Article

AFM study of complement system assembly initiated by antigen-antibody complex. Centr Eur J Chem 4:194-206

Sector of Immunoanalysis and Informatics, Institute of Immunology, Vilnius University, Moletu pl. 29, 08409 Vilnius 21, Lithuania
Central European Journal of Chemistry (Impact Factor: 1.33). 02/2006; 4(1):194-206. DOI: 10.1007/s11532-005-0015-8

ABSTRACT

The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM).
The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab
complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and
30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation
of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information
that can be used to complement other well-established techniques.

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    • "The TIRE experimental set-up; PMT – photomultiplier tube, BK7 – glass prism, 11-MUA – SAM of mercaptoundecanoic acid, EDC-NHS – antibody linking groups that were formed by the EDC/NHS activation, frag-Ab – fragmented-antibody based layer, intact-Ab – intact-antibody based layer, Ag – antigen involved in the formation of the Ab–Ag complex. crystal microbalance [24] [25], atomic force microscopy (AFM) [26] [27], capacitive micromachined ultrasound transducer (cMUT) [28] and some others were used to show the advantages in application of frag-Ab vs. randomly oriented intact-antibodies [29] [30] [31]. "
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    ABSTRACT: a b s t r a c t Total internal reflection ellipsometry (TIRE) technique was used to investigate biological recognition lay-ers of immunosensors in order to estimate orientation of immobilized intact-and fragmented-antibodies. Two differently prepared biological recognition layers formed on gold-substrate were investigated: one layer was based on intact-antibodies (intact-Ab), second layer was based on chemically fragmented specific-antibodies (frag-Ab), which were obtained by reduction of intact-Ab. It was shown that TIRE enables to resolve differences in nanostructures of intact-Ab and frag-Ab layers. A multilayer model applied in our calculations shows that the distance between fragmented-antibody active sites and gold-surface after the immobilization process is lower than theoretical dimension of fragmented-antibodies. Moreover, it was calculated that analytical sensitivity of the () parameter of TIRE was 5.89 times better if compared to the sensitivity of the « () parameter, which is in fact similar to the sensitivity of surface plasmon resonance (SPR) based immunosensors.
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    • "In present study we have applied the BLV virus proteins [16] and antibodies against these proteins [31] as well as HRP-labeled antibodies against gp51/anti-gp51-Ab as a model system [32]. Before this study, the interactions of the same set of affinity agents were investigated by mean of atomic force microscopy [31], by constantpotential voltammetry [32]. The major biosensing and reference layer preparation protocols, treatments with BLV infected and not infected bovine blood samples, as well as affinity-based interactions, were the same as in previously published study based on detection of fluorescence of HRP-labeled antibodies, which were quenched by Ppy if these antibodies were nonspecifically adsorbed on the Ppy [33]. "
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    ABSTRACT: Polypyrrole (Ppy) has been shown as a matrix for label-free electrochemical immunosensor based on electrochemical impedance spectroscopy (EIS) measurements. The immunosensing system model presented here was based on bovine leukemia virus (BLV) protein (gp51) entrapped within electrochemically-synthesized polypyrrole (Ppy/gp51). This Ppy/gp51 layer interacted with antibodies against gp51 (anti-gp51-Ab) that are present in significant concentration in the blood serum of BLV infected cattle. After this interaction protein complex (Ppy/gp51/anti-gp51-Ab) was formed. The horseradish peroxidase (HRP) labeled secondary antibodies (Ab⁎) against anti-gp51-Ab were applied as agents interacting with Ppy/gp51/anti-gp51-Ab and forming the large protein complex (Ppy/gp51/anti-gp51-Ab/Ab⁎). The EIS study was performed for electrodes modified with different Ppy layers described here and an optimal equivalent circuit was adopted for evaluation of EIS spectra, it was a major outcome of this study.
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    ABSTRACT: The atomic-force microscopy-based method of irreversible chemical AFM-fishing (AFM-IFchem) has been developed for the detection of proteins existing at ultra-low concentrations in solution. Using this method, a very low concentration of horseradish peroxidase (HRP) protein (10−17 M) was detected in solution. A theoretical model that allows the description of obtained experimental data has been proposed. This model takes into consideration both transport of the protein from the bulk solution onto the AFM-chip sur-face and its irreversible binding to the activated area.
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Questions & Answers about this publication

  • Arunas Ramanavicius added an answer in Transmission Electron Microscopy (TEM):
    TEM images for protein complex
    I would like to take TEM images for protein complex , complex size is around 85 Kd , Could you please let me know that what is size limitation for protein complex to take TEM images ? please site the any good reference if you have it ? Thanking for your kindly help in advance !
    Arunas Ramanavicius
    In addition some AFM images, probably they will be interesting in this topic. For more detail read attached publication.
    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM). The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and 30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information that can be used to complement other well-established techniques.
      Full-text · Article · Feb 2006 · Central European Journal of Chemistry
  • Arunas Ramanavicius added an answer in Bioinformatics and Computational Biology:
    What are tools available to check the accuracy of model protein structure?
    I wish to know the details on how to rate the structure modeled using modeller.
    Arunas Ramanavicius
    AFM could be applied for some visualizations, for more detail read attached publication:
    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM). The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and 30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information that can be used to complement other well-established techniques.
      Full-text · Article · Feb 2006 · Central European Journal of Chemistry
  • Arunas Ramanavicius added an answer in Protein NMR:
    How to calculate the number of water molecules present on the binding interface of a protein complex ?
    I would like to calculate the number of water molecules replaced before and after complex formation on the binding surface? How we can do this? Any suggestions?
    Arunas Ramanavicius
    If YOU have the structure YOU can try to apply some programs of "Molecular Modeling", otherwise YOU can to try somehow to estimate from, SPR, QCM and AFM.
    There is one article attached:
    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM). The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and 30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information that can be used to complement other well-established techniques.
      Full-text · Article · Feb 2006 · Central European Journal of Chemistry
  • Arunas Ramanavicius added an answer in Biomaterials:
    Detection of peptides immobilized onto polymer surfaces
    I'm planning to introduce short peptide sequences (e.g. RGD) onto the surface of polymer material (polyurethane). Could anyone suggest some techniques to confirm the succesful immobilization of the peptide?
    Arunas Ramanavicius
    AFM could be applied, e.g.:
    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM). The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and 30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information that can be used to complement other well-established techniques.
      Full-text · Article · Feb 2006 · Central European Journal of Chemistry
  • Arunas Ramanavicius added an answer in Polymer Nanocomposites:
    What is the best way to get silica surface modification efficiency with silane modifiers?
    silica nanoparticles: aerosil 200, silane modifiers: APTES
    Arunas Ramanavicius
    In this publication one option for silanization, which further is applied for immobilization of biomolecules is presented:
    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM). The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and 30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information that can be used to complement other well-established techniques.
      Full-text · Article · Feb 2006 · Central European Journal of Chemistry
  • Almira Ramanaviciene added an answer in Nanobiotechnology:
    How to functionalize Sio2 with strepatvidin. Any method other than EDC/NHS coupling?
    We are trying to functionalize nanooxides with streptavidin to use them for detection.
    Almira Ramanaviciene
    For such purpose we performed silanization, read this publication:
    • Source
      [Show abstract] [Hide abstract]
      ABSTRACT: The shape and size of complement system C1 components assembled on a SiO2 surface after classical activation by antigen-antibody complex was determined by tapping mode atomic force microscopy (AFM). The SiO2 substrate was silanized and bovine leukemia virus proteins gp51 were covalently bound to the SiO2 substrate. Self-assembly of complement system proteins was investigated by AFM. Uniform coating of silanized surface by gp51 proteins was observed by AFM. After incubation of gp51 coated substrate in anti-gp51 antibody containing solution, Ag-Ab complexes were detected on the substrate surface by AFM. Then after treatment of Ag-Ab complex modified substrate by guinea-pig blood serum containing highly active complement system proteins for 3 minutes and 30 minutes features 2–3 times and 5–8 times higher in diameter and in height if compared with those observed after formation of Ag-Ab complex, were observed respectively on the surface of SiO2. This study revealed that AFM might be applied for the imaging of complement system assembly and provides valuable information that can be used to complement other well-established techniques.
      Full-text · Article · Feb 2006 · Central European Journal of Chemistry