Indian Journal of Ophthalmology

ORIGINAL ARTICLE
Year
: 2002  |  Volume : 50  |  Issue : 1  |  Page : 41--48

Development of an immunoanalytical method for the detection of β- and γ- Crystallins and anti-crystallin antibodies. A molecular biomarker for cataract


S Nayak, RB Sashidhar, K Seetharam Bhat 
 Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007, India

Correspondence Address:
S Nayak
Department of Biochemistry, University College of Science, Osmania University, Hyderabad-500 007
India

Abstract

Purpose: To develop and evaluate an immunoanalytical method for the detection of β - and g-crystallins and anti-crystallin antibodies. Materials and Methods: Beta and g-crystallins isolated from rat lens were used as immunogens to raise polyclonal antibodies in rabbits. Antibody capture assay and western blot analysis showed that the antibodies to β - and g-crystallins were specific. An indirect competitive enzyme linked immunosorbent assay (ELISA) developed to quantitate β - and g-crystallin showed an IC50 value of 70 ng and 65 ng, respectively, based on regression analysis. Spiking studies with purified β -crystallin antibodies showed that 33 ng of the purified antibody gave an absorbance of 1.1 at 450 nm, indicating the sensitivity of the method. Results: Antibodies to β - and g-crystallins were not detected in serum samples of the cataractous CFY/NIN rats (used as an animal model for induction of experimental cataract by feeding high galactose diet). However, the cataractous rat serum samples effectively displaced β - and g-crystallin antibodies, indicating that these crystallins leak during cataract formation. The concentration of β - and g-crystallins in the rat serum, as analysed by indirect competitive ELISA, was found to be in the range of 17.6 - 81.6 mg/l and 12.4- 19.6 g/ml, respectively. Conclusions: The methodology developed in the present study may find application as a biochemical tool in molecular epidemiology of cataract



How to cite this article:
Nayak S, Sashidhar R B, Bhat K S. Development of an immunoanalytical method for the detection of β- and γ- Crystallins and anti-crystallin antibodies. A molecular biomarker for cataract.Indian J Ophthalmol 2002;50:41-48


How to cite this URL:
Nayak S, Sashidhar R B, Bhat K S. Development of an immunoanalytical method for the detection of β- and γ- Crystallins and anti-crystallin antibodies. A molecular biomarker for cataract. Indian J Ophthalmol [serial online] 2002 [cited 2020 Mar 31 ];50:41-48
Available from: http://www.ijo.in/text.asp?2002/50/1/41/14825


Full Text

Cataract is one of the major causes of blindness in all developing countries including India. Recent studies have shown that in India blindness due to cataract is around 50%.[1],[2] The pathogenic mechanism underlying cataract is still not clear. Cataract appears to be a multifactorial disease. The major predicted risk factors for cataract include diabetes, renal failure, diarrhoea, obesity (abdominal adiposity), solar radiation (UV-B), nutrient(s) inadequacy, excessive smoking, certain drugs, and genetic predisposition, in addition to aging.[3],[4]

Limited studies have suggested that cataract may be an autoimmune disorder.[5][6][7]

The lens consists of two cell types, epithelial and fibre cells. The continuous growth of the lens occurs through mitosis of the lens epithelial cells and differentiation of these cells into elongated fibre cells.[8] Crystallins are the major structural proteins of the eye lens (most of them are present in the fibre cells) and account for approximately 90% of the total proteins. All vertebrate lenses contain α- β- and γ-crystallins. In the human lens, α-crystallins (molecular mass 800-1000 kDa) make up 40% and comprise two related proteins, αA- and &# 945;B-crystallins. The β-crystallins (molecular mass 40-200 kDa) are of two types - acidic βA-species and basic βB-species, based on overall charge.[9] Eye lens crystallins have always been considered as proteins. However, presence of αA-, αB- and βB2- has been shown in tissues other than lens.[5] It has been suggested that anti-crystallin auto-antibodies are specifically directed against those crystallins that appear to be lens restricted, while immunological tolerance would exist for the crystallins that occur outside the lens. It has been reported that more than 98% of the cataract patients showed elevated antibody titre against lens crystallin.[10]

It was speculated that age-related cataract might be an autoimmune disease resulting from antibody mediated injury to lens epithelial cells (LECs). Further, older mice were more susceptible to the lens epithelial cell damage than younger ones.[11]

Normally, ocular tissues are secluded from the immune system by an anterior chamber associated immune deviation (ACAID).[12] With age, however, the blood-aqueous barrier may deteriorate and antibodies may gain access to the anterior chamber. Cytotoxic anti-LEC antibodies might enter the anterior chamber, change cellular functions, kill LECs, and cause cataract formation. Complement is necessary for the cytotoxic effect on LECs in culture.[13]

Currently, surgery is the only cure for cataract. The blindness-related financial losses associated with cataract are significant. If the development of cataract could be delayed, the costs and consequences of cataract could be greatly reduced. The major goal of current cataract research is focused on developing strategies to detect cataract in the early stages so that preventive measures can be taken. Studies of human and experimental cataracts have indicated that supplementation of antioxidant micronutrients and certain dietary components in the initial stages of cataract could be beneficial.[14] In view of the importance of early detection of cataract, the objective of the present work was to develop a simple, rapid and sensitive immunodiagnostic method for the detection of β and γ-crystallins and anti-crystallin antibodies. If successful, this could be used as a molecular biomarker to identify populations at risk of developing cataract.

 Materials and Methods



Bovine serum albumin (BSA-fatty acid free, RIA grade), Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), fish gelatin, anti-rabbit immunoglobulin G (IgG) labeled with horseradish peroxidase raised in goat (whole molecule), tetra methyl benzidine (TMB), urea H2O2, coomassie brilliant blue R-250, P-nitro blue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl phosphate toluidine (BCIP), nitrocellulose paper and polystyrene micro titre ELISA plates were from Sigma (St. Louis, MO, USA). Sepharose 6B, Protein-G affinity column and marker proteins - aldolase, ferritin, thyroglobulin, blue dextran, ribonuclease A, chymotrypsinogen A, ovalbumin and BSA were obtained from Pharmacia (Upsala, Sweden). Galactose (LR grade) was procured from Thomas Baker Chemicals (Mumbai, India). Other reagents were of analytical grade.

 Experimental Animals



One month old male, Wistar/NIN strain rats of 35-40 gm body mass and adult male New Zealand White (NZW) rabbits of 2-2.5 kg body mass maintained on the cereal-pulse based semi-synthetic stock pellet diets [Table:1], for rodents and rabbits respectively were obtained from National Center for Laboratory Animal Sciences (NCLAS), National Institute of Nutrition, Hyderabad, India. On slitlamp examination (Kowa portable, handheld slitlamp,Japan), lenses were clear and free of any ocular abnormalities. Rats were decapitated by guillotine and the eyes were immediately enucleated; lenses were removed using the posterior approach and placed in preweighed glass vials (5 ml) kept on ice.

 Induction of cataract in rats



Sixteen weanling male CFY/NIN strain rats obtained from NCLAS, NIN were distributed equally into two groups and fed either control (zero galactose) or experimental (50% galactose) diets using a randomised block design. Briefly, the control diet consisted of 70% sucrose, 20% casein, 5% peanut oil, 4% salt mixture, 0.6% vitamin mixture and 0.4% choline chloride.[15] The carbohydrate source in the experimental diet was 50% galactose and 20% sucrose. Weekly ophthalmic examinations (after dilation of the pupil with 1-2 drops of 1% atropine sulphate) were carried out using the slitlamp biomicroscope (animals were conscious). At seven weeks of age animals were killed and blood was collected.

Extreme care was taken in the maintenance and killing of the animals (Guidelines prepared by the National Committee under the aegis of Indian National Science Academy) were followed. Animals were housed individually in screen bottomed cages in a room with 12-hour light (standard fluorescent light)/ 12-hour dark cycles and maintained at about 22 C and 55% humidity. Food and water were provided ad libitum.

 Isolation and purification of β- and γ-crystallins



Lens α- and γ-crystallins were isolated and purified from one-month-old Wistar/NIN rats by the method standardised earlier using Sepharose 6B gel chromatography.[16] A 10% lens homogenate was prepared in 20 mM phosphate buffer (pH 7.4) containing 100 mM sodium chloride and 25 mM EDTA (Ethylene-diaminetetraacetic acid). Soluble proteins were isolated by centrifugation of the lens homogenate at 10,000 g for 15 minutes at 4 C. The protein concentration was determined by the Lowry method.[17] The supernatant fraction was subjected to gel filtration on Sepharose 6B column, 140 x 2.5 cm (Pharmacia, Uppsala, Sweden) at 10C. Proteins were eluted with the same homogenate buffer. Fractions of 2.5 ml were collected at a flow rate of 20 ml/hour. The elution was monitored at 280 nm. The relative amounts of these proteins were determined by measuring the peak areas using planimetry. Fractions corresponding to β and γcrystallins were pooled separately and dialysed against deionised water, lyophilised and stored until further use[16]

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a slab gel (10% acrylamide) by Laemmli's method under reducing conditions.[18] Proteins (β and γcrystallins and the standard markers) at a concentration of 1mg/ml were dissolved in 1 ml of treatment buffer[SDS, 2% (w/v); β mercaptoethanol,2% (v/v); glycerol, 20% (v/v); bromophenol blue, 0.001% (w/v) in tris-HCl buffer (125mM, pH 6.8)] After boiling the samples at 100 C for 2 minutes electrophoresis was carried out with a constant current of 25 mA for 1-1.5 hours using vertical gel electrophoretic system (Sigma, St. Louis, MO, USA). The gels were stained with coomassie brilliant blue R-250 for 16-18 hours and were destained using the mixture containing 7% acetic acid and 5% methanol.

 Production and characterisation of antibodies



 Immunization



Two rabbits were used for the production of antibodies and were allowed to acclimatise for 3-4 weeks. Blood was collected from the marginal ear vein. Prior to the first injection pre-immune serum was collected from each animal, lyophilised and stored at -20C. The antigen was prepared under aseptic conditions using Laminar flow system (Micro-filt, Pune, India). Further, the antigens were filter sterilised using 0.45 μm Millipore membrane filter. A primer dose of about 200-250 μg/kg body mass of the purified antigens (β- or γ-crystallin) was given by multiple site epidermal injection. It was dissolved in sterile saline and emulsified with Freund's complete adjuvant in a 1:1 ratio. Later, three booster doses were given (100-150 μg/kg) intramuscularly in Freund's incomplete adjuvant. Blood was also collected from the animals after 30 days of primer dose and 10-12 days after the booster doses. At the end of the immunisation schedule, the animals were killed and blood was collected by cardiac puncture. Serum was separated, lyophilised and stored at -20 C till further use.

 Characterisation of the antiserum



The serum from each animal was analysed for the presence of antibodies by using double diffusion Ouchterlony technique.[19] Cross-reactivity of the antisera raised against β- or γ-crystallins was also checked. Sterile, polycarbonate petriplates were pre-coated with 1% agar and air dried at 37C. The plates were then recoated with 1.2% agar in 10mM phosphate buffered saline (pH 7.2) containing 0.05% sodium azide. The plates were stored at 4C. Wells (2 mm dia.) were punched into the gel using a gel puncher. Different dilutions of antigen and antisera were loaded into the wells.

 Antisera titers by antibody capture assay



Antisera titres for pre-immune primer, I, II, and III boosters were determined by checkerboard analysis using antibody capture assay. Microtitre plates were coated with 50 μl/well of 100 mM carbonate buffer, pH 9.6 containing different concentrations of the antigens (β- or γ- 100, 250, 500 and 1000 ng). The plates were dried overnight at 37C and washed thrice with washing buffer [phosphate buffered saline (PBS) T 10 mM, pH 7.2 with 0.05% tween -20 and 0.01% sodium azide]. The wells were blocked for nonspecific binding with 100 μl/well of 0.1% fish gelatin in PBS (10 mM, pH 7.2). The plates were washed three times with washing buffer and different dilutions of antisera (50 μl/well) were added and incubated for two hours at 37C. The plates were washed three times and incubated at 37C for one hour with 50 μl/well of 1: 6,000 diluted (0.5% fish gelatin in PBS) horseradish peroxidase labeled anti-rabbit IgG raised in goat. The plates were washed thrice and 150 ml/well of substrate [450μl of TMB (10 mg /ml of dimethyl sulfoxide) in 15 ml of 10 mM acetate buffer pH 5, containing 0.25% (w/v) β-cyclodextrin and 0.015% (w/v) of urea-hydrogen peroxide] was added. The reaction was terminated after 10 minutes by adding 50 μl/well of 1.25 M H2SO4. The absorbance was taken at 450 nm using SLTspectra ELISA reader (SLT Lab Instruments, Salzburg, Austria).

 Development of indirect competitive ELISA



The protocol for the indirect competitive ELISA is shown in [Figure:1]. It was similar to antibody capture assay except that after blocking, the plates were incubated for 2 hours with a mixture of 25 μl of different concentrations of antigen (β- or γ-, 1 - 1000 ng) and 25μl of diluted antibody (β- 1: 3,500 or γ- 1: 5,000). Percent binding of antibodies versus concentration of the analyte was used to generate inhibition plot using linear regression analysis. The cross-reactivity of the antibody (β-) with γ-, α- and high molecular weight (HMW) crystallins was evaluated by indirect competitive ELISA where these respective antigens were used as analytes to displace the antibody. The cross-reactivity studies were carried out at a 50% inhibition level of β- crystallin.

 Western blot analysis



Western blot analysis was carried out according to the Towbin method.[20] Proteins separated by SDS-PAGE were electrophoretically transferred to a nitrocellulose membrane with the Milliblot-Graphite Electroblotter I system (Millipore Corporation, Bedford, MA, USA). The transfer was carried out with 100 mA current for 30 minutes. The membrane was washed in PBS and incubated in a blocking solution (3% fish gelatin in PBS) for 30 minutes and once again washed with PBS. It was then incubated overnight with antibody buffer (3% fish gelatin in PBS) containing 1: 1,000 diluted polyclonal antibody to α- or γ-crystallin. The membrane was washed in PBS to remove unbound antibody and incubated for 2 hours in a conjugate solution (3% fish gelatin in PBS) containing 1: 2,500 diluted goat anti-rabbit IgG coupled with alkaline phosphatase. The membrane was washed and incubated with alkaline phosphatase color developer (23.06 mmol/l NBT and 183.4 mmol/l BCIP) until purple bands appeared. Immersing the membrane in distilled water arrested the colour development. The membrane was stored after incubating for 10 minutes in glycerol and dried. It was photographed using gel documentation system (GDS-5000 Ultra Violet Product, Cambridge, UK).

 Validation of the ELISA method



The ELISA method developed was validated by spiking affinity-purified antibodies to control rat serum. Beta-crystallin antibodies were affinity purified by using commercial Protein -G column. 2 mg of purified antibody was spiked to 1 ml of control rat serum. Then 1 ml of serum diluted with 1 ml of equilibrating buffer (20 mM phosphate buffer, pH 7.0) was loaded on to the column. Unbound protein was removed by washing the column with 5-6 bed volumes of equilibration buffer. The flow rate of the column was maintained at 0.5 ml/minutes. The bound IgG was eluted with 4 bed volumes of 100 mM glycine buffer, pH 2.7. The eluate (900 μl) was collected in tubes containing 100 μl of 1M tris-HCl buffer, pH 9.0. The column was washed with 10 bed volumes of double distilled water and 10 bed volumes of 20% ethanol. Presence of antibody was analysed by antibody capture assay as described earlier.

The ELISA method was also validated by using serum samples obtained from rats in which cataract was induced by feeding galactose high diet. Serum samples were collected and analysed for the presence of β- and γ-antibodies as well as circulating levels of β- and γ-crystallins by indirect competitive ELISA.

 Results



 Isolation and purification of β- and γ-crystallins



Gel filtration of soluble lens proteins from rat lens showed four major fractions [Figure:2]. High molecular weight protein was eluted with the void volume followed by α-, β- and γ-crystallins. The β- and γ-crystallins formed the major fractions (about 40 % each of the total soluble proteins), while HMW protein and

α-, β-, γ-crystallin constituted about 3.5% and 16% respectively, as reported earlier.[16] Upon electrophoresis four distinct bands (36, 31, 26 and 23 kDa) were observed with β-crystallin and a single band (20 kDa) with γ-crystallin, similar to the one reported earlier.[21]

 Characterisation of the antisera



Purified antigen (β- or γ-) was used to produce polyclonal antibodies in rabbits. Both the animals showed good response. Antiserum titre after the second booster was 1: 8 against β- and 1: 16 against γ-, as determined by the Ouchterlony double diffusion technique. The antibodies raised against β- did not show cross-reactivity with γ-crystallin. Similarly, antibodies raised against γ- did not show cross-reactivity with β- crystallin. These results suggested that the antibodies were specific.

 Antisera titres by antibody capture assay



Titre determination by checkerboard analysis at different concentrations of the coating antigen (100, 250, 500 and 1000 ng) showed that the optimal concentration of the antigen was 500 ng for both β- and γ-crystallins. Beta-crystallin antibody titre curve as determined by antibody capture assay showed that a titre of 1: 7,000 dilution of antisera at 500 ng/well of β-crystallin concentration gave an absorbance of 0.8 (after appropriate blank correction) at 450 nm, when horseradish peroxidase labeled second antibody was used as an enzyme label. γ-crystallin antibody titre curve as determined by antibody capture assay showed that a titre of 1: 10,000 dilution of antisera at 500 ng/ well of β-crystallin concentration gave an absorbance of 1.56 (after appropriate blank correction) at 450 nm, when horseradish peroxidase labeled second antibody was used as an enzyme label. Antibody titre profile of rabbit serum (pre-immune to III booster) against the antigen β-crystallin as well as γ-crystallin showed that the third booster of (β-crystallin) and II booster of (γ-crystallin) antibodies showed high absorbance. ([Figure:3] and [Figure:4]).

 Indirect competitive ELISA



The standard displacement curve for various concentrations of β- and γ-crystallins (1-1000 ng) is shown in [Figure:5] and [Figure:6]. The inhibitory concentration at 50% antibody binding (IC50)for β- and γ-crystallins was found to be 70 ng and 65 ng, respectively [based on regression analysis, β-crystallin, y = 115.5 + (-35.17)x, r2= 0.952); γ-crystallins, y = 103.5 + (-29.68)x; r2= 0.997]. When HMW proteins, α-, and γ-crystallins were competed with bound β-crystallin, the γ-crystallin antibody cross-reacted with α-crystallin by 78%; while HMW proteins and γ-crystallin did not show any cross-reactivity.

 Western blot analysis



Western blot analysis, after SDS-PAGE of β-crystallin yielded two stained bands with β-crystallin antibody (indicating that of the four bands visualised by coomassie blue staining, only two bands cross-reacted with the antibody) and no bands with γ- crystallin antibody [Figure:7]. Similarly, Western blot analysis after SDS-PAGE of γ-crystallin yielded a single stained band with γ-crystallin antibody and no bands with β-crystallin antibody [Figure:8].

 Validation of ELISA



Spiking of affinity purified β-crystallin antibody to control rat (Wistar/ NIN) serum showed that 33 ng of the purified antibody gave an absorbance of 1.1 (after appropriate blank correction) at 450 nm, when horseradish peroxidase labeled second antibody was used as an enzyme label.

As expected, slitlamp examination of the eyes of galactose-fed rats showed progressive lens changes with increasing feeding period. The observed lens changes at the end of one week were vacuolisation and haziness in the cortex; at the end of two weeks: severe vacuolisation, haziness and in a few rats opacity; and at the end of four weeks nuclear opacity with moderate cortical turbidity.

The crystallin-specific antibodies were not detected in the serum samples of galactosaemic rats. However, the cataractous rat serum samples displaced β- and γ-crystallin specific rabbit antibodies in the immunoassay, indicating the leakage of these crystallins during cataract formation. The concentrations of β- and γ-crystallins in serum from the experimental rats were found to be in the range of 17.6 - 81.6 μg/ml and 12.4 - 19.6 μg/ml respectively.

 Discussion



Cataract is the commonest cause of blindness. To date, neither prophylactic nor pharmacological treatment has been effective in treating cataract. The current focus of lens research is on the development of an effective method for the treatment and prevention of cataract. In the recent past, development of molecular biomarkers for human diseases has gained importance. The potential utility of the measurement of biomarker lies in the identification of high-risk populations and utilising the preventive strategies.

Several studies have implied that cataract is an autoimmune disorder.[5],[22][23][24][25][26] In mature cataracts longstanding leakage of crystallins through the damaged capsule is likely to occur. The immune system is thus exposed to the crystallins, which are normally sequestered in the lens. However, those crystallins that normally occur in small amounts outside the lens would be recognised as self, and therefore normally not evoke an auto-immune response. Only the truly lens-specific crystallins would be recognized as non-self antigen, thus triggering auto-antibodies formation. Circulating antibodies against β- and γ-crystallins have been observed in patients with mature cataract, whereas no antibodies were observed against αA-, αB- and βB2-crystallins. This indicates that auto-antibodies are specifically directed against those crystallins that appear to be lens-restricted, while immunological tolerance would exist for crystallins that occur outside the lens.[5] Circulating auto-antibodies against lens antigens are prevalent in patients with age-related cataract.[6] It has been shown that auto-antibodies against lens antigens are cytotoxic to LECs and that cell death may involve complement-mediated pathways. A recent study showed that rat lens epithelial cell damage induced by immunological response to bovine lens membrane protein, leads to cataract development.[27] Oral administration of lens homogenate suppressed antibody production significantly in mice immunized with β-crystallins.[7]

In the present study polyclonal antibodies raised against β- and γ-crystallins were found to be specific. Western blot analysis and ELISA demonstrated that the β-crystallin antibody did not recognise γ-crystallin and γ-crystallin antibody did not recognise βcrystallin. The high antibody titre observed in the present investigation for γ-crystallin indicates that the γ-crystallin could be a more immunogenic protein than β-crystallin. Gamma-crystallin, being a monomeric protein (20 kDa), may leach out much earlier than β-crystallin (20 - 200 kDa), during cataractogenesis. Hence, γ-crystallin may be an earlier biomarker than β-crystallin.

None of the serum samples from the rats fed on high galactose diet showed the presence of antibodies against β- and γ-crystallins. This could be attributed to the short duration of the experimental period, which may have led to an insufficient response time for the activation of immune system. Further, it is pertinent to note that, (i) the type of experimental induction of cataract in rat models (diabetic[28], tryptophan deficient[29], riboflavin deficient[16] etc.), (ii) duration of cataract development and (iii) molecular mechanism(s) may have an important role in the leakage of lens crystallins, leading to specific antibody formation to lens specific crystallins, during the course of cataract development.

The methodology developed in the present study may find application in screening natural and synthetic anti-cataractogenic compounds for their ability to intervene metabolically or to delay or prevent the onset of cataractogenesis, in animal models. Further, this methodology could be extended to developing an immunoassay specifically for human circulating β- and γ-crystallins /β- and γ-crystallin specific antibodies as a biochemical marker for early detection of cataract.

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