An antibody binds to an antigen to start an immune response. Certain antibodies get attached to specific antigens. This reaction forms the basis of identification tests. Such a reaction can be viewed only under the microscope by a labeled antibody. A label can be an enzyme, a fluorophore or colloidal gold. The enzyme can produce a chromogenic reaction making identification possible. The fluorescent microscope is used to view a fluorophore label. The electron microscope is used to view colloidal gold.
Antibody labeling can be done by two methods: Direct labeling and indirect labeling. As the name implies direct labeling uses a single primary antibody for the labeling process. This antibody is covalently bonded to a labeling molecule. Then the primary antibody is added to the antigen complex and incubated. Primary antibody attaches to the specific antigen. Excess antibody is washed off. Attachment of the label to the primary antibody can lead to a little loss in specificity. Indirect labeling exercises two antibodies. The primary antibody is used for the identification and detection. The secondary antibody is used for labeling and amplification of the label. Secondary antibodies also have a property to bind nonspecifically to other molecules.
A label is a molecule which aids in the identification of the antigen-antibody complex. In order to fill this task, the label should be covalently bonded to the primary or secondary antibody. The antibody is generally a protein molecule which is built by multiple amino acids with chain lysine group. All the labeling procedure targets this lysine group for attaching the label. Four kinds of covalent bonds are used. NHS bond is formed in labels which contain this active side chain. In a case of labels, which are protein or enzyme, a heterobifunctional reagent is used for bonding purpose. Both the antibody and the label contains many lysines. So, one of the protein molecules is modified having a modified active group X and the other protein molecule is modified having active group Y. Then the modified label and antibody side chains react to form the labeled antibody. Carbodiimides bonds are used for labels like dye. These labels contain carboxyl group which reacts with the amine group of the antibodies.
Radioimmunodiagnosis have been practiced since 1988. The radioisotope is used for the labeling purpose in this approach. There are many numbers of radioisotopes used. The choice of isotope varies depending on the application. Broadly based on the target and pharmacokinetic action antibodies can be classified into four types. Agonistics antibodies which mimic the endogenous ligand and leads to the death signal. Blocking antibodies which bind to the ligand or changes the receptor. Antibodies that initiates the effector functions. Conjugated antibodies which are conjugated with cytotoxic substances.
Selection of radioisotope
The choice of a radionuclide for antibody labeling depends on the target and application. The following properties are the basis of selection of an isotope.
Pharmacokinetics of the antibody and half-life period of the radioisotope should be the same.
120-200 keV energy photons should be emitted from the radioisotope for imaging purpose.
For imaging and therapy application, isotopes which emit gamma radiation is used. For therapy alpha and beta radiation emitting isotope are also used.
Halogens and metal cations are used for radiolabeling. Radioiodination of proteins is an electrophilic substitution reaction. Radiolabel is covalently bond with tyrosine residue of the protein molecule. In the case of radiolabeling by metal cations, the antibody should be modified by a chelating agent. Pentetic acid (DTPA,diethylenetriaminepentaacetic acid) is used as a chelating agent. ANtibodies retain their immunoreactivity for one or two acid molecules per a single antibody. Acid to protein ratio is to be strictly maintained in this method. The reactivity of antibody decreases with increase in the acid-antibody ratio.
Preparation of antibodies for labeling: Antibodies are to be purified from a serum sample. The antibodies are stored in a neutral pH. The protein solution is stored at 10mg/mL.
Preparation of Immunoconjugates: Mix derivative of pantetic acid and antibody solution in the ratio of 1:5. Allow the reaction for 2 h at room temperature. Ultracentrifuge the sample and remove the unreacted pantetic acid.
Attachment of DTPA: Prepare an antibody solution at 5 mg/mL in a sodium carbonate buffer. Dissolve cyclic anhydride of DTPA in anhydrous dimethyl sulfoxide. Add 5molar equivalents of cyclic anhydride to 1 mL of the antibody solution. Allow the reaction to proceed at room temperature for 2 hours. Ultracentrifuge the mixture to remove unreacted DTPA.
Radiolabeling with Yttrium-90 or Indium-111: Thaw an appropriate volume of antibody-DTPA solution. Add the desired amount of Indium chloride/Yttrium chloride and gently tap to mix them. Allow the reaction to proceed at room temperature for 1.5 hours. Terminate the reaction by adding DTPA to the solution.
Quality control in radiolabeling
Determination of residues conjugated to an antibody molecule
- To a vial containing 10 p.L of the crude mixture containing antibody to DTPA mixture in a 1:2 ratio add 4pL of nonradioactive Indium chloride and 1 pL of radioactive indium chloride.
- Allow the reaction to proceed for 1h at room temperature.
- Prepare 3 cm long ITLC places. Place 2 FL of the labeling mixture about 1 cm from the bottom of each plate. Air dry the plate. Elute it with 0.9% saline solution.
- Determine the radioactivity associated with the protein at the origin. Measure radioactivity of unreacted DTPA at every 0.5 cm.
- Calculate the number of DTPA resides per protein. Multiply the fraction of counts associated (protein-bound radioactivity) at the origin by the total DTPA to antibody ratio used in the conjugation reaction. For e.g. if our DTPA: Ab ratio is 2:1 and the fraction of radioactive bound protein are 0.5, then the DTPA residues per protein are (2*0.5) 1 per protein.
Determination of the radiochemical purity of the radio-labeled antibody(ITLC method)
- Dilute the sample to be tested to a concentration of about 2 mCi/mL using PBS as the solvent. Divide the diluted sample into two portions.
- To one portion add 10 mL of 0.01 M DTPA,pH 6.5. Apply each sample to a separate plate. Allow the samples to air dry.
- Elute both the plates with either 0.9% saline or 0.06 M acetate buffer.
- Stop eluting when the solvent front is about 1 cm from the top. Seal the plates in a tight glass jar and allow them to air dry. Scan the plates for radioactivity.
- Divide radioactivity associated with the origin by the total radioactivity of the plate. This gives the radiochemical purity.
- Analyze both the test samples for radioactivity. It is done for 5-7 days with 24 hour time interval. This gives the stability of the immunoconjugate.
Covalent binding of Antibody – Procedure
1)Bicarbonate buffer: Dissolve 17.3 g of sodium bicarbonate and 8.6 g of sodium carbonate in 1 liter of distilled water.
2) Take 1 mg of purified antibody and dialyze it against bicarbonate buffer.
3) Make 1 mg/mL antibody solution and measure OD at 280 nm.
4a) Fluorescein isothiocyanate(FITC): Make 1mg/mL FITC in DMSO solution. Add 45 ml per milligram of protein. This should generate a molar ratio of 5:1. Mix and incubate at room temperature for 2 hours.
4b) Biotin: Makeup Sulfo-NHS-LC-Biotin solution to 1mg/ml concentration in pure water. Mix and incubate at room temperature at least 2 hours
5) Dialyse the solution in phosphate buffered saline (PBS) to remove unconjugated reagents. This process is incubated for 2 hours at room temperature.
6) For FITC conjugates measure OD at 280 nm and 493 nm.
Non-covalent labeling of antibodies
Covalent labeling requires a lot of purified antibodies for labeling purpose. Non-covalent labeling works with a few antibodies. Noncovalent labeling joins the Fab region of the antibody. Simply mixing of monovalent Fab fragments with an unconjugated complex of antibody produces labeling complex. For single color immunolabeling, it is not necessary to remove free uncomplexed Fab fragments. In the case of multiple labeling, residual Fab fragments are also necessary.
Protocol for generating Antibody-Fab fragment complex
- Incubate unconjugated primary antibodies with labeled monovalent Fab fragments against IgG ratio at 1:2. It is mixed in a small volume and kept in a small tube for 20-30 min at room temperature.
- Staining buffer serum is prepared to contain 10%-20% primary antibody in the concentration of 1 mg/mL.
- Dilute the primary antibody with-Fab fragment with staining buffer solution and incubate for 15-30 min at room temperature to block unbound labeled monovalent Fab fragments.
- Prior to application the resultant antiboy-Fab fragment complexes to tissue section, preincubate the antibody solution for 5-10 minutes.
Enzyme labels for light microscopy
Enzyme labels can be seen in light microscopy. It has been in use for more than 300 years. Enzymes labels are used for screening in indirect methods. Enzyme labels are usually added to the secondary antibody. Few examples of enzyme labels include HRP (Horse Radish Peroxidase) and calf intestinal alkaline phosphate(AP). Glucose oxidase and b-galactosidase are rarely used in the application. All enzymes are protein molecules so they use the covalent bonding technique of heterobifunctional reagent. They are visualized by histochemical methods and chromogenic reactions. They produce a water insoluble during the reaction. Peroxidases convert phenols and amines into an insoluble pigment in the presence of hydrogen peroxide. Alkaline phosphates reaction is identified by a coupling mechanism. This phenomenon is targeted to release a chromogenic pigment on hydrolysis. It is based on either the reduction of tetrazolium salts or production of colored diazo compounds.
Basic protocol for immuno enzymatic labeling
- Deparaffinize and rehydrate tissue section. It is rinsed using distilled water.
- Extract antigen from the tissue solution. Place small portions of tissue in a Coplin jar with a retrieval solution of choice (e.g. Citrate acid, pH 6, conc 10mM) and heat at 90-110 deg. Celsius in a microwave or an autoclave.
- Wash with PBS solution for 2-3 minutes.
- Place a hydrophobic barrier on top of the tissue section for tests. It should be kept in wet condition.
- Block the enzyme and wash in PBS solution.
- Add primary antibody to the blocking solution and incubate at room temperature for 60 minutes.
- Add HRP labeled or AP labeled antibodies and incubate for 30-60 minutes at room temperature. Wash for excess antibody removal.
- Incubate the sample sections with enzyme substrate and incubate until the color develops. Counterstain in case of multiple labeling.
Key considerations for antibody labeling
Any antibody solution or mixture may contain substances other than the antibody. They can be anything from buffer solution, salts, other proteins, and additives. In every labeling procedure, the antibody mixture is purified before labeling process. In certain cases, it is necessary to purify the mixture more than once. Purification will involve the removal of high molecular weight like proteins and low molecular weight compounds like buffer and salts. Different labeling methods may have different effects of additives. Since most of the labeling procedure exploits the lysine residue for labeling, substances with primary amines should be avoided for additives. Dialysis is used for the removal of unwanted low molecular additives. Frequent change in buffer (as much as 3-4 times) is recommended for effective removal. Instead of making one huge buffer dialysis system, many small scale dialysis system is recommended for better results.
Antibody concentration and purity
For many labeling reactions, the antibody should have purity levels from 90-95%. Concentrations of antibody used also should be in the range of 0.5-1 mg/mL.
Application – Characterizing Higher order Monoclonal Antibodies
This section deals with how biologically useful and functionally relevant information about IgG1 is investigated. It is done by using two covalent labeling approaches: Hydroxyl radical based footprinting (HRF) and carbodiimide based carboxy group labeling by glycine ethyl ester (GEE) tagging. This system helps in understanding the sites for monoclonal labeling and the potential structure assessment. Discovery and development of monoclonal antibodies (mAbs) are important for a wide variety of molecular immunology studies. Methods like X- ray crystallography and nuclear magnetic resonance have a relatively high resolution but are complex. Other biophysical techniques such as circular dichroism (CD), fluorescence and infrared have low resolution. Resolution of mass spectroscopy lacks specificity so have a medium resolution. This limitation is overcome by covalent labeling (CL)- Mass spectroscopy. The digested sample is subjected to LCMS. Tandemly MS (MS/MS) is used to identify the peptides and localize sites of modification. The intensities from the selected ion chromatograms are integrated to compute the abundance of each peptide form. Dose-Response (DR) curves are generated to monitor the loss of the unmodified fraction as a function of oxidation exposure time. The amount of labeling of a given region is proportional to solvent accessibility, the inherent reactivity of the constituent residues and the solution condition. Various MS approaches have been employed in the characterization of primary structure and various post-translational modifications. Application of hydroxyl radical based protein footprinting and mAb characterization. These methodologies find their application in conformation variation, the conformational difference in mAb and IgG2 isomers. The goal of this two reversible CL procedure is to define the sites of labeling and thus revealing the potential resolution for the structure assessment. Secondly to determine the reproducibility of this technique to find its suitability in the characterization of mAb.
In HRF approach, synchrotron X-ray radiation was used to irradiate the solution containing IgG1. This process produces a high flux of radicals on a millisecond timescale. These reactive hydroxyl groups, present in the solvent, attacks the amino groups (side chains) of protein molecule present in solvent accessible portion. The end product of this reaction is a stable modified side chain. In GEE approach the label attacks the carboxyl group of the protein molecule. As earlier, it can only react with the solvent accessible portion only. The two techniques complement each other. HRF approach gives the data about the amino groups while GEE tagging gives information about the C-terminal carboxyl group. Replicates of these samples are tested for verifying the reproducibility of tests. Labeling information is coupled with protein generated computational models. These labeling patterns produce the distinctive features of the structure of IgG1. Thus this technique can be used for the pharmaceutical application of protein fingerprinting.
The mAb sample was buffer exchanged into 10 mM phosphate buffer, 150 mM NaCl, pH 7.4.
For HRF experiments, a 5 ml sample of the mAb (5 mM) was exposed to an X-ray source. The exposure was done for 0-15 milliseconds at ambient temperature. The experiments were performed at a ring energy of 2.8 GeV of beam current of 212 and 200 mA. To quench secondary oxidation of methionine, 10 mM Met-NH2 X HCl buffer (pH 7.0) was added immediately after irradiation.
For GEE tagging, 10 mg of the mAb at the concentration of 1mg/ml was taken for labeling reaction. A stock solution of GEE and EDC are made at a concentration of 1M and 0.025 M respectively. The solvent used for this buffer is 10 mM phosphate buffer, 150 mM NaCl, pH 7.4. The stock solution of GEE and EDC are added to their reaction vial to make a concentration of 210 mM and 7mM of them respectively. the labeling reaction is carried out at room temperature for 0 to 10 min. The reaction was quenched by addition of 1% formic acid. It is added till the final solution becomes 0.1% (v/v) of formic acid.
Deglycosylation and digestion
Radiolyzed mAb samples at 3.7 mg of total protein per sample are deglycosylated using 1 mL of peptide-N-Glycosidase F enzyme or 30 min at 37 deg. Celsius. It is precipitated with 10% trichloro acetic acid/ acetone overnight. These samples were washed 3 times with acetone and air dried. Protein samples were then reconstituted in 50 mM Tris, 8 M urea buffer (pH 7.8), reduced with 10 mM dithiothreitol (DTT) for 45 min at 37 °C and alkylated with 25 mM iodoacetic acid (IAA) for 45 min at room temperature in the dark. Protein samples were digested with Lys-C for 3 h at 37 °C at 1:20 w/w enzyme to protein ratio followed by trypsin digestion at 1:20 w/w enzyme to protein ratio at 37 °C overnight.
The GEE-labeled mAb samples were buffer exchanged two times with 20 volumes excess of 8 M urea and 40 mM DTT using a 0.5 mL 3000 MWCO filter. The protein samples are concentrated to approximately 50 μL and then deglycosylated at 37 deg. Celsius for 1 hour using 2 μL of PNGase F. Next, protein samples were buffer exchanged two times with 20 volumes excess of 50 mM Tris, 2 M urea buffer (pH 7.8), concentrated to approximately 50 μL, and then reduced and alkylated with DTT and IAA, respectively, on the filter. Finally, mAb samples were transferred to Eppendorf Tubes and digested with Lys-C and trypsin at 37 °C overnight.
Peptide mixtures derived from the hydroxyl-labeled and GEE-labeled proteins were separated by reversed phase high-pressure liquid chromatography (HPLC) The separation is done by a gradient formed with mobile phase A (100% water with 0.1 % formic acid) and mobile phase B (100% acetonitrile [ACN] with 0.1% formic acid). The gradient program consisted of ramping mobile phase B from 2 to 40% over a period of 60 min, 40% to 43% over a period of 5 min and 43% to 83% over a period of 10 min at ambient temperature and a flow rate of 300 nl/min. A full MS1 scan was obtained for eluted peptides in the range of 380 to 1800 m/z followed by twenty data-dependent MS/MS scans. MS/MS spectra were generated for peptides with a minimum signal of 2000 by collision-induced dissociation of the peptide ions at a normalized collision energy of 35%, an isolation width of 2.5, and an activation time of 30 ms to generate a series of b- and y-ions as major fragments.
Data analysis for both hydroxyl radical labeling and the carboxyl group labeling experiments was performed using a commercial software package specifically for the automated analysis of covalent labeling experiments. The software uses the tandem MS data for identification and localization of labeled residues. Every single ion chromatograms from the MS data is analyzed for quantifying the extent of modification. Areas under the SIC plots are used to construct DR curves for each labeled peptide as a function of label exposure time.