Editing Peptide Conjugation to KLH

== Coupling Peptides to KLH or BSA ==

m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is water insoluble and sensitive to moisture, stored at -20 oC, soluble in DMF (Dimethylformamide), DMSO (Dimethylsulfoxide) its NHS ester and maleimide reactive groups react with amino and sulfhydryl moites, respectively.

'''http://excellgen.net/eln/images/e/ec/Mbs_linker.png'''

1. Dissolve <s>5 mg</s> of KLH or BSA in<s>0.5</s> mL of 0.01 M phosphate buffer (pH 7).

25 mgKLH--> 2.5 ml Buffer

2. Dissolve<s>3 mg</s> of MBS in<s>200 µL</s> DMF.&nbsp; 30 mg-->2 ml

3. <s>Add 70 µL of MBS solution to 0.5 mL of KLH solution.</s> Add 700 µl dropwise, or 10&nbsp;µl&nbsp; while mixing

4. After stirring or rotating for 30 min at room temperature, the KLH/MBS or BSA/MBS solution is passed through a PD-10 column using 0.05 M phosphate buffer (pH 6). Collect the 3.5 mL of purified KLH/MBS or BSA/MBS. Add 0.5 mL of water.

5mg/4 ml, ~ 1mg/ml

5. Dissolve 5 mg of peptide in 100 µL of DMF. Rapidly add 1 mL of purified KLH/MBS or BSA/MBS. Shake rapidly and immediately add 11 µL of 2 N NaOH.

5 mg peptide to ~1 mg&nbsp;activated&nbsp;KLH

6. Check the pH with pH paper. It should be 7.0–7.2. Too high pH or too low pH will stop the reaction between KLH/MBS or BSA/MBS and peptide. If needed, add immediately an appropriate amount of 0.5 N HCl or 2 N NaOH to change the pH.

7. Stir or rotate the solution 3 h or overnight at 4°C. Finally, add 3 mL of ammonium bicarbonate (0.1 M) before lyophilizing the reaction solution

This type of conjugation requires either a N- or C-terminal cysteine.

A total of 3 mg m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is dissolved in 200 ul dimethyl formamide (DMF).

Next, 70 ul MBS/DMF is added to a KLH solution consisting of 5 mg KLH in 0.5 ml 10 mM phosphate buffer, pH 7.0, and it is stirred gently at room temperature for 30 min.

The free crosslinker is removed using a Sephadex G25 size exclusion column. First, the column is equilibrated in 50 mM phosphate buffer (pH 6.0). The KLH reaction mixture is then loaded, and eluted using 50 mM phosphate buffer (pH 6.0). Collect the 3.5 mL of purified KLH/MBS or BSA/MBS. Add 0.5 mL of water.



Conjugation to Peptides



'''Protocol 1'''

A total of 5 mg synthetic peptide is dissolved in 100 μL of DMF. Rapidly add 1 mL of purified KLH/MBS or BSA/MBS. Shake rapidly and immediately add 11 μL of 2 N NaOH to adjust the pH between 7.0-7.2. The reaction will stop if the pH is not in the right range.

The mixture is stirred for 3 hours at room temperature or overnight at 4°C.

Add 3 mL of ammonium bicarbonate (0.1 M) before Lyophilization.

'''Protocol 2'''



1. Reconstitute one vial of the maleimide-activated protein by adding 200 ul of ultrapure water to make a 10 mg/ml solution.

Note: Vortex mixing or heating will cause the protein to precipitate.

2. Dissolve the sulfhydryl-containing hapten in a volume of Conjugation Buffer equal to 1.0-2.5 times the volume of reconstituted activated protein. For example dissolve 2 mg of peptide in 200-500 ul of buffer and add it to the reconstituted protein. Alternatively, if the peptide is freely soluble it may be added as a solid to the protein solution.

Note: For haptens with limited solubility, '''DMSO''' may be used for solubilization. Use approximately '''30'''% DMSO in the final conjugation solution or the carrier protein may irreversibly denature.

3. Immediately mix the peptide and activated protein and react for 2 hours at room temperature.

4. Purify the conjugate by desalting or dialysis to remove EDTA and sodium azide.






'''Note:'''

1. DMF was used as the solvent to solubilize peptides instead of PBS or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7).

2. The last desalting or dialyzing step to remove uncoupled peptides in the traditional method was eliminated.

3. Three milliliters of 0.1 M ammonium bicarbonate was added to the carrier protein conjugated peptide solution before lyophilization, which helped the lyophilization process.

A. Checking Peptide Thiol Groups (see Hint #2)

1. Weigh out about 1 mg of peptide of interest into a tared 50 ml polypropylene conical tube (see Hint #3).

2. Add 0.5 ml of Ellman's Reagent. The solution should turn bright yellow.

3. Dilute the mixture 1:50 in 0.1 M Sodium Phosphate Buffer. Read the absorbance at the 412 nm (Abs412) wavelength, comparing the reading to the Abs412 of the reagent alone diluted to the same concentration.

4. Calculate the apparent molecular weight of the peptide based on thiol groups, using a molar extinction coefficient (εmax) of 14,000. Compare this to the expected molecular weight of the peptide. They should agree within a factor of three, with the estimated molecular weight usually higher (see Hint #4).

== Coupling of Peptide to KLH ==

This protocol will yield enough peptide-KLH for the immunization of two rabbits with approximately five injections per rabbit (see Hint #5).

1.Weigh out 100 mg of Keyhole Limpet Hemocyanin (KLH) and dissolve the solid in 2 ml of ddH2O.

2. Sonicate and vortex to encourage the KLH to dissolve (takes approximately 4 hours), and mix the solution by continuous rotation using a "nutator" rotating platform or similar device at 4°C.

3. Once the KLH is dissolved, dialyze the KLH solution against 2 liters of 0.1 M Sodium Phosphate, pH 7.8, overnight at 4°C (see Protocol on Dialysis of Proteins). This removes any contaminating thiols or amino compounds.

4. Centrifuge 10 minutes at full speed in a microcentrifuge to remove aggregates (see Hint #6).

5. Split the dialyzed KLH solution into 2 aliquots: one for thiol- and one for amino- coupling.

C. Amino Coupling

1. For amino coupling, add 5 mg of peptide to one aliquot of KLH. Add the peptide as a solid if it is soluble in water; otherwise, add the peptide directly from a 100 mg/ml stock of peptide dissolved in DMSO. Precipitation does not matter and often happens.

2. Add Glutaraldehyde to a final concentration of 0.1% (CAUTION! see Hint #1).

3. After adding the Glutaraldehyde, check the pH of the peptide-KLH-Glutaraldehyde reaction by spotting 2 μl onto pH paper. If the pH is low, adjust to pH 7.8 using NaOH.

4. Incubate for 8 to 12 hr at 4°C, rotating gently on a nutator or equivalent.

5. Add a tiny pinch (a few milligrams) of Sodium Borohydride (NaBH4) to quench the remaining Glutaraldehyde. Make sure the sample is in a larger tube (i.e. 15 ml conical) since it tends to fizz up.

6. Incubate the sample for 8 to 12 hr at 4°C with gentle rotation. This is the "glut conjugate".

D. Thiol Coupling

1. For the thiol coupling, warm the second aliquot of KLH solution to room temperature.

2. Add 0.11 volume of IAA-NHS ester.

3. Incubate at room temperature. After 10 min, the KLH-IAA-NHS reaction will start to get a little cloudy.

4. Load the reaction onto a P-10 (Bio-Rad) column equilibrated with 0.1 M Sodium Phosphate, pH 7.8. Make sure the column is at least 10 times the volume of the sample.

5. Elute the KLH-IAA-NHS with 0.1 M Sodium Phosphate, pH 7.8.

6. Pool the KLH containing fractions by color (greyish green).

7. Add 5 mg of peptide to the KLH containing fractions. Add the peptide as a solid if it is soluble in water; otherwise add peptide directly from a 100 mg/ml stock of peptide dissolved in DMSO. Precipitation does not matter and often happens.

8. Add Glutaraldehyde to a final concentration of 0.1%.

9. After adding the Glutaraldehyde, check the pH of the peptide-KLH-glutaraldehyde reaction by spotting 2 μl onto pH paper. If the pH is low, adjust to pH 7.8 using NaOH.

10. Incubate for 8 to 12 hr at 4°C, rotating gently on a nutator or equivalent.

11. Add a tiny pinch (a few milligrams) of NaBH4 to quench the remaining Glutaraldehyde. Make sure the sample is in a larger tube (i.e. 15 ml conical) since it tends to fizz up.

12. Incubate the sample for 8 to 12 hr at 4°C with gentle rotation. This is the "glut conjugate".

13. Pool the coupled peptides (glut conjugates) from the amino and thiol couplings.

Dilute to 5 ml and add NaCl to 0.15 M final concentration. If there is a precipitate, sonicate vigorously to break it up.

14. Split the peptide-KLH into 1 ml aliquots (each aliquot will immunize two rabbits) and freeze them to store.





== Protocol 4 ==

1. Dissolve KLH in 10 mM NaPO4 buffer, pH 7.2 to final conc 16 mg/ml<br/>&nbsp;2. Make 280 mg/ml of MBS in Dimethylformamide freshly<br/>&nbsp;3, Add 10 ul of MBS while stirring to avoid a high local concentration and contine to stire for 30 min at room temperature<br/>&nbsp;4, Centrifuge at 15000 rpm at 4 oC for 5 min. Take a supernatant<br/>&nbsp;5. Separate the MBS-activated KLH from the free MBS by gel filtration on&nbsp;&nbsp;G25 column equilibrated with 50 mM sodium phosphate buffer, pH 6.0<br/>&nbsp;6. Pool MBS-activated KLH and divide equally into 10 microcentrifuge tubes. Store at -80oC until use<br/>&nbsp;7. Thaw MBS-activated KLH in one tube<br/>&nbsp;8. Add 0.5 vol of 0.2 M Na2HPO4 (pH should be 7.3 to 7.5)<br/>&nbsp;9. Dissolve 1 mg of the peptide containing cystein residue at the N- or C-terminus in 0.5 ml of&nbsp;&nbsp;distilled water, and add to MBS-activated KLH<br/>&nbsp;10 Stir the reaction for 3 hours at room temperature<br/>&nbsp;11. Divide equally into 10 microcentrifuge tubes. Store at -80oC until use

=== &nbsp;Deoxidation of Peptide ===

<br/>&nbsp;1. Use Ellman's reagent to determine if there are free sulfhydryls on the terminus of the pep<br/>&nbsp;2. Dissolve 1 mg of the peptide containing cysteine residue at the N- or C-terminus in 0.5 ml of 20 mM NaPO4, pH 8.5<br/>&nbsp;2. Add NaBH4 to a final concentration of 0.1 M<br/>&nbsp;3. Stand the reaction for 45 min at room temperature<br/>&nbsp;4. Adjust the final pH to 4.0 by adding 0.1 N HCl<br/>&nbsp;5. Stand the reaction for 5 min at room temperature<br/>&nbsp;6. Adjust the final pH to 7.5 by adding 0.2 M Na2HPO4<br/>&nbsp;7. Add to MBS-activated KLH<br/>&nbsp;8. Stire the reaction for 3 hour at room termperature<br/>&nbsp;9. Dialyze against PBS at 4 oC overnight<br/>&nbsp;10 Divide equally into 10 tubes, store at -80 Oc until use

=== &nbsp;Immunization ===

&nbsp;2 or more female rabbits (weight 2.5 Kg) are immunized subcutaneously&nbsp;&nbsp;by standard protocol with ~100 ug of peptide-KLH conjugate emulsified with<br/>&nbsp;Freund's complete adjuvant (FCA) 4 times at biweekly intervals, and boosted with&nbsp;&nbsp;peptide-KLH conjugate in Freund's incomplete adjuvant.&nbsp;&nbsp;One week after boost, bleed abou 70 ml from marginal ear vein of rabbits

4 or more female Balb/c mice (4-5 wk) are immunized at the footpad with 10-20 ug&nbsp;&nbsp;of peptide-KLH conjugate emulsified with Freund's complete adjuvant (FCA) twice at&nbsp;&nbsp;4-d intervals. 3 d after last immunization, boosted with ~20 ug of&nbsp;&nbsp;peptide-KLH conjugate in Freund's incomplete adjuvant

==  ==

== Chemical coupling of synthetic peptides to KLH or BSA ==

The synthetic peptides of figures 1-8 were reduced with lOmM DTT, or with immobilized DTT (Reduetacryl, Calbiochem, San Diego, California, U.S.A., 10 mg Reductacryl per mg peptide) overnight at 4°C, pH8 in lOmM borate or other buffers depending upon peptide solubility, with gentle mixing. KLH was dissolved at a concentration of 10-30 mg/ml in 10 mM phosphate buffer pH 7.2., and BSA was dissolved at a concentration of 20-80 mg/ml for treatment with heterobifunctional linkers MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester), SPDP [N-Succinimidyl 3-(2 pyridyldithio)propionate], or Sulfo-SMCC [Sulfo- Succinimidyl 4(N-maleimidomethyl)-cyclohexane-l-carboxylate]. The hetero- bifunctional linkers were added to the protein solutions in a molar excess of linker to BSA of 100, or of linker to KLH of 1000. MBS was dissolved in DMF at lOmg/ml, SPDP was dissolved in absolute ethanol at lOmg/ml and each of these was added dropwise with mixing into the protein solution, whereas the Sulfo- SMCC dry powder was dissolved into the protein solution. Each protein-linker mixture was reacted at room temperature for 15 minutes to 1 hour and then free linker was separated from BSA-linker or KLH-linker by gel chromatography with Sephadex G25 using 50mM phosphate, pH 6 for KLH-MBS, lOmM acetate, 100 mM NaCl, pH 4.6 for BSA-SPDP, or 100 mM phosphate, 1 mM EDTA, pH 6.5 for BSA- SMCC. The reduced peptides were separated from DTT by Sephadex G10 column chromatography when soluble DTT was used, or by centrifugation followed by filtration through sa 0.22 micron filter when Reductacryl was used. Reduced peptides were combined with linker treated KLH or BSA in molar ratios of 100- 1000 peptide/KLH-MBS, 1-50 peptide/BSA-SMCC, and 0.1-10 peptide/BSA-SPDP. Each reduced peptide was added to the linker-treated protein with mixing, overlayed with N2, and then mixed gently at room temperature for one hour, and then overnight at 4°C. The SMCC-BSA conjugates were further treated with a 1 m final concentration of mercaptoethylamine for 4 hours, and then dialyzed overnight against 100 volumes of buffer (20mM phosphate, 1 mM EDTA, 0.05% sodium azide, pH 6.5) at 4°C. All other conjugates were adjusted to a final concentration of sodium azide of 0.05%. Optimal molar ratios of peptide/protein for the conjugates were determined by solubility and antigenicity (as evaluated by EIA). For example, the best ratios were found to be approximately 1:1 (SPDP- BSA), 10:1 (SMCC-BSA), and 100:1 (MBS-KLH).



== Immunization ==

The rabbit was immunized subcutaneously with 1 mg peptide/carrier conjugate in 1 mL PBS mixed 1:1 with complete Freunds adjuvant. The rabbit was boosted with 0.5 mg peptide/carrier conjugate in 1 mL PBS mixed 1:1 with incomplete Freunds adjuvant at 4 wks and again at 8 wks. The rabbit antiserum was collected 10 d after the last immunization, and the serum was tested using enzyme-linked immunosorbent assay (ELISA).

== ELISA ==

Synthetic peptide (2.5 μM) was prepared in carbonate buffer (pH 9.6) and incubated on a microtiter plate overnight at 4°C. Wells were washed three times with PBS containing 0.05% Tween-20. The remaining sites on the wells were blocked by BSA (10 mg/mL) in PBS/Tween-20 at 37°C for 1 h. After subsequent washing, 300 μL of 1:1000 dilutions of antipeptide antiserum was added to wells and incubated for 2 h at 37°C. The wells were washed three times with PBS/Tween-20 to remove the unbound antibody and then incubated with 1:500 dilution of alkaline phosphatase conjugate of goat anti-rabbit IgG in PBS/Tween-20 for 2 h at 37°C. Wells were washed three times with PBS/Tween-20, and 50 μL of enzyme substrate (p-nitrophenyl phosphate) prepared in water was added for 10 min at 37°C. The plate was read at λ = 405 nm with a micotiter plate reader (StatFax-2100, Awareness Technology, Palm City, FL).

&nbsp;<br/>The improved procedure for producing peptide/carrier protein conjugates used in this study consisted of three major modifications of the traditional protocol.

DMF was used as the solvent to solubilize peptides instead of PBS or 6 M guanidine-HCl/0.01 M phosphate buffer (pH 7).<br/>The last desalting or dialyzing step to remove uncoupled peptides in the traditional method was eliminated.<br/>Three milliliters of 0.1 M ammonium bicarbonate was added to the carrier protein conjugated peptide solution before lyophilization, which helped the lyophilization process.
[[Category:Antibodies]]