TITLE:  Preparation of a protein-LG conjugated to horseradish peroxidase by the periodate method.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SpLG comprises of 4 Ig-binding domains of SpL and 2 IgG Fc-binding SpG domains [1]. This hybrid molecule was found to bind many intact human Ig molecules and Ig fragments [1]. It proved a powerful tool for the binding, detection and purification of antibodies [2].It was reported that chimeric SpLG was a potent mitogen for mouse splenic B cells, and induced cell differentiation and the production of immunoglobulins. Inhibition experiments demonstrated that the Ig-binding capacity of both SpG and SpL in the chimeric molecule are independent of each other. It was also shown that SpLG selectively absorbed Igs present in the sera of humans, rabbits, mice and rats [1-3]. The preparation of SpLG-HRP by the periodate method is a novel application [4].</div><div class = "text-block">References</div><div class = "text-block"><span>1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.</span><span style = "font-style:italic;">West Indian Med J</span><span>. 2013;62(1):12-20.</span></div><div class = "text-block">2. Kihlberg BM, Sjöbring U, Kastern W, Björck L. Protein LG: a hybrid molecule with unique immunoglobulin binding properties. J Biol Chem 1992, 267(35): 25583-8.</div><div class = "text-block">3. Justiz-Vaillant AA, McFarlane-Anderson N, and Smikle M. “Bacterial Immunoglobulin (Ig)-Receptors: Past and Present Perspectives.” American Journal of Microbiological Research, vol. 5, no. 2 (2017): 44-50. doi: 10.12691/ajmr-5-2-4.</div><div class = "text-block">4. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175. </div></div>

[STEPS]
?. Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
?. Mix 500 µg of staphylococcal protein-A (SpL) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate. On the other hand mix 500 µg of recombinant protein-L with an equal amount (500 micrograms) of the mix of horseradish peroxidase-sodium periodate.
?. The two mixtures are incubated separately for 3 hours at 4°C with gentle agitation.
?. Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added separately  to the preparations, which are centrifuge (13,000rpm., 10 minutes at RT). Add to each preparation cold saturated amonium sulphate solution and centrifuge again (10000rpm, 25 minutes at 4°C).
?. Now mix the SpL-HRP preparation with SpG-HRP and incubate the mixture for 90 min at 4°C in the dark with gentle agitation.
?. The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
?. The pellet (SpLG-HRP) is re-suspended in 500 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
?. An equal volume of glycerol is added to the dialysate followed by 200 µl of bovine serum albumin, BSA (20 mg/ ml).
?. The conjugate is then stored at -20°C until further used.