of 0.005 to 150 μg/l (R1) and 0.007 to 200 μg/l (R2), which corresponds to more than four orders of magnitude for both. This is quite remarkable for a competitive immunoassay, which is often believed to have a narrow dynamic range. The limit of detection was calculated to 0.6 ng/l (R1) and 1.5 ng/l (R2), which is up to 100 times improvement in relation to the assay of Zeck et al. (1999) on the basis of a monoclonal antibody. The excellent selectivity of the polyclonal antibodies was comprehensively examined by determining the cross-reactivity to common explosives and other nitroaromatics including nitro musk components. The widely held belief that polyclonal antibodies generally display higher cross-reactivities than monoclonals could be disproved.     

Production and characterization of a panel of monoclonal antibodies to Toxoplasma gondii antigens

A representative collection was obtained containing 68 monoclonal antibodies (MAB) to Toxoplasma gondii antigens, which was characterized by the binding with the below fractions of tochizoites in the immune-enzyme assay (IEA) and immunoblotting (IB): membrane (MEM), somatic (water-soluble, SOM) and excretory-secretory (ES). Most of MABs were produced to MEM antigens (43), 6 MABs reacted with the somatic fraction, and 3 MABs reacted with both fractions. Two MABs to ES antigen were detected in the latter group. An analysis of MABs in concurrent IEA and IB revealed the immune-dominant proteins of the MEM and SOM fractions of antibodies to T. gondii tochizoites (p30 and p27, respectively). The presence of 2 non-overlapping antigenic determinants was shown for p30. Further research would detect MABs that could be used in the diagnosis of toxoplasmosis.     

An antisense oligodeoxynucleotide-doxorubicin conjugate: preparation and its reversal multidrug resistance of human carcinoma cell line in vitro

An antisense oligodeoxynucleotide-doxorubicin conjugate was synthesized by an aminocaproic acid linker. The synthetic conjugate was identified by HPLC analysis and UV-vis spectra. Properties of the conjugate in vitro conditions were investigated. The results demonstrated that the conjugate was remarkably stabilized by doxorubicin. When incubated in Dulbecco Phosphate-Buflered Saline (pH 7.4) at 37 degrees C, the conjugate was more stable than doxorubicin or the mixture of doxorubicin and antisense oligodeoxynucleotide. When incubated in 10% fetal serum at 37 degrees C, the conjugate showed a remarkable stabilization as compared to the unmodified oligodeoxynucleotide. Melting experiments demonstrated that the covalent attachment of doxorubicin strongly stabilized the binding of the oligodeoxynucleotide to its complementary sequence. In addition, in vitro reversion of multidrug resistance by the conjugate was assayed in a human carcinoma cell line (KB-A-1) resisting to doxorubicin. The result showed that the conjugate displayed very high reversal multdrug resistance activity in KB-A-1 cells in vitro. The conjugate lowered the IC50 value from 21.5 microM to 2.2 microM with a fold-reversal factor of 10. In contrast, a slight decrease of the IC50 value was observed when they combined with the "free" antisense oligodeoxynucleotide: the IC50 value was down from 21.5 microM to 16.8 microM. This study suggested that antisense oligodeoxynucleotide-doxorubicin conjugate might be helpful in multidrug resistance reversal.