Production and Characterization of Antibodies against Fumonisin B1

1996 ◽  
Vol 59 (9) ◽  
pp. 992-997 ◽  
Author(s):  
FENG-YIR YU ◽  
FUN S. CHU
Keyword(s):  
Detection Limit ◽  
Polyclonal Antibodies ◽  
Fumonisin B1 ◽  
Correlation Coefficients ◽  
Keyhole Limpet Hemocyanin ◽  
Hplc Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Indirect Competitive Elisa ◽  
Antibody Titers

Polyclonal antibodies against fumonisin B1 (FmB1) were produced in rabbits after immunizing the animals with either FmBl-keyhole limpet hemocyanin (KLH) or FmB1 bovine serum albumin (BSA). A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were used for the characterization of the antibodies and for analysis of the toxin in corn samples. The antibody titers in the serum of rabbits immunized with FmBl-KLH were considerably higher than in those immunized with FmBl-BSA. The antibodies from the rabbits immunized with FmBl-KLH were further characterized. The concentrations causing 50% inhibition of binding of FmB1-horseradish peroxidase (HRP) to the antibodies by FmB1, FmB2 and FmB3 in the ELISA were found to be 0.45, 0.72, and 25 ng/ml, respectively. The detection limit of FmBl, based on 95% confidence at 5% of inhibition of binding of FmBl-HRP conjugate, in buffer of the dc-ELISA was found to be 0.05 ng/ml. In the presence of a matrix such as corn, the detection limit was less than 50 ppb. The overall analytical recoveries of FmBl (50 to 1,000 ng/g) added to the ground corn and then extracted with CH3CN/H2O (1/1, vol/vol) with cleanup and without cleanup in the dc ELISA were found to be 70.5 and 85.9%, respectively. A good correlation was found between the FmBl levels in 2 starch and 10 naturally contaminated corn samples analyzed by the dc-ELISA and the high-pressure liquid chromatography (HPLC) method. The correlation coefficients between ELISA and HPLC were found to be 0.955 (y [ELISA] = 1.3 1x [HPLC] + 77 ppb; P < 0.001) and 0.811 (y = 1.13x + 34 ppb; P < 0.01) for the sample without and with cleanup treatment, respectively.

scholarly journals Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50

2006 ◽  
Vol 72 (12) ◽  
pp. 7634-7643 ◽  
Author(s):  
Raquel Criado ◽  
Jorge Gutiérrez ◽  
María Martín ◽  
Carmen Herranz ◽  
Pablo E. Hernández ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Polyclonal Antibodies ◽  
Keyhole Limpet Hemocyanin ◽  
Enzyme Linked Immunosorbent Assay ◽  
C Terminus ◽  
Bacteriocin Producer ◽  
Agric Food ◽  
Different Temperatures ◽  
Enterocin P

ABSTRACT Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47°C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32°C, but production becomes negligible when the growth temperature is above 37°C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47°C. Maximum EntL50A and EntL50B production was detected at 25°C, while EntP and EntQ are maximally produced at 37 and 47°C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.

Polyclonal Antibody-Based ELISA and HPLC Methods for the Determination of Fumonisins in Corn: A Comparative Study

1996 ◽  
Vol 59 (8) ◽  
pp. 893-897 ◽  
Author(s):  
ERIC W. SYDENHAM ◽  
SONJA STOCKENSTRÖM ◽  
PIETER G. THIEL ◽  
JOHN P. RHEEDER ◽  
M. BRUNO DOKO ◽  
...  
Keyword(s):  
Comparative Study ◽  
Polyclonal Antibody ◽  
High Performance ◽  
Linear Regression Analysis ◽  
Correlation Coefficients ◽  
Hplc Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Regression Slopes ◽  
The Individual ◽  
The Comparative Study

The performance of an experimental polyclonal antibody (PAb)-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) developed for the analysis of fumonisins in corn was assessed by comparison with an established high-performance liquid chromatography (HPLC) method. The comparative study was conducted using a series of 20 corn samples naturally contaminated with combined fumonisin levels ranging from <0.05 to >5 μg/g (ppm). Linear regression analysis between the results generated by HPLC and CD-ELISA provided correlation coefficients (r) and regression slopes (b) of r = 0.960, b = 1.493 (P < 0.001); r = 0.865, b = 3.903 (P < 0.001); and r = 0.832, b = 0.107 (P < 0.001) for the individual fumonisins B1 (FB1), B2 (FB2) and B3 (FB3), respectively, while corresponding values of r = 0.967, b = 1.059 (P < 0.001) were obtained for the combined FB1, FB2, and FB3 concentrations. In 3 of 18 fumonisin-positive corn samples, combined fumonisin levels determined by CD-ELISA were between 85 and 100% higher than those determined in the same extracts by HPLC, while in 13 other samples, CD-ELISA results were between 1.8 and 53% higher than those determined by HPLC. Conversely, in 2 of 18 samples, CD-ELISA results were lower than those determined by HPLC. The differences recorded between HPLC and the experimental PAb-based CD-ELISA were far less than those previously recorded for other mono- and polyclonal antibody-based CD-ELISA systems. The results indicate that the experimental PAb-based CD-ELISA may be effectively applied for the initial screening for fumonisins in corn.

scholarly journals Pneumococcal Type 22F Polysaccharide Absorption Improves the Specificity of a Pneumococcal-Polysaccharide Enzyme-Linked Immunosorbent Assay

2001 ◽  
Vol 8 (2) ◽  
pp. 266-272 ◽  
Author(s):  
Nelydia F. Concepcion ◽  
Carl E. Frasch
Keyword(s):  
Correlation Coefficient ◽  
Immunosorbent Assay ◽  
Pneumococcal Vaccination ◽  
Correlation Coefficients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Functional Activities ◽  
Antibody Titers ◽  
The Common ◽  
Elisa Inhibition

ABSTRACT The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.

Development of Enzyme Immunoassay for Captan and Its Degradation Product Tetrahydrophthalimide in Foods

1993 ◽  
Vol 76 (2) ◽  
pp. 381-386 ◽  
Author(s):  
W Harvey Newsome ◽  
Jupiter M Yeung ◽  
Peter G Collins
Keyword(s):  
Detection Limit ◽  
Human Serum Albumin ◽  
Degradation Product ◽  
Protein Concentration ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Compound ◽  
Coefficients Of Variation ◽  
Spacer Arm ◽  
Related Compounds

Abstract A simple, sensitive, and precise enzyme-linked immunosorbent assay (ELISA) is described for the quantitation of captan as its degradation product tetrahydrophthalimide (THPI) in foods using polyclonal antibodies. Three hapten analogues of THPI with different alky I spacer arm lengths were synthesized. Immunogens and coating proteins were prepared by coupling these haptens to human serum albumin and ovalbumin, respectively. A 5-carbon spacer arm appeared to be optimum for the production of antibodies. Heterologous coating proteins did not improve the sensitivity, but reduction of homologous coating protein concentration did improve the sensitivity, resulting in a concentration of test compound required to inhibit binding by 50% of 15.5 ng/mL The antiserum is specific for captan, captafol, and THPI, but not other structurally related compounds. The minimum detection limit was 1 ng/mL; the linearity was 1-200 ng/mL. The overall recoveries of captan and THPI from 11 commodities spiked at 4 levels were 92 and 100%, respectively. The intra-assay and interassay coefficients of variation were 9.1 and 16.8% for apple blanks and 5.9 and 4.2% for apple spiked with 3 ppm THPI, respectively. The ELISA described is suitable for measuring captan and THPI at levels comparable to those typically found in fruit.

scholarly journals A Modified Enzyme-Linked Immunosorbent Assay for Measurement of Antibody Responses to Meningococcal C Polysaccharide That Correlate with Bactericidal Responses

1998 ◽  
Vol 5 (4) ◽  
pp. 479-485 ◽  
Author(s):  
Dan M. Granoff ◽  
Susan E. Maslanka ◽  
George M. Carlone ◽  
Brian D. Plikaytis ◽  
George F. Santos ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Geometric Mean ◽  
Correlation Coefficients ◽  
Antibody Responses ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Avidity ◽  
Igg Antibody ◽  
Standard Assay ◽  
Bactericidal Antibody ◽  
Antibody Titers

ABSTRACT The standardized enzyme-linked immunosorbent assay (ELISA) for measurement of serum immunoglobulin G (IgG) antibody responses to meningococcal C polysaccharide has been modified to employ assay conditions that ensure specificity and favor detection primarily of high-avidity antibodies. The modified and standard assays were used to measure IgG antibody concentrations in sera of toddlers vaccinated with meningococcal polysaccharide vaccine or a meningococcal C conjugate vaccine. The results were compared to the respective complement-mediated bactericidal antibody titers. In sera obtained after one or two doses of vaccine, the correlation coefficients, r, for the results of the standard assay and bactericidal antibody titers were 0.45 and 0.29, compared to 0.85 and 0.87, respectively, for the modified assay. With the standard assay, there were no significant differences between the geometric mean antibody responses of the two vaccine groups. In contrast, with the modified assay, 5- to 20-fold higher postvaccination antibody concentrations were measured in the conjugate than in the polysaccharide group. Importantly, the results of the modified assay, but not the standard ELISA, paralleled the respective geometric mean bactericidal antibody titers. Thus, by employing conditions that favor detection of higher-avidity IgG antibody, the modified ELISA provides results that correlate closely with measurements of antibody functional activity that are thought to be important in protection against meningococcal disease.

scholarly journals Production of Monoclonal Antibodies Specific for the i and 1,2 Flagellar Antigens of Salmonella typhimurium and Characterization of Their Respective Epitopes

1998 ◽  
Vol 64 (12) ◽  
pp. 5033-5038 ◽  
Author(s):  
N. de Vries ◽  
K. A. Zwaagstra ◽  
J. H. J. Huis in’t Veld ◽  
F. van Knapen ◽  
F. G. van Zijderveld ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Salmonella Typhimurium ◽  
Immunosorbent Assay ◽  
Specific Antibody ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Minimal Area

ABSTRACT Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC(H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

Production and Characterization of Antibodies Against Neosaxitoxin

1992 ◽  
Vol 75 (2) ◽  
pp. 341-345 ◽  
Author(s):  
Fun S Chu ◽  
Xuan Huang ◽  
Sherwood Hall
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Antibody ◽  
Antibody Titers ◽  
Antibody Elisa

Abstract Antibody against neosaxitoxin (neo-STX) was obtained from rabbits after immunization with neo- STX conjugated to either keyhole limpet hemocyanln (KLH) or bovine serum albumin (BSA). An indirect enzyme-linked Immunosorbent assay (ELISA), In which either neo-STX-BSA or neo-STXKLH was coated to the mlcroplate, was used to monitor the antibody titer. Although high antibody titers were obtained from rabbits after immunization with both immunogens, only antibody obtained from rabbits immunized with neo-STX-KLH was useful for Immunoassay. Competitive indirect ELISA revealed that the antibodies obtained from rabbits Immunized with neo-STX-KLH are specific for neo-STX but also have good cross-reactivity with STX. The concentrations causing 50% inhibition binding of neo-STX-BSA to the anti-neo-KLH by neo-STX, STX, and decarbamoyl-STX (DC-STX) were 0.9,8.0, and 53.1 ng/mL, respectively. Saxitoxin conjugated to polylyslne (STX-PLL) was also used as the coating reagent In the indirect ELISA. The concentrations causing 50% inhibition binding of antl-neo-STX-KLH to STX-PLL coated on the mlcrotiter plate by neo-STX, STX, and DC-STX were 1.2,4.1, and 36.1 ng/mL, respectively. With this newly developed antibody, ELISA could be a very effective method for monitoring seafood for both neo-STX and STX.

Production and Characterization of Monoclonal Antibodies against Acridine Ester

2014 ◽  
Vol 1010-1012 ◽  
pp. 101-106
Author(s):  
Li Xin Zhu ◽  
Yan Fan ◽  
Xue Mei Qiu ◽  
Ren Rong Liu ◽  
Long Xu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Keyhole Limpet Hemocyanin ◽  
Absorption Method ◽  
Weakly Alkaline ◽  
Indirect Competitive Elisa ◽  
Traditional Procedure ◽  
Inhibition Concentration ◽  
Alkaline Conditions

AE (Acridine ester) artificial antigens were obtained by acridine ester coupled with KLH (keyhole limpet hemocyanin) and BSA (bovine serum albumin) in weakly alkaline conditions, respectively. These two kinds of antigens were identified by the ultraviolet absorption method, which showed that they were synthesized successfully. Balb/c mice were immunized with the AE-KLH for producing the McAb (monoclonal antibody) against AE, and then cell fusion was used. Two monoclonal antibodies against AE were acquired according to traditional procedure. One of anti-AE antibodies produced from the 1E8 cell lines was the IgG3 subclass with a kappa-type light chain. An indirect competitive ELISA assay was developed. A linear relationship was observed over the concentration range from 0.25 to 10 ng/mL, and the detection limit was 0.125 ng/mL. The 50% inhibition concentration was 2.3 ng/mL. The monoclonal antibody against AE was successfully obtained.

scholarly journals Characterization of Serological Responses to Pertussis

2006 ◽  
Vol 13 (3) ◽  
pp. 341-348 ◽  
Author(s):  
Mineo Watanabe ◽  
Beverly Connelly ◽  
Alison A. Weiss
Keyword(s):  
Adenylate Cyclase ◽  
Serological Diagnosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vaccine Antigens ◽  
History Of ◽  
Antibody Titers ◽  
Iga Antibody ◽  
Conventional Vaccine ◽  
Culture Positive

ABSTRACT We have compared the use of five nonvaccine antigens to the use of conventional vaccine antigens, pertussis toxin (PT), and filamentous hemagglutinin (FHA) for the serological diagnosis of pertussis by enzyme-linked immunosorbent assay (ELISA). The nonvaccine antigens included the catalytic region of adenylate cyclase toxin (CatACT), the C-terminal region of FHA (C-FHA), lipooligosaccharide (LOS), the peptidoglycan-associated lipoprotein (PAL), and the BrkA protein. The serological responses of individuals with culture-confirmed pertussis were compared to those of adults with no recent history of a coughing disease. An immunoglobulin G (IgG) ELISA for PT was the most sensitive (92.2%) test for the serodiagnosis of pertussis. Of the nonvaccine antigens, ELISA for IgG responses to CatACT (sensitivity, 62.8%), C-FHA (sensitivity, 39.2%), and LOS IgA (sensitivity, 29.4%) were less sensitive but could also distinguish culture-positive individuals from control individuals. The use of a combination of multiple ELISA targets improved the sensitivity of the assay for serological diagnosis. Elevated IgG and IgA antibody titers persisted for more than a year in the individuals with culture-confirmed pertussis.

Development of an Analytical Protocol for the Determination of Polybrominated Biphenyls in Water by Enzyme-Linked Immunosorbent Assay

2011 ◽  
Vol 347-353 ◽  
pp. 1537-1541 ◽  
Author(s):  
Han Yu Chen ◽  
Hui Sheng Zhuang
Keyword(s):  
Detection Limit ◽  
Quantitative Determination ◽  
Incubation Time ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Concentration ◽  
Polybrominated Biphenyls ◽  
Analytical Protocol ◽  
Optimized Conditions

A novel immunoassay has been developed for the quantitative determination of polybrominated biphenyls (PBBs) using indirect competitive format. A new method was developed to synthesize PBBs congener (PCB15) hapten and its artificial immunogen, then the polyclonal antibodies. The assay was optimized concerning the coating conjugate and antibody concentration, incubation time and temperature, the tolerance to organic solvents and so on. Under optimized conditions, PBB15 can be determined in the concentration range of 0.01-100 μg L-1 with a detection limit of 0.02 μg L-1. The cross-reactivities of the assays were below 8%. While water samples could be analyzed directly.

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