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Storage and stability of IgG and IgM monoclonal antibodies dried on filter paper and utility in Neisseria meningitidis serotyping by Dot-blot ELISA.

Ferraz AS, Belo EF, Coutinho LM, Oliveira AP, Carmo AM, Franco DL, Ferreira T, Yto AY, Machado MS, Scola MC, De Gaspari E - BMC Infect. Dis. (2008)

Bottom Line: The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20 degrees C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper.The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year.The application of meningococcal typing methods and designations depend on the question being asked.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology Section, Adolfo Lutz Institute, São Paulo, São Paulo, Brazil. alineseneme@hotmail.com

ABSTRACT

Background: A simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs) at room temperature or -20 degrees C after spotting on filter paper, for subsequent serotyping of outer membrane antigens of N.meningitidis by dot-blot ELISA.

Methods: Monoclonal antibodies (Mabs) were spotted within a 0.5-1 cm diameter area of Whatman grade 903 paper, which were stored individually at room temperature or at -20 degrees C. These MAbs were stored and analyzed after periods of one week, 4 weeks, 12 months, or 13 years in the case of frozen Mab aliquots, or after 4 weeks at -20 degrees C or at room temperature (RT) in the case of Mabs dried on filter paper strips. Assays were performed in parallel using dot-blot ELISA. In addition to the MAbs specific for serotyping class 1, 2 or 3, we used a larger number of Mabs for polysaccharides, lipooligosaccharides (LOS), class 5 and cross-reactive antigens for native outer membrane of N.meningitidis. The Mabs dried on filter paper were eluted with phosphate-buffered saline (PBS) containing 0.2% gelatin.

Results: Mabs of the isotypes IgG and IgM dried on filter papers were not affected by duration of storage. The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20 degrees C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper.

Conclusion: The use of filter paper is an inexpensive and convenient method for collecting, storing, and transporting Mab samples for serotyping studies. In addition, the samples occupy little space and can be readily transported without freezing. The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year. The application of meningococcal typing methods and designations depend on the question being asked.

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Dot- blot ELISA using monoclonal antibodies for serogroups A, B and C eluted from filter paper stored for one year at -20°C. Dot-blot ELISA results of analysis of 323 case strains of N meningitidis . The strains was dotted (1 μL) as whole cells suspension.A) Reactivity of the Mab WRAIR 2D7B5B5B2 at dilution 1:25,000(serogroup A); B) Reactivity of the Mab WRAIR 5C1-3H7 at dilution 1:50,000 (serogroup B) and C) Reactivity of the Mab WRAIR 7H94 at dilution 1:25,000 (serogroup C).
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Figure 1: Dot- blot ELISA using monoclonal antibodies for serogroups A, B and C eluted from filter paper stored for one year at -20°C. Dot-blot ELISA results of analysis of 323 case strains of N meningitidis . The strains was dotted (1 μL) as whole cells suspension.A) Reactivity of the Mab WRAIR 2D7B5B5B2 at dilution 1:25,000(serogroup A); B) Reactivity of the Mab WRAIR 5C1-3H7 at dilution 1:50,000 (serogroup B) and C) Reactivity of the Mab WRAIR 7H94 at dilution 1:25,000 (serogroup C).

Mentions: The results of the serological tests performed with ascites or culture supernatant samples and dried Mab spots on paper kept at -20°C or at room temperature are presented in Tables 1, 2, 3, 4. The titers were mostly equal and the differences did not alter the final results when the dried Mab spots were stored at -20°C or at room temperature for a period of one year. After 13 years storage, the titers of IgG and IgM antibody isotypes did not interfere with the specificity (Figure 1) of the antibodies when analyzed by dot-blot ELISA. However, none of the differences resulted in a change in serotyping with epidemic strains when we used Mabs of serogroups A, B or C eluted after one year at -20°C (Figure 1). We also included in assays control culture supernatant lacking antibodies to N.meningitidis to look for false positives. In all cases where Mab samples were antibody-negative, the corresponding Mab samples dried on dot-Blot ELISA paper tested negative after one week, 4 weeks, 1 year or 13 years of storage at -20°C. Most of the Mabs used in the present investigation are included at the website of, (University of Oxford, UK) and were produced by Dr.W.D Zollinger and Dr. J.T Poolman. Comparison between filter paper Mab and freezer Mab can be seen in Tables 1, 2, 3, 4. The application of these MAbs for serogroup identification of meningococci was demonstrated by their abilities to correctly identify 323 clinical isolates in Brazil by dot blot-ELISA (Figure 1). Serogrouping of N.meningitidis (or meningococci) is important because the disease caused by some serogroups can be prevented by active immunization [24].


Storage and stability of IgG and IgM monoclonal antibodies dried on filter paper and utility in Neisseria meningitidis serotyping by Dot-blot ELISA.

Ferraz AS, Belo EF, Coutinho LM, Oliveira AP, Carmo AM, Franco DL, Ferreira T, Yto AY, Machado MS, Scola MC, De Gaspari E - BMC Infect. Dis. (2008)

Dot- blot ELISA using monoclonal antibodies for serogroups A, B and C eluted from filter paper stored for one year at -20°C. Dot-blot ELISA results of analysis of 323 case strains of N meningitidis . The strains was dotted (1 μL) as whole cells suspension.A) Reactivity of the Mab WRAIR 2D7B5B5B2 at dilution 1:25,000(serogroup A); B) Reactivity of the Mab WRAIR 5C1-3H7 at dilution 1:50,000 (serogroup B) and C) Reactivity of the Mab WRAIR 7H94 at dilution 1:25,000 (serogroup C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2311311&req=5

Figure 1: Dot- blot ELISA using monoclonal antibodies for serogroups A, B and C eluted from filter paper stored for one year at -20°C. Dot-blot ELISA results of analysis of 323 case strains of N meningitidis . The strains was dotted (1 μL) as whole cells suspension.A) Reactivity of the Mab WRAIR 2D7B5B5B2 at dilution 1:25,000(serogroup A); B) Reactivity of the Mab WRAIR 5C1-3H7 at dilution 1:50,000 (serogroup B) and C) Reactivity of the Mab WRAIR 7H94 at dilution 1:25,000 (serogroup C).
Mentions: The results of the serological tests performed with ascites or culture supernatant samples and dried Mab spots on paper kept at -20°C or at room temperature are presented in Tables 1, 2, 3, 4. The titers were mostly equal and the differences did not alter the final results when the dried Mab spots were stored at -20°C or at room temperature for a period of one year. After 13 years storage, the titers of IgG and IgM antibody isotypes did not interfere with the specificity (Figure 1) of the antibodies when analyzed by dot-blot ELISA. However, none of the differences resulted in a change in serotyping with epidemic strains when we used Mabs of serogroups A, B or C eluted after one year at -20°C (Figure 1). We also included in assays control culture supernatant lacking antibodies to N.meningitidis to look for false positives. In all cases where Mab samples were antibody-negative, the corresponding Mab samples dried on dot-Blot ELISA paper tested negative after one week, 4 weeks, 1 year or 13 years of storage at -20°C. Most of the Mabs used in the present investigation are included at the website of, (University of Oxford, UK) and were produced by Dr.W.D Zollinger and Dr. J.T Poolman. Comparison between filter paper Mab and freezer Mab can be seen in Tables 1, 2, 3, 4. The application of these MAbs for serogroup identification of meningococci was demonstrated by their abilities to correctly identify 323 clinical isolates in Brazil by dot blot-ELISA (Figure 1). Serogrouping of N.meningitidis (or meningococci) is important because the disease caused by some serogroups can be prevented by active immunization [24].

Bottom Line: The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20 degrees C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper.The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year.The application of meningococcal typing methods and designations depend on the question being asked.

View Article: PubMed Central - HTML - PubMed

Affiliation: Immunology Section, Adolfo Lutz Institute, São Paulo, São Paulo, Brazil. alineseneme@hotmail.com

ABSTRACT

Background: A simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs) at room temperature or -20 degrees C after spotting on filter paper, for subsequent serotyping of outer membrane antigens of N.meningitidis by dot-blot ELISA.

Methods: Monoclonal antibodies (Mabs) were spotted within a 0.5-1 cm diameter area of Whatman grade 903 paper, which were stored individually at room temperature or at -20 degrees C. These MAbs were stored and analyzed after periods of one week, 4 weeks, 12 months, or 13 years in the case of frozen Mab aliquots, or after 4 weeks at -20 degrees C or at room temperature (RT) in the case of Mabs dried on filter paper strips. Assays were performed in parallel using dot-blot ELISA. In addition to the MAbs specific for serotyping class 1, 2 or 3, we used a larger number of Mabs for polysaccharides, lipooligosaccharides (LOS), class 5 and cross-reactive antigens for native outer membrane of N.meningitidis. The Mabs dried on filter paper were eluted with phosphate-buffered saline (PBS) containing 0.2% gelatin.

Results: Mabs of the isotypes IgG and IgM dried on filter papers were not affected by duration of storage. The detection by serotyping Mabs was generally consistent for dried filter paper MAb samples stored frozen for over 1 year at -20 degrees C, and although decreased reactive antibody titers were found after storage, this did not interfere with the specificity of the Mabs used after 13 years as dry spots on filter paper.

Conclusion: The use of filter paper is an inexpensive and convenient method for collecting, storing, and transporting Mab samples for serotyping studies. In addition, the samples occupy little space and can be readily transported without freezing. The efficiency of using immunoglobulin G (IgG) or M (IgM) eluted was found to be consistent with measurement of IgG or IgM titers in most corresponding, ascites Mabs stored frozen for over 1 year. The application of meningococcal typing methods and designations depend on the question being asked.

Show MeSH
Related in: MedlinePlus