About Authors
*1M Prasad Naidu, 2T Madhu Chaithanya, 3N Mallikarjun Rao, 4Muneer Bhanu , 5Dr Madhu Sudan Reddy
1Medical Biochemistry, NMCH
2Medical Pharmacology, NMCH
3Biochemistry, NMCH
4Biotechnology, NMCH
5MD Pharmacolgy, NMCH
*www.prasadnaidu.com@gmail.com
Introduction
Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen-specific monoclonal or polyclonal antibody in which neither the antigen-combining site nor the active site of the enzyme is functionally altered. The chemistry of cross-linking HRPO or urease to immunoaffinity purified monoclonal or polyclonal antibodies (IgG) is presented in. The chemistry of cross-linking alkaline phosphatase to antibodies is presented in. The enzyme most commonly used in the immunoreagent (the antibody enzyme conjugate) preparation is horseradish peroxides. This enzyme is cheap and can be attached to the immunoreagent by a variety of methods. Moreover many chromogenic substrates for it are also available.
REFERENCE ID: PHARMATUTOR-ART-1808
Background Information
The conjugation of HRPO (Nakane and Kawaoi, 1974) to antibody is dependent on the generation of aldehyde groups by periodate oxidation of the carbohydrate moieties on HRPO. Combination of these active aldehydes with amino groups on the antibody forms Schiff base upon reduction by sodium borohydride, become stable. For urease conjugation (Healey et al., 1983), cross-linking the enzyme and antibody with MBS is achieved through benzoylation of free amino groups on antibody. This is followed by thiolation of the maleimide moiety of MBS by the cysteine sulfhydryl groups of urease. The advantages of urease conjugates are their stability in solution at normal working dilutions, the rapid turnover rate of the enzyme, the easily discernible color change when substrate is added, and the fact that urease is not found in most mammalian or bacterial systems. The disadvantage is that since no precipitable substrate is available, urease conjugates cannot be used for immunohistology or western blotting. Alkaline phosphatase conjugates are useful for all types of immunological assays depending on the alkaline phosphatase substrate used (i.e., p-nitrophenyl phosphate in diethanolamine is the preferred substrate for ELISA with colorimetric detection, 4-methylumbelliferyl phosphate is useful for ELISA with fluorimetric detection, and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate is the preferred substrate for western blotting). Alkaline phosphatase conjugates are as stable as urease conjugates and more stable than HRPO conjugates. Endogenous phosphatases can cause false positive reactions. However, levamisole will inhibit alkaline phosphatase in many mammalian tissues but not the alkaline phosphatase (i.e., bovine intestinal) used in the conjugates and for this reason levamisole may be added to the substrate solution. The one-step glutaraldehyde method (Voller et al., 1976) is the simplest available procedure for preparing alkaline phosphatase–antibody conjugates. Various alternative procedures for preparing alkaline phosphatase conjugates have been compared (Jeanson et al., 1988). The sensitivity that can be achieved with HRPO, urease, or alkaline phosphatase conjugates is comparable and between 1ng/ml and 10ng/ml of antigen can be detected.
Principle
The most commonly used method for labeling IgG molecules with HRP exploits the glycoprotein nature of the enzyme. The saccharide residues of HRP are oxidized with sodium meta-periodate to produce aldehydes groups that can react with the amino groups of the IgG molecule and the Schiff’s bases formed are then reduced by borohydride to give a stable conjugate. Desalting is done using a gel filtration column to remove the borohydride and to store the conjugate in PBS.
The efficacy of the enzyme-labeled antibody is tested by direct Dot-ELISA and is expressed as the titer value of the conjugate. Here nitrocellulose membrane having an appropriate antigen immobilized on it (human IgG) is incubated with various dilutions of the conjugate. The amount of HRP-labeled antibody bound is assayed by adding the substrate, tetra methyl benzidine–H2O2 (TMB/ H2O2). HRP acts on hydrogen peroxide to release nascent oxygen that oxidizes TMB, a chromogen, giving a blue colored product, TMB oxide.
Oxidation of HRP:
0.2ml of distilled water was added to the oxidation tube containing HRP and sodium meta-periodate. The solution was stirred for 20 min at room temperature. (Note: colour changes from orange to green).
Coupling:
0.2ml of carbonate buffer was added to antibody coupling tube and the contents were mixed thoroughly. Immediately after mixing, oxidized HRP was added to the antibody solution and the contents were stirred for 2 h at room temperature. After 2 h, 10μl of reductant solution (borohydride) was added and the solution was mixed for 10 min at room temperature.
Desalting:
The desalting column was first equilibrated with 20ml of 1X PBS. After allowing the reaction mix to settle down the supernatant was loaded on to the desalting column that was previously equilibrated with PBS. After running down the reaction mixture into the column 0.1ml of 1X PBS was added along the walls of the column to wash down the reaction mixture sticking onto the column. Once the PBS entered the bed of the column, 1ml of 1X PBS, was added at a time till the entire colored fraction (HRP conjugate) eluted out of the column completely. Immediately after collecting the HRP conjugate 0.5ml of stabilizer was added to the HRP conjugate and the conjugate was stored at 4oC until use. After eluting the HRP conjugate, the column was washed with 5ml of 1X PBS and stored at 4oC.
Titration:
The HRP conjugate was diluted with 1X assay buffer. Four antigen (Fraction no. 2 from serum sample 1, 2 and 3) spotted nitrocellulose strips were labeled 1:1000, 1:2000, 1:4000 and 1:8000 and then the strips were agitated in appropriately diluted conjugate (i.e. 1:1000, 1:2000, 1:4000 and 1:8000 diluted anti human IgG HRP conjugate. The strips were then incubated at room temperature for 30 min with constant shaking. After incubation, the strips were washed 5 times with 2-3ml of rinse buffer (1min each wash). After each wash the old buffer was replaced with fresh buffer. After the fifth wash the strips were washed with distilled water once. After washing, the strips were agitated in 2ml of substrate solution (TMB/H2O2) till blue/ grey colored spots were observed. Soon after the spots developed the strips were removed before the background turned dark. The highest dilution at which the spot are seen is the titer value of the conjugate.
Purification of human IgG employing protein-A affinity chromatography
Principle
Protein-A agarose column is an immunoaffinity column in which protein-A is coupled to cross linked agarose using cyanogenbromide activation method. Protein-A binds to the Fc regions of immunoglobulin’s through interactions with heavy chain. The binding of protein-A has been well documented for immunoglobulin’s from a variety of mammalian species and for immunoglobulin’s IgM and IgA as well. Protein-A agarose column has been used as powerful tool to isolate and purify classes, sub-classes and fragments of immunoglobulins from biological fluids and cell culture media. Since, only the Fc region is involved in the binding, Fab region is available for binding antigen. Hence, protein-A agarose column is extremely useful for isolating immune complexes.
Chemicals and reagents
1. 10N sodium hydroxide
2. 2. Phosphate buffer saline (PBS) pH 7.4 (equilibration buffer): (20x/1000ml)
3. Elution buffer: (0.1M citrate buffer pH = 4.6)
4. Neutralization buffer: (2 M Tris (hydroxy methyl) amino methane)
5. Storage buffer: (equilibration buffer containing 0.05 % (w/v) sodium azide)
6. Protein A affinity column: (column size-2ml)
7. Serum samples
Methods: Serum sample was mixed with equilibration buffer in 1:1 ratio in an eppendorf tube. After mixing, the sample was subjected to centrifugation at 6000 rpm at 40Cfor 10 min to remove particulate matter.
Protein A Column Chromatography (Bed volume 2ml):
After the column was brought to ambient temperature, the column was equilibrated with 10 bed volumes of equilibration buffer. After equilibration, 0.9ml of the 1:1 diluted clear serum sample was loaded on to the column. While loading, the flow rate of the column was adjusted to 1ml/5 min. Once the serum sample passed through the column, the column was washed with equilibration buffer till the A 280 of the equilibration buffer was less than 0.1 O.D. (About 10 bed volumes of equilibration buffer was required for washing). After the unbound materials were removed by washing, the bound proteins were eluted with 15 bed volumes of elution buffer. 1ml fractions were collected in tubes containing neutralizing buffer (25μl/ml). After collecting the fractions, A280 for each fraction was measured at 280nm in a UV-VIS spectrophotometer. Serum samples 2 and 3 were treated similarly. After taking the absorbance value a graph was plotted with fraction number on x-axis vs. absorbance values on y-axis.
Enzyme linked immunosorbent assay (ELISA)
Principle
ELISA can qualitatively or quantitatively determine antibodies. Enzyme labeled Horse Radish Peroxidase (HRP) specific antibody is added to an antigen-coated micro titter plate well and it is allowed to react with the antigen. Any free enzyme labeled antibody is washed away and a substrate along with a chromogen is added to micro titter plate well. If the antibody is specific to the antigen, the enzyme present on the antibody reacts with the substrate to give a colored product, whose intensity can be read in an ELISA reader.
Chemicals and reagents
1. Antigen:
a. Pure human IgG was purchased from Bangalore Genie Pvt Ltd, Bangalore India.
b. Pure Rabbit IgG was purchased from Bangalore Genie Pvt Ltd, Bangalore India.
c. Serum samples 1, 2, 3
d. Fraction no 2 ( serum samples 1, 2 and 3)
Samples were prepared at a concentration of 5μg/ml individually in coating buffer (0.1M sodium carbonate and bicarbonate buffer pH 9.6).
2. Antigen coating buffer:(0.1 M sodium carbonate bicarbonate buffer pH 9.6)
3. Phosphate buffer saline (PBS) pH 7.4 (equilibration buffer): ( 20x/1000ml)
4. Rinse buffer: 50ml of 20X PBS was diluted to 1000ml with distilled water. After mixing, 1ml of tween 20 was added to it.
5. Serum diluent buffer: 1gm of egg albumin was dissolved in 100ml of rinse buffer.
6. Labeled antibody: Anti-humanIgG HRP conjugate prepared in our laboratory was used.
7. Substrate solution: Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2):
TMB/H2O2 was purchased from Bangalore Genie Pvt Ltd, Bangalore India. TMB is a noncarcinogenic chromogen used in assays involving Horseradish Peroxidase (HRP) enzyme. TMB/H2O2 for ELISAproduces a soluble blue coloured product with HRP. The reagent is supplied as 20 X concentration. Just before use 1 volume of the substrate solution was mixed with 19 volumes of distilled water.
Other requirements
1. 96 well micro titer plates.
2. Micro pipettes 2-20 and 200-1000µl and tips.
3. Glass pipettes 5ml and 10ml.
4. Glass beakers and conical flasks
5. Measuring cylinders
6. Wash bottle
7. Incubator set at 370 C.
8. Moist chamber (plastic box with a layer of wet cotton)
9. ELISA Reader.
Method
All assays were carried out in 96 well flat bottomed polystyrene plates.
1.Antigen coating:
100µl of antigen at a concentration of 5µg/ml in 0.1 M sodium carbonate–bicarbonate buffer, pH 9.6 was coated onto each well and the plate was incubated for 30 min at 37°C. (Few wells coated with rabbit IgG was used as negative control). After incubation, excess antigen was removed from the well by washing 5 times with rinse buffer (PBS with 0.1%).
2. Blocking non specific sites:
After washing, non-specific sites were blocked by addition of 300 ml serum diluent buffer (SDB, containing PBS with 0.1% tween-20 and 1% egg albumin) into each well and plate was incubated for 30 min at 37°C. After incubation, plate was washed 5 times with rinse buffer.
3. Labeled antibody (Anti-human IgG HRP-conjugate and goat anti-human IgG HRP conjugate):
After washing, 100µl of 1:2000 diluted anti-human IgG -HRP conjugate was added to each well and the plate was incubated for 30 min at 37o C. Excess antibody enzyme conjugate was removed by washing the well 5 times with rinse buffer.
4. Substrate solution:
After washing, 100µl of the chromogenic substrate (TMB/H2O2)was added to each well and the plate was incubated for 10 min at room temperature till colour developed. Colour developed was read at 450n.m in a micro titter plate auto reader ELISA reader.
Western Blotting
Principle
Identification of proteins separated by gel electrophoresis is limited by the small pore size of the gel, as macromolecular probes for protein analysis cannot permeate the gel. The separated proteins are buried in the polyacrylamide gels and therefore further analysis of proteins or their recovery is cumbersome. However, the proteins can be effectively transferred from the gel to the supporting medium by blotting. The transfer of protein bands from a polyacrylamide gel on to a more stable and immobilizing support is called as protein blotting or immunoblotting.
Chemicals and reagents
1. Blotting buffer
2. Phosphate buffer saline (PBS) pH 7.4 (equilibration buffer): (20x/1000ml)
3. Rinse buffer: 50ml of 20X PBS was diluted to 1000ml with distilled water. After mixing;
1ml of tween 20 was added to it.
4. Serum diluent buffer: 1 gm of egg albumin was dissolved in 100ml of rinse buffer.
5. Labeled antibodies: Anti-human IgG HRP conjugate was prepared in our laboratory.
6. Substrate solution: Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) was purchased from Bangalore Genie India. TMB is a noncarcinogenic chromogen used in assays involving Horseradish Peroxidase (HRP) enzyme. The reagent is supplied as 20 X concentration. Just before use 1 volume of the substrate solution was mixed with 19 volumes of distilled water.
7. Fraction no 2 from serum samples 1, 2 and 3
Other requirements
1. Nitro cellulose membrane (pore size 0.22-0.45 micro meter)
2. Glass pipettes
3. Beakers
4. Conical flask
5. Petri dish
6. Scalpel blade
7. Glass tray or Plastic tray
8. Glass plate platform 25x20 cm
9. Tissue paper rolls
10. Whatman filter paper
11. Scissors
12. Forceps
13. Gloves
14. Small electrophoresis unit
15. Electrotransfer unit or small electro blotting system.
16. Power supply unit
17. Connecting cords
18. Shaker.
Method
Western blotting is essentially a combination of three techniques electrophoresis (PAGE), blotting (protein blotting) and immunochemical detection (blot development).
Stage I: Separation of proteins on SDS-PAGE
The proteins to be analyzed (fraction no 2 from serum samples 1, 2 and 3) by western blotting was first subjected to separation on a 10-12% separating gel and 6% stacking gel. Samples were electrophoresed at 80-100 volts.
Stage II: Electro transfer of the separated protein onto the nitro cellulose membrane
Blotting is the transfer of resolved proteins from the gel to the surface of a suitable membrane. The separated proteins are transferred out of the gel either by the capillary action of the buffer or in an electric field (known as electro blotting).
After separation of proteins by SDS-PAGE, the stacking gel was discarded and the required portion of the gel was cut with a scalpel blade, if the whole gel was not to be blotted. Similarly, the nitro cellulose membrane (one layer) and the filter paper (six layers) were cut to the exact size of the gel. The gel, the NC membrane and the filter papers were then soaked in a tray containing the blotting buffer for 10 min. After 10 min, the blotting sandwich was assembled. During sandwich assembly preparation care was taken to avoid air bubbles between the gel and NC membrane. After assembling the blotting sandwich, the cassette was inserted into the apparatus filled with the blotting buffer. The gel was placed towards the cathodic end; since proteins are negatively charged (due to presence of SDS) their migration will be towards anode. After inserting the cassette into the apparatus lid was placed over the buffer tank and with the help of connecting cords the apparatus was connected to power supply and electro transfer was carried out overnight at 50 volts. The presence of SDS facilitates the migration of proteins. Once out of the gel, the proteins come in contact with the nitrocellulose membrane, which binds the protein very strongly on to the surfaces as a band, thus producing a replica of original gels. However, the protein location and detection can only be assessed after immunodetection.
Stage III: Immunodetection
The transferred proteins are bound to the surface of the nitrocellulose membrane and are accessible for reaction with immunochemical reagents.
1. Blocking non specific sites: After electrotransfer, the NC membrane was placed in a petridish and the non specific sites i.e. the unadsorbed sites on NC membranes were blocked by agitating the membrane for 1 h in serum diluent buffer. After incubation, the membrane was washed thrice with rinse buffer.
2. Labeled antibody (anti-human IgG HRP-conjugate): After washing, the membrane was agitated for 1 h in 10ml of 1:1000 diluted anti-human IgG HRP conjugate Excess antibody enzyme conjugate was removed by washing the membranes thrice with rinse buffer.
3. Substrate solution: After washing, the membrane was agitated in substrate solution (TMB/H2O2) till blue/ grey colored bands were observed. The blot was photographed for permanent record.
Immunofluorescence (dot blot method)
Principle
Immunofluorescence is a process in which dyes called fluorochromes are exposed to UV, violet, or blue light to make them fluoresce or emit visible light. Dyes such as rhodamine B or fluorescein isothiocyanate (FITC) can be coupled to antibody molecules without changing the antibody’s capacity to bind to a specific antigen. Fluorochromes also can be attached to antigens. Immunofluorescence can qualitatively determine antibodies. FITC labeled specific antibody is added to an antigen-spotted nitrocellulose membrane and it is allowed to react with the antigen that is adsorbed on to the nitrocellulose membrane. Any free fluorescent labeled antibody is washed away. If the antibody is specific to the antigen, the location of the fluorescein-tagged antibody will be visible by its red-orange color spot.
Chemicals and reagents
1. Antigen:
a. Fraction no 2 ( serum samples1, 2 and 3)
2. Phosphate buffer saline (PBS) pH 7.4 (equilibration buffer): (20X/1000ml)
3. Rinse buffer: 50ml of 20X PBS was diluted to 1000ml with distilled water. After mixing, 1ml of tween 20 was added to it.
4. Serum diluent buffer: 1 gm of egg albumin was dissolved in 100ml of rinse buffer.
5. Labeled antibody: Goat anti-human fluorescein isothiocyanate (FITC) was purchased from Bangalore Genie Pvt Ltd Bangalore India.
Other requirements
1. Nitrocellulose membrane strips
2. Micro pipettes 2-20 and 200-1000µl and tips.
3. Glass pipettes 5ml and 10ml.
4. Glass beakers and conical flasks
5. Measuring cylinders
6. Wash bottle
7. Petridish
8. Shaker
9. UV cabinet
Method
2-5µl of the antigen (Fraction no.2, serum samples1, 2 and 3) was spotted on nitrocellulose membrane strips. After air drying, the unadsorbed (non-specific) sites on the membrane was blocked by agitating the membrane in 10ml blocking buffer (taken in a petridish) for 1 h on a shaker. After incubation, excess blocking buffer was removed by washing the membrane 3 times with rinse buffer (3 x 5 min each). After washing with rinse buffer, the membrane was agitated in 1:200 diluted goat anti-human IgG-FITC for 30 min on a shaker. Excess goat anti-human IgG-FITC conjugate was removed by washing the membranes thrice with rinse buffer (3 x 5 min each). After air drying, the membrane was placed in a UV cabinet to observe the fluorescence spot developed.
References:
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2.Nakane, P.K. and Kawaoi, A. 1974. Peroxidase-labeled antibody. A new method of conjugation. J.Histochem. Cytochem. 22:1084-1091.
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9.Principles of applications: Hermanson , G.T.Krishna Mallia and Smith P.K(1992) immobilized affinity ligand techinques, academic press INC, London and New York (exhaustive coverage of affinity chromatography producers including immunoaffinity methods deals with numerous procedures for isolation of antibodies e.t.c
10.P.Metej and Schuk, editor, affinity separations; a practical approach (1997) , IRL press (Oxford) Porath, J., Maisano, F., and Belew, M. (1985) Thiophilic adsorption—A new method for protein fractionation. FEBS Lett. 185, 306–310.
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