Affinity binding studies of anti-3-nitro tyrosine antibody to nitrated peptides by

The purpose of this study was the detection and identification of possible cystic fibrosis sputum protein tyrosine nitration. In this study two anti-3-nitro tyrosine antibodies were used, a) monoclonal antibody (MAB5404) (Chemicon International) and, b) Mouse Monoclonal Nitrotyrosine (39B6) (Santa Cruz Biotechnology) by dot blot experiments using nitro-peptides.

Before describing the antibody specificity a brief consideration of antibody structure and function is necessary. Antibodies are the secreted form of the B-cell receptor. An antibody is identical to the B-cell receptor of the cell that secretes it except for a small portion of the C-terminus of the heavy-chain constant region. In the B-cell receptor the C-terminus is a hydrophobic membrane-anchoring sequence, while in antibody it is a hydrophilic sequence that allows secretion. Since they are soluble, and secreted in large quantities, antibodies are easily obtainable and easily studied. For this reason, most of what is know about the B-cell receptor comes from the study of antibodies.

Antibody molecules are roughly Y-shaped molecules consisting of three equal-sized portions, loosely connected by a flexible tether. The two arms of the Y end in regions that vary between different antibody molecules are the V regions.

These are involved in antigen binding, whereas the stem of the Y, or the C region, are far less variable and they are parts that interact with effector cells and molecules (Figure 2.48). All antibodies are constructed in the same way from paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins. Within this general category, however, five different classes of immunoglobulins, IgM, IgD, IgG, IgA, and IgE can be distinguished by their C regions. More subtle differences confined to the V region account for the specificity of antigen binding. IgG antibodies are large molecules, having a molecular weight of approximately 150 kDa, composed of two different kinds of polypeptide chain. One, of approximately 50 kDa, is termed the heavy or H chain, and the other, of 25 kDa, is termed the light or L chain.

Figure 2.48. General scheme of an antibody showing the constant and variable regions together with the light and heavy chain. Disulfide bonds are links the different heavy and light chains (www.abcam.com)

The antigen binding domain of the molecule is highly variable and, therefore it is referred to as the variable (V) domain, while the remainder of the molecule is a relatively constant sequence and, therefore referred to as the constant regions. The complementary determining regions CDRs confer the specificity (recognition) and the affinity binding of the antibodies for their target antigens [206]. The constant region of both the H and L chains can consist of several distinct classes. Light chains are composed of 220 amino acid residues, while heavy chains are composed of 440-550 amino acids. The heavy and light chains are linked by cysteine disulfide bonds [207].

Antigen-antibody complexes are non-covalent, formed by electrostatic interactions, hydrogen bonds, hydrophobic and Van der Waals forces contributing to the binding specificity. There are two categories of antibodies: polyclonal and monoclonal.

Polyclonal antibodies are produced in response to a vaccine, which represents multiple clones of B-lymphocytes in response to an antigen with each clone produces an antibody molecule with a slightly different specificity and affinity [208]. Monoclonal antibodies are produced by a single clone of antibody-producing cells that is a tumor cell line (myeloma or hybridoma). The high degree of specificity of monoclonal antibodies and their specific uniformity make them ideal for pharmaceutical

applications [209]. Both anti-3-nitro tyrosine antibodies were monoclonal. The characteristics of anti-3-nitro tyrosine antibodies are summarized in Table 2.7.

Table 2.7. Characteristics of monoclonal antibodies against anti-3-nitro tyrosine

Supplier Antibody details

Chemicon International (1) Mouse Anti-Nitrotyrosine Monoclonal Antibody (MAB5404) Concentration: 1 µg/µl

Immunogen: Nitrated KLH (Keyhole-limpet hemocyanin) Santa Cruz (2) Mouse Monoclonal Nitrotyrosine (39B6)

Concentration: 0.2 µg/µl

Immunogen: 3-(2-(4-hydroxy-3-nitrophenyl)acetamido) propionic acid-bovine serum albumin conjugate

In order to test the specificity of the antibodies, nitrated vs. non-modified peptides were used. In addition to these two peptides a third nitrated peptide having adjacent amino acids replaced by alanine was tested to evaluate the importance in binding of the neighbouring amino acid residues. The tested peptides are shown in Table 2.8.

Table 2.8. The characteristics of PCS peptides used for testing the anti-3-nitro tyrosine antibody (MAB5404) specificity

No. Peptide code Sequence MALDI-TOF^c m/z [M + H]⁺calc/exp

Dot blot Affinity MS

1 PCS-Y^a DFYKDGKRLKNYSL-OH 1746.91/1747.72 - -

2 PCS-NO2a DFYKDGKRLKNY(NO2 )SL-OH

1791.90/1792.96 + +

3

PCS-NO2(RR)^b DFY(NO2

)KDGRRLKNYSL-OH 1819.90/1822.13 - 0

a Tyr-430 of Prostacycline synthase (PCS)

b Tyr-421 of Prostacycline synthase, Lys⁴²⁵ was replaced by an Arg

c MALDI-TOF mass spectra were recorded with a Bruker Biflex^TM linear TOF mass spectrometer 0 – not tested

+ positive response (affinity binding to anti-3-nitro tyrosine antibody (MAB 5404)) Negative response (no affinity to the anti-3-nitro tyrosine antibody).

The key feature of Dot blotting is the use of immunodetection to identify a specific protein, such as a protein marker for a disease. Once the proteins/peptides are immobilized on a protein binding membrane, they can be probed with a primary antibody; ie. an antibody specific for the protein of interest. Once bound the antibody is visualized, either with a specific tag coupled to the primary antibody or with a

secondary antibody. The secondary antibody is a general antibody that recognizes the constant domain of immunoglobulin G and it is species specific. If the primary antibody is a mouse antibody, the secondary antibody used will recognize all mouse antibodies. If a secondary antibody is used, then this will carry the tag that allows visualization of the protein (Figure 2.49).

PVDF membrane PCS peptides

HRP

Anti-3-nitrotyrosine antibody

Detection antibody

Coupled enzyme

PVDF membrane PCS peptides

HRP HRP

Anti-3-nitrotyrosine antibody

Detection antibody

Coupled enzyme

Figure 2.49. General scheme of a Dot blot experiment

For testing the anti-3-nitro tyrosine antibodies specificity, the three peptides mentioned above were spotted on a PVDF membrane, followed by blocking of the unspecific sites with Roti®-Block solution. After washing steps with PBS-Tween buffer, the membrane was incubated with anti-3-nitro tyrosine antibody for 1 h at room temperature. After further washing steps, a second enzyme-conjugated antibody (horse radish peroxidase goat anti-mouse IgG), which recognizes the Fc (crystallizable fragment) fragment of the first antibody was added and incubated for 45 minutes. A mixture of ECL-solutions was added in order to develop the membrane and expose it on a film. Figure 2.50a and b show the comparison of the two antibodies’s specificity. Chemicon antibody proved to be highly specific as it shows stronger signal in the native peptide sequence, while in the mutated peptide the immune response was weaker probing the importance in binding of the neighbouring amino acid residues. Dot blot experiment shows that Santa Cruz antibody (39B6) is less specific.

PCS-NO₂

Y(NO₂)SL NO₂

PCS-RR PCS-Ala

PCS-Y

H-DFYKDGKRAAAY(NO₂)AL-NH₂ PCS-Ala

PCS-?

H-DFY(NO₂)KDGRRLKNYSL-NH₂

H-DFYKDGKRAAAY(NO₂)AL-NH₂ PCS-Ala

H-DFYKDGKRAAAY(NO₂)AL-NH₂ PCS-Ala

Figure 2.50. Dot blots analysis of using synthetic PCS peptides employed for testing the specificity of two different anti-3-nitrotyrosine antibodies (A) antibody (Santa Cruz) (39B6) antibody and (B) MAB5404 (Chemicon) to the synthetic PCS peptides.

2.6.3 Detection and identification of oxidatively modified proteins using