Detection of anti-lens crystallin antibody in dogs with and without cataracts (original) (raw)
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Purpose: The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract. Methods: Polyclonal antibodies were produced against human total lens proteins (40-80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat β-, β-glycated, γ-, and γ-glycated lens crystallins was also analyzed. A noncompetitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+α, HMW+α-glycated, β-, and β-glycated crystallins from humans and γ-and γ-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group. Results: The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat γ-and β-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+α (16.45 ng), (iii) human HMW+α-glycated (273 ng), (iv) human β-(37.82 ng), (v) human β-glycated (260 ng), (vi) rat γ-(105.34 ng), and (vii) rat γ-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating β-glycated and γ-glycated crystallins in the age group of 40-80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW +α-glycated crystallins in the age group of 40-80 years as compared to their age-matched controls. Notably, the levels of serum γ-glycated crystallins were found to be threefold higher than that of HMW+α-glycated and β-glycated crystallins in the age group of 70-80 years. Circulating autoantibodies to HMW+α-glycated, β-glycated, and γ-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40-80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to γ-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to β-glycated and HMW+α-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA. Conclusions: During the course of aging, leakage of lens crystallins (HMW+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40-80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lensspecific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.
THE ANALYSIS OF a-CRYSTALLINE PROTEIN IN WHITE AND BRUNESCENT CATARACT
Asian Journal of Pharmaceutical and Clinical Research, 2021
Objectives: The objectives of the study were to determine the difference of concentration and expression of a-crystalline protein in white and brunescent cataract lenses. Methods: The design of this study is cross-sectional comparative. The subject was cataract patients who underwent cataract surgery in Puskesmas Pariaman, West Sumatra, Indonesia. Lens examination was carried out at the Microbiology Laboratory of FK Unand from July 2019 to February 2020. The samples consisted of 36 subjects who met the inclusion criteria. ELISA examination was used to determine the concentration of a-crystalline protein and Western Blot examination was performed to see the expression of the a-crystalline protein in all subjects. Results: The difference in the concentration of a-crystalline protein in white cataract and brunescent cataract was not statistically significant, with p=0.129 (p>0.05). The result of Western blot examination was normal expression of a-crystalline protein in white cataract and under expression of a-crystalline protein in brunescent cataracts. Conclusion: The expression of a-crystalline protein appeared to be different between white and brunescent cataract lenses. In brunescent cataract, under expression of a-crystalline proteins was related to the decrease of chaperone activity. This change occurred allegedly because of photochemical reaction that happened inside the lens.
Local antibody formation within the eye: a study of immunoglobulin class and antibody specificity
Investigative Ophthalmology Visual Science, 1975
Primary immunogenic uveitis was induced in the rabbit eye with a single injection of antigen into the vitreous, and secondary booster uveitis responses were induced two months later by intravenous administration of the same antigen. The distribution of immunoglobulin classes and the specificity of the antibodies produced were assessed early and late in the primary response and early and late in the secondary response, and were compared with the analogous responses in the spleen and regional lymph nodes. At each of these stages of intraocular antibody response, IgG formation was higher and IgM formation lower than that seen in organized lymphoid tissues, while the proportion of IgA-forming cells was similar to the low levels usually found in the spleen. A significant proportion of IgA-forming cells was found in the perilimbal conjunctiva, and even greater levels in the lacrimal glands. At each stage of the response, the proportion of immxinoglobulin-forming cells making antibody specific for the inciting ovalbumin antigen was surprisingly low, reaching only seven per cent during the late primary reaction and 18 per cent during the late secondary reaction.
Beta B1-Crystallin: Identification of a Candidate Ciliary Body Uveitis Antigen
Investigative Opthalmology & Visual Science, 2003
PURPOSE. Perineuclear anti-neutrophil cytoplasmic antibody (pANCA), a marker antibody present in 12% of patients with anterior uveitis, recognizes cytoplasmic antigens in the nonpigmented ciliary body epithelium, a probable site of immunologic reactivity in this inflammatory disease. In this study, a recombinantly isolated pANCA monoclonal antibody was used to identify the corresponding antigenic target(s) in the ciliary body. METHODS. Proteins from microdissected eye bank ocular ciliary body tissue were used to identify the corresponding ANCA antigen. Parallel two-dimensional protein gels were used for simultaneous identification of candidate antigenic protein spots by Western blot analysis and as a source of material for proteomic analysis. Multiple independent methods including Western blot analysis, confocal microscopy, and RT-PCR were used to provide additional characterization of the candidate protein. RESULTS. Proteomic analysis suggested that beta B1 (B1)-crystallin is the primary ciliary body antigen. The presence of B1-crystallin in the human ciliary body was confirmed by Western blot with a B1 specific anti-peptide antibody. Confocal microscopy revealed colocalization of the antigenic reactivity of both anti-B1 antibody and monoclonal pANCA. RT-PCR confirmed the presence of B1-crystallin RNA in the ciliary body tissues. CONCLUSIONS. This study identified B1-crystallin as a new cytoplasmic ciliary body antigenic target of a marker autoantibody associated with uveitis. This characterization of B1crystallin outside the lens raises questions about its extralenticular expression, intracellular role, and potential target of inflammation in uveitis.
The localization of a 43K polypeptide in normal and cataractous lenses by immunofluorescence
Experimental Eye Research, 1981
The distribution of the 43K polypeptide within the human lens has been examined by immunofluoresrence. The results indicate that in the mature lens Bber this polypeptide is located in the region of the inner surface of the fiber cell membrane. It is not present in the epithelial cell but is found throughout the developing fiber cell. In fetal lenses it is located throughout the fiber cytoplasm in the first trimester but by six months the distribution of the polypeptide is similar to that found in the adult. In cataractous lenses there are amorphous areas devoid of 43K and globules containing 43K throughout their structure. The study indicates that immunofluoreseence provides a valuable tool for examining cataract and the distribution of lens polypeptides. The location of the 43K in the mature fiber cell at the intracellular membrane surface suggests that it may be involved in some aspect of membrane biology.