• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.527-533
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• 2013

Polyclonal antibodies labeled with a tracer have been commonly used as secondary antibodies in immunochemical assays to quantify the concentration of antibody-antigen complexes. The majority of these antibodies conjugated with a tracer are commercially available, with the exception of few untouched targets. This study focused on the production and application of mouse anti-quail IgY as an intermediate antibody to link between quail egg yolk IgY and goat anti-mouse IgG-HRP as primary and secondary antibodies, respectively. Subsequently, the produced mouse anti-quail IgY was labeled with horseradish peroxidase (HRP) and its efficiency on enzyme linked immunosorbent assay (ELISA) was compared with that of commercial rabbit anti-chicken IgY-HRP. As an intermediate antibody, mouse anti-quail IgY was successfully produced with good affinity and sensitivity (1:10,000) to the primary and secondary antibodies. Subsequently, mouse anti-quail IgY was effectively conjugated with HRP enzyme, resulting in a secondary antibody with good sensitivity (1:10,000) to quail anti-V. parahaemolyticus and V. vulnificus IgY. The detection limit was $10^5$ CFU/ml for both V. parahaemolyticus and V. vulnificus. The efficiency of the produced conjugate to detect quail IgY on ELISA was comparable to that of the commercial rabbit anti-chicken IgY-HRP, and hence the produced and labeled mouse anti-quail IgY-HRP can be used as a secondary antibody to detect any antibody produced in quail.

• Food Science and Biotechnology
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• v.17 no.3
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• pp.547-552
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• 2008

The detection of carbamate (carbofuran, carbaryl, benfracarb, thiodicarb, and methomil) and organophosphate (diazinon, cadusafos, ethoprofos, parathion-methyl, and chlorpyrifos) pesticide residues with very low detection limits was carried out using surface plasmon resonance (SPR) based equipment. The capacity to develop a portable SPR biosensor for food safety was also investigated. The applied ligand for the immunoassays was polyclonal goat anti-rabbit immunoglobulin (IgG) peroxidase conjugate. Concentration tests using direct binding assays showed the possibility of quantitative analysis. For ligand fishing to find a proper antibody to respond to each pesticide, acetylcholinesterase (AChE), and glutathione-S-transferase (GST) were tested. The reproducibility and precision of SPR measurements were evaluated. With this approach, the limit of detection for pesticide residues was 1 ng/mL and analysis took less than 11 min. Thus, it was demonstrated that detecting multi-class pesticide residues using SPR and IgG antibodies provides enough sensitivity and speed for use in portable SPR biosensors.

• Archives of Pharmacal Research
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• v.21 no.2
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• pp.135-139
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• 1998

High titer rabbit polyclonal antibodies (pAbs) which show a specificity for saikosaponin a (SSA), have been generated. The immunogen used was a conjugate of SSA linked through its glucose moiety to bovine serum albumin by periodate oxidation method. The antibody titers obtained from two rabbits, innoculated with the immunogen, reached a plateau after the fourth and third booster injection, respectively. The specificity of the pAbs was determined by hapten inhibition assays using several SSA-like structures. SSA competitively inhibited the binding of the rabbit anti-SSA pAbs to SSA-ovalbumin on solid phase, a coated antigen on the well. The antibodies showed high specificity to SSA, exhibiting no significant cross-reactivity with any of SSA analogues tested.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.2
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• pp.211-218
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• 1993

The effect of active immunization against porcine somatostatin (SRIF-14) on somatostation and somatotropin secretion profile in 18 gilts was investigated. Gilts were assigned to the following treatments: control (sham injection, n = 6); bovine serum albumin (BSA) (injection of BSA with bacterial protein adjuvant, n = 6); SRIF (injection of BSA-SRIF-14 conjugate with bacterial protein adjuvant n = 6). Serum SRIF and pST were assayed from the blood samples taken on day 7 after the last immunization injection. Anti-SRIF antibody titres were assayed in weekly samples two weeks after the initial immunization to one week after the last immunization. Results revealed that the immunization protocol used in the present investigation failed to produce antibodies capable of neutralizing endogenous somatostatin. In addition, the porcine somatotropin assay revealed no significant differences in baseline pST concentration, mean peak amplitude and number of peaks during a 24 h secretory period among SRIF, BSA and control treatment. There were also no differences in SRIF baseline concentration, peak amplitude, and number of peaks during a 24 h secretory period among any of the three treatments. Circulating concentrations of pST and pSRIF were highly correlated (r = -0.09). Furthermore, anti-SRIF antibody titre was not detected in the serum of the gilts actively immunized against SRIF. These data, collectively, suggest that the protocol employed in the present investigation for active immunization against SRIF is not an effective method for changing SRIF and pST secretion profiles of the gilt and thus to enhance performance.

• BMB Reports
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• v.30 no.5
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• pp.326-331
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• 1997

To construct a competitive ELISA standard curve for the detection of glucose-6-phosphate debydrogenase (G6PD), we used highly purified native G6PD (nG6PD) as both immobilized and soluble antigens and anti-G6PD serum raised against nG6PD as antibody. The polystyrene cuvettes coated with nG6PD were challenged with a mixture of a limiting amount of anti-G6PD serum and various doses of nG6PD as competitors followed by incubation with alkaline phosphatase-anti-IgG conjugate. The competitive ELISA did not exhibit the typical sigmoidal dose-response curve characteristic of competition immunoassays under the optimal concentrations of antigen and antibody. The soluble nG6PD used as competitor failed to effectively inhibit the binding of antibodies to the immobilized nG6PD. The addition of NADP, a cofactor of G6PD enzyme, to coating buffer used for immobilizing nG6PD to the cuvettes and PBS-Tween-BSA buffer for diluting competitors did not improve the inhibition of antibody binding to immobilized nG6PD by soluble n/G6PD. The addition of BSA to coating buffer did not increase inhibition, either. Surprisingly, when partially active G6PD (paG6PD), obtained by repeated freeze-thawing, was used as competitor, the antibody binding to either immobilized nG6PD or immobilized paG6PD was inhibited 49-58%. We conclude that an effective competitive ELISA system with nG6PD enzyme and anti-G6PD serum for the detection of G6PD may not be established due to the poor inhibition of antibody binding to immobilized nG6PD by soluble nG6PD under the present assay conditions and that the inhibition may be improved by using an inactivated enzyme as competitor regardless of the type of immobilized antigen used. These results imply that the immobilized nG6PD may undergo denaturation upon binding to the polystyrene cuvettes and that our anti-G6PD serum may recognize denatured enzyme better than active enzyme.

• Korean Journal of Veterinary Research
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• v.29 no.4
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• pp.567-575
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• 1989

This study was conducted to evaluate the possibility of application of a microenzyme-linked immunosorbent assay(micro-ELISA) for the serodiagnosis of specific toxoplasma antibodies in swine sera and this test was performed as a microplate system by coating the polystyrene plates with toxoplasma soluble antigen, incubated serially diluted sera, then added horse radish peroxidase labelled goat anti-swine IgG(r) conjugate followed by o-phenylenediamine as substrate. The color development by enzyme-substrate reaction was determined by the photometric reading [ELISA reader at 490nm (OD)] and visual reading. The soluble antigen was prepared from the tachyzoites in mouse peritoneal cavity. A total of 1,200 swine sera from pig slaughter-house and a total of 116 swine sera from pig breeding station (S-C farm) were tested for the detection of antibodies to Toxoplasma gondii. The results obtained were summarized as follows: 1. The optimal reactions of indirect ELISA for the test sera were determined by the dilution of antigen 1:256 and 1:3,200 of horse radish peroxidase conjugate [anti-swine IgG(r)]. 2. The specific togoplasma antibody(IgG) in pigs infected with Tp artificially were detected as the serum titers of 1:64 or 1:128 at one week postinfection. 3. Of a total of 1,200 swine sera from pig slaughter-house 505 samples of sera were detected as positive (42.1%) and of a total of 116 swine sera from S-C pig breeding station 68 samples of sera as positive (58.6%). 4. The specific antibody(IgG) detection rates against a total of 1,200 test sera from pig slaughter-house were not significant between male (43.1%) and female (40.7%). 5. The indirect ELISA was proved to be a sensitive and specific procedure for the serodiagnosis of swine toxoplasmosis and also evaluated as an effective screening test for the large scale of test samples in laboratory.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.30 no.1
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• pp.47-52
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• 1985

A radioimmunoassay technique has been developed for the quantitative analysis of Abscisic acid (ABA). The antibody, obtained by immunizing rabbits against a conjugate of ABA with human serum albumin, had a high affinity (Ka=3.28x10$\^$13/l/mol) for ABA. The use of $^3$H-labelled ABA as tracer and of dextran-coated charcoal for separation of free ABA from antibody-bound ABA permitted detection of as little as 0.5x10$\^$-12/ mol ABA. The measuring range extended to 14x10$\^$-12/ mol. Because of the high specificity of this immunoassay, no extract purification steps were required prior to analysis. And then, only 2 hr in radioimmunoassay was required to ABA analysis. From these results, it is suggested using this assays that more than hundreds samples can be assayed sensitive and simple per day for ABA.

• Journal of Food Hygiene and Safety
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• v.13 no.2
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• pp.129-134
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• 1998

To develop an enzyme-linked immunosorbent assay (ELISA) for nivalenol (NIV), we produced polyclonal antibodies against tetraacetyl nivalenol (Ac4-NIV) and established ELISA conditions. Ac4-NIV-hemisuccinate conjugated to bovine serum albumin (Ac4-NIV-HS-BSA) was immunized with Freund's adjuvants into rabbits subcutaneously several times. By use of the antiserum showing the highest titer and Ac4-NIV-HS-HRP conjugate, we established competitive direct ELISA (cdELISA). Standard curve of cdELISA showed that the detection range of Ac4-NIV was about 10~5,000 ng/ml (ppb). The cross-reactivities of the polyclonal antibody towards Ac4-NIV and acetyl T-2 were 100 and 70% respectively, and those towards NIV, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, triacetyl deoxynivalenol, fusarenonX, and T-2 were less than 0.1%. When cdELISA was applied to NIV-spiked corns followed by extraction with 70% acetonitrile and acetylation with acetic anhydride in pyridine, the recovery rates of the Ac4-NIV were 108, 143, and 70% (average, 107%) in the levels of 100, 300, and 1,000 ng/g (ppb), respectively.

• Biomedical Science Letters
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• v.4 no.1
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• pp.57-63
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• 1998

The objectives of this study were to produce polyclonal antibody to adipocyte plasma membrane (APM) proteins isolated from pig, and to investigate its tissue specificity. Plasma membrane proteins from adipocyte, brain, heart, kidney, liver and spleen were isolated using a self-forming Percoll gradient. Sheep (40kg) was immunized three times at three week interval with the purified APM proteins. Blood was taken from non-immunized sheep (NS) and from immunized sheep at 10 (AS-1), 12 (AS-2), and 14 (AS-3) days after the third immunization. Antisera titers and cross-reactivity against other tissues were determined by enzyme-linked immunosorbent assay (ELISA). Antisera reacted strongly to APM proteins showing detectable amounts of antibody at 1：81,000 dilution. And antisera showed much stronger reactivity to APM proteins than any other tissue plasma membrane proteins. Furthermore, tissue specificity of antisera against APM was reconfirmed by immunoblotting using anti-sheep immunoglobulin G-horseradish peroxidase conjugate as a secondary antibody Antisera to APM proteins showed adipocyte specificity compared with other tissues. In conclusion, polyclonal antibody against APM proteins isolated from pig was developed successfully in our laboratory, and these antisera showed tissue specificity with APM.

• Bulletin of the Korean Chemical Society
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• v.27 no.3
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• pp.407-412
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• 2006

Bioluminescence single-site immunometric assay for methamphetamine (MA) using the native aequorin, a photoprotein, as a signal generator was developed for the first time. MA is a potent sympathomimetic amine with stimulant effects on the central nervous system. MA abuse induces hallucinations and, thus, may cause a serious social problem. The single-site immunometric MA assay was optimized and its dose-response behavior was examined. The dose-response curve shows that the detection limit is 1.1 ${\times}$ $10^{-10}$ M and a dynamic range is four orders of magnitude with 15 $\mu$g/mL BSA-MA conjugate and 1.0 ${\times}$ $10^{-8}$ M anti-MA antibody-biotin conjugate. In order to evaluate this assay, the structurally similar compounds, amphetamine, ephedrine, norephedrine, benzphetamine and N-4-(aminobutyl)methamphetamine were examined for their crossreactivity. None of these five compounds showed any cross-reactivity. Additionally, an artificial urine solution spiked with MA was analyzed by the MA assay, and the result of the analysis demonstrated the usefulness of the present assay for the determination of MA in urine.