Preparation of enzyme conjugated antibodies

Antibody conjugation protocol, enzyme conjugated antibody, antibody conjugation methods, enzyme-conjugated antibody elisa, conjugated enzymes, function of enzyme conjugate in elisa, antibody production steps, mechanism of antibody production pdf

Aim: Preparation of enzyme conjugated antibodies

BACKGROUND

The enzyme-antibody conjugation is important tool in immunochemistry. Although certain enzymatic conjugations, like sortase-based protein terminal conjugation or even through protein engineering, are becoming more and more prominent, antibody conjugation is often accomplished through chemical reactions.

The process of conjugating enzymes to antibodies entails the creation of a strong, covalent bond between an enzyme (such as alkaline phosphatase, urease, or horseradish peroxidase) and an antibody.

Either a monoclonal or polyclonal antibody that is specific for the antigen. Neither the enzyme’s active site nor the antigen-combining site is functional changes.

The chemistry of HRPO or urease cross-linking purified monoclonal or polyclonal antibodies to immunoaffinity (IgG) Alkaline phosphatase cross-linking chemistry is presented.

The roots of the horseradish plant frequently contain the enzyme horseradish peroxidase (HRP). This multi-isoform metalloenzyme is frequently used in numerous biochemical processes. The oxidation of hydrogen peroxide to water and oxygen gas is essentially catalysed by the enzymes.

Horseradish peroxides is the enzyme that is most frequently utilized in the immunoreagent (the antibody enzyme conjugate) manufacture. There are numerous ways to bind this inexpensive enzyme to the immunoreagent.

Through a number of intermediates, HRP is known to catalyse the oxidation of luminol to 3-aminophthalate. There is the production of 428 nm low intensity light during this reaction. However, when specific chemicals are introduced, the emission of light can be multiplied by a factor of 1,000, making it much simpler for spectrophotometers to detect the light.

Additionally, the substances may make the reaction more sensitive. Enhanced chemiluminescence refers to this increased light production. There are several different sorts of enhancers available now that can be used in the HRP reaction. Most of these enhancers are phenols that have been changed (chiefly indophenols).

When creating a conjugate, one wants to produce something that is not only highly active but also reasonably priced. To put it another way, one will pick the conjugate that, in an enzyme immunoassay, produces a predetermined optical density (OD) value at the lowest initial antibody and enzyme concentrations.

REQUIREMENTS

S. no.
1	1 mg/ml antibody solution (monoclonal or polyclonal antibodies),
2	0.1 M phosphate buffer,
3	0.1 M carbonate buffer,
4	Horseradish peroxidase,
5	Freshly prepared Sodium periodate (NaIo4),
6	Freshly prepared Sodium borohydride (NaBH4),
7	Ammonium sulphate [(NH4)2SO4] solution,
8	Tris-EDTA-NaCl (TEN) buffer,
9	Bovine serum albumin (BSA),
10	Glycerol,
11	Dialysis membrane,
12	Sephadex G-25,
13	Pipet,
14	Glass wool

PROCEDURE

1. Dialyse 1 mg/ml antibody solution against 2 lit. of 0.1 M phosphate buffer (pH 6.8) for overnight at 4˚c with gentle stirring,¹
2. Dissolve 10 mg HRPO in 1ml 0.1 M carbonate buffer (pH 9.2),
3. Mix 0.25 ml freshly prepared NaIO₄ solution with 0.25 ml of 10mg/ml HRPO-carbonate mixture, incubate at room temperature for 2 hr in dark condition.
4. Take a pipet fitted with glass wool and blocked the tip with parafilm,
5. Add 1 ml of the dialyzed 1 mg/ml antibody solution to 0.5 ml of the 10 mg/ml HRPO solution, add 0.25 g Sephadex G-25 to the antibody-HRPO mixture, incubate in the dark for 3 hr at room temperature, ³
6. To elute conjugate, wash the column with 0.75 ml of carbonate buffer,
7. Add 38µl freshly prepared NaBH₄ solution to the elute and incubate in dark for 30 min at room temperature,
8. Add 112µl freshly prepared NaBH₄ and incubate in dark for 1hr at room temperature,
9. Add 0.9 ml of saturated (NH₄)₂SO₄ solution and stir gently for 30 min at 4˚c, centrifuge at 10,000×g for 15 min at 4˚c,
10. Discard supernatant and resuspend pellet in 0.75 ml TEN buffer, ¹
11. Dialyse resuspended pellet overnight in 2 lit TEN buffers at 4˚c, in the morning change the TEN solution and continue dialysing for 4 hr,
12.  Remove the conjugate from dialyse membrane and add sufficient Bovine serum albumin to bring the conjugate solution to a final concentration of 20 mg BSA/ml, ²
13. Add an equal volume of glycerol and store at -20˚c.

CONCLUSION

Enzyme-antibody conjugate are successfully prepared with the help of horseradish peroxidase and store up to 2-4 months at -20˚c.

[ps2id id=’references’ target=”/][ps2id id=’1′ target=”/]

REFERENCES

1. Scott E. Winston, Steven A. Fuller, Michael J. Evelegh and John G.R. Hurrell, Current Protocols in Molecular Biology, 2000.
2. Ramesh Kumar K, Sneha Xiavour, Swarna Latha, Vijay Kumar and Sukumaran, “Anti-human IgG horseradish peroxidase conjugate preparation and its use in ELISA and Western blotting experiments”, Journal of chromatography separation techniques, 2014.
3. Antoinette Jeanson, Jean-Michel Cloes, Mireille Bouchet and Bernard Rentier, “Comparison of conjugation procedures for the preparation of monoclonal antibody-enzyme conjugates”, Journal of immunological methods, vol. 111, issue 2, 1988.
4. M.J. O’Sullivan, E. Gnemmi, D. Morris, G. Chiregatti, M. Simmons, A.D. Simmonds, J.W. Bridges and V. Marks, “A Simple Method for the Preparation of Enzyme-Antibody Conjugates”, North-Holland Biomedical Press, vol. 95, November 1978.
5. J. Burns, Maria Hambridge and C.R. Taylor, “A Comparative study on three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates”, J. clin. Path, 1974, 27, 548-557
6. https://www.labome.com/method/Antibody-Conjugation.html
7. https://michigandiagnostics.squarespace.com/use-of-horseradish-peroxidase-and-alkaline-phosphatase-in-chemiluminescence-the-pros-and-cons
8. https://www.pharmatutor.org/articles/preparation-of-anti-human-immunoglobulin-g-horseradish-peroxidase-conjugate-by-western-blot-technique

Also read:

• Purification of Immunoglobulin by Precipitation
• Microplate based Alamar Blue Assays
• Isolation of Genomic DNA from E. coli
• Estimation of DNA and its purity check using UV spectrophotometer (A260 measurement)
• Study of Polyploidy in Onion Root Tip Bycolchicine Treatment

FAQs

1. Can we use any other simple catalyse instead of horseradish peroxidase?

 Ans: Alkaline phosphatase is use instead of horseradish peroxidase.

2. How can we confirm enzyme-antibody conjugate?

 Ans: Enzyme-antibody conjugate confirm by ELISA and western blot.

 

🔴 Would you like to attempt Labmonk Daily quiz? Click here

🔵 Check out Jobs & Exam Notices. Labmonk Notice Board

🔴 Labmonk Scholarships. Click here

🔵 Labmonk Blog. Click here

🔴 Do you need notes? Click here

Watch Career related videos on Youtube: Watch now !!