N-Homocysteinyl-Proteins - Homocysteine in Protein Structure/Function and Human Disease - page 96

5.5.3 Immunoassays with Rabbit Polyclonal

Anti- N -Hcy-Protein Antibodies

N-Hcy-proteins were first found to be immunogenic in 1998 when anti-N-Hcy-
protein IgG antibodies were generated by immunizing rabbits with homologous
low-density lipoprotein modified with Hcy-thiolactone [356]. Similar antibodies
were subsequently prepared by immunizing rabbits with Hcy-thiolactone-modified
keyhole limpet hemocyanin (KLH) [172] and rabbit serum albumin [168].
Rabbit polyclonal anti-N-Hcy-protein antibodies, generated with rabbit serum
N-Hcy-albumin as an antigen, were used in clinical studies of plasma N-Hcy-
protein as a risk factor for coronary heart disease in a group of 254 patients and
308 controls [168]. For an N-Hcy-protein ELISA immunoassay, tested plasma is
mixed with anti-N-Hcy-protein antiserum and added to wells of microtiter plates
coated with N-Hcy-albumin. Bound antiserum is detected with anti-rabbit IgG
secondary antibody conjugated with HRP by reading A 492 after reaction with
OPD solution. The plasma level of N-Hcy-protein adducts was significantly higher
in coronary heart disease patients than in controls (40.65 10.87 units/mL
vs. 30.58 10.20 units/mL, P < 0.01), with odds ratio of 7.34 (95 % confidence

interval 4.020-13.406, P < 0.01), and increased according to the number of ath-

erosclerotic coronary arteries: 35.59 10.34 units/mL (n ¼ 76), 41.88 8.83

(n ¼ 70), and 43.13 11.47 (n ¼ 108) in subjects with 1, 2, and 3 affected

arteries, respectively (r ¼ 0.01).

Rabbit polyclonal anti-N-Hcy-protein IgG antibodies, generated with N-Hcy-
KLH as an antigen [172], are useful for the immunohistochemical detection of N-
Hcy-protein in human and mouse tissues with good sensitivity and specificity [357].
This antibody detects N-Hcy-protein in a dot blot assay with the signal depending
on the amount of the antigen, but the magnitude of the signal is protein dependent.
The rabbit anti-N-Hcy-protein IgG specifically detects Nε

0.174, P <

-Hcy-Lys epitopes in
human tissues, as shown by positive immunohistochemical staining of myocardium
and aorta samples from cardiac surgery patients, and a lack of staining when the
antibody was pre-adsorbed with N-Hcy-albumin. An increased immunohistochemi-
cal staining for N-Hcy-protein is also observed in aortic lesions from ApoE /
mice, and the staining is increased in ApoE / animals that are fed with a
hyperhomocysteinemic high-methionine diet [357].

5.5.4 Western Blot Immunoassay for N -Hcy-ApoAI

N-homocysteinylation introduces a free thiol group into a protein. Such thiol group
can be easily detected in proteins that do not contain cysteine residues with a free
thiol group. This principle has been exploited for the detection and quantification of
N-Hcy-ApoAI [303], a major protein component of HDL particles that does not
contain free cysteine residue thiols. The (N-Hcy-SH)-ApoAI in serum or HDL is