Commercial Kits:  Pierce Chicken IgY Purification Kit   Gallus Immunotech  Promega Eggstract®

Ammonium sulfate or sodium sulfate precipitation: has widely been used as an initial purification step in the isolation of immunoglobulin isotypes from serum bile and egg yolk. A high concentration of ammonium sulfate competes with the proteins in a solution for water molecules, reducing the solubility of the proteins and thus causing precipitation (Beetham, Avian Diseases 37: 1026-1031, 1993).

Particular Schemes

 Water dilution – AMS: 

Akita (J food science, 57(3), pp. 629634, 1992) discloses isolation of IgY by first a simple dilution of egg yolk with water in low pH follwoed by ammonium sulfate with 60% saturation.

Larsen (US 12/299670 and WO2007/079755) discloses a classic protocol for isolating antibody from egg yolk with Ammonium Sulphate:   First, separate egg white and yolk, wash the yolk with water, puncture the yolk and dissolve it in  1:10 weight volumn water, reeze and dry the solution, add solid ammonium sulphate to 25% saturation, incubate at room temp for 20 min, centrifuge, transfer supernatant to a new glass and add solid ammonium sulphate to 40% saturation and incubate at room temeprature for 20 min, centrifuge. The pellet is redisosolved.

Henderson (US14/020469) discloses a method for purifying IgY antibodies by (a) contacting a sample with a precipitating agent such as ammonium sulfate and (b) differentially precipitating the IgY antibodies obtained in (a) using the same or different precipitating agent to separate IgY(Fc) and IgY(deltaFc) antibody isoforms. In one embodiment, the method comprises a first precipitation by (a) contacting the sample containing the IgY antibodies with a precipitating agent at a first concentraiton to obtain a first precipitate containing the IgY antibodies (b) resuspending this first precipitate and contacting it with the same or different precipitating agent at a second concentration to obtain a second precipitate containing a majority of one IgY isoform and a supernatant containing a majority of the other IgY isoform (c) contacting the supernatant obtained in (b) with the same or different precipitating agent at a third concentration to obtain a third precipitate containing a majority of the other IgY isoform. The precipitated IgY(Fc) and IgY(deltaFc) antibodies can be further purified ushing other chromatographic techniques. In one embodiment and prior to the differential precipitation above eggs laid from immunized geese are collected, after gently making an opening in the egg shell, egg shite is removed and the yolk rinsed with several columnes of ice cold deionzied water, the washing egg yold is then transferred to an absorbent paper towel to remove residue egg white, the yold sac is then punctured and the yolk contents transferred to a 1L beaker and ice cold deionized water is added 90x the yolk weight. The diluted yold is then gently stirred at 250-300 rpm for 15 min at room temperature and the yolk suspension is acidified by adding 1N HCl until pH 5.0 and then further mixed for 5-10 min and frozen at around -20C. The frozen yolk suspension can be thawed, centrifuged and filtered prior to precipitation above with ammonium sulfat at concentraiton of about 50%.

Water dilution- AMS -Affinity purification: Cook, (J. Bioscience and Bioengineering, 91(3), 305-310, 2001) discloses obtaining IgY from egg yolks by mixxing six fold volume of chilled water and allowing the yolks to settle overnight. The speurnatant was decanted and AMS was added in the amount of 200 g/l supernatant. After one half hour stirring at 4C, the preciptiate containing IgY was recovered by centrifugation and resolubilized in phosphate buffered saline for the final AMS preparation. For a more specific antibody preparation, the AMS precipitate can be passed over an affinity column to give the final affinity purified product. 

Water dilution -AMS – alcohol precipitation: Akita (“Immunoglobuilins form egg yolk: isolation and purification” 57(3), Journal of Food science, 1992, pp. 629) discloses separating egg yolk from the white, washing with distilled water to remove albumen, rolling on paper towels to remove adhering egg white, puncturing the membrane and flowing the yolk into a cylinder without the membrane, deluting with distilled water (acidified with 0.1 N HCL) and held for at least 2  hr before centrifugation for 1 hr or filtration through filter paper. The resulting immunoglobuilin containing filtrate is further purified by salt precipatation (AMS). Then either UF followed by DEAE-Sephacel/gel filtration or alcohol precipitation followed by DEAE-Sephacel/gel filtration or salt precipitation (sodium sulfate) can be used to future purify. 

Bizhanov (Scand. J. Lab. Anim. Sci. 3(31), 2004) discloses replacing the sodium sulfate with lithium sulfate in the ater dilution of Akita above. In the method, yolk diluted 1:9 with distilled water, acidified with HCL to pH 5.0 is incubated overnight at 4C. After futher centrifugation, the precipitate containing Igy was resupended in TBS, precipitated twice in either sodium citrate, lithium sulfate or ammonium sulfate (comparison of all 3 methods) and dialyzed against TBS.

Caprylic acid -ammonium sulfate –DF:

Bhanushall discloses purifying IgG from chicken serum by diluting chicken serum with acetate buffer and adding caprylic acid, removing the precipitate, collecting the supernatant and adding ammonium sulfate (NH4)SO4. The pellet was resupsended in PBX and dialyzed against PBS.

Sodium acetate buffer – PEG – AMS precipitation: Duan (CN101787080, published 7/28/2010) discloses a three step method of preparing egg yolk IgY by (1) removing yolk and stirring in acetic acid-sodium acetate buffer followed by sufficient mixting and then (2) stirring in 40% PEG and centrifuging for 30 min. The supernatant is decanted and the precipitate contacts antibody IgY crude extract was subjected to staturation of 33% ammonium sulfate precipitation and purified by SDS-PAGE analysis with a purity of 90-95%. 

Sodium acetate – ammonium sulfate: Wang, (US 13/956511) teaches extraction of IgY from yolk by diluting the yolk with a buffer solution with sodium acetate at pH from 4.6 to 5.4 stirring the mixture, centrifuging and then adding an inorganic salt such as ammonium sulfate solution to the sueprnatant to salt out the IgY. 

Wu (CN101955532A, published 1/26/2011) teaches a method for rxtracting chicekn egg yolk antibodies mixing in a sodium acetate buffer solution to the yolk at pH of 4.8-5.2 and leeting the mixture stand. To the supernatant, ammonium sulfate is added at 25-30%, then centrifuging. The precipitate is resuspended in PBS and ammonium sulfate solution volume of 0.7 times the begging is stirred in, let stand and then the precipitate was resupended in PBX to give a solution of yolk antibodies. 

Natural Gums – Ammonium suophate precipitation: 

Natural gum zlpah-carrageenan has been used to isolate IgY (IgG) fraction from the yolk of chicken eggs (Hatta, Agric. biol. Chem, 54: 2531-2535 1990). The mechanism is not clear but Hatta showed that the natural gums that isolated the highest percentage immunoglobulin fraction were acidic and that the method is due to the ionic binding between the negative charge of the alpha-carrageenan and the net positive charge of the immunoglobulin fraction.

Tan (J Immunological Methods, “A novel, cost-effective and efficient chicken egg IgY purificaiton procedure” 2012) disclose a method of IgY purification from chicken eggs yolks using the common plant gums pectin and kappa-carrageenan in the presence of calcium chloride to delipidate egg yolk mixtures while maintaining IgY in solution and then ammonium sulphate to subsequently precipitate the resulting IgY antibodies to higher purity. 

PEG-AMS

Rajic (“protein depletion using IgY from chickens immunised with human protein cocktails” Prep Biochem Biotechnol 2009, 39(3) 221-47) discloses IgY extraction procedure using water dilution to deplet lipids and lipoproteins followed by sequential precipitation with 31% AMS and 12% pooy ethylene glycol (PEG).

 Stationary phase (silicate, carbon, cellulose, synthetic fiber)- Ammonium sulfate (first concentration 15-24% -AMS (second concentration 25-40%): 

Chiou ( EP1371665B1; see also US20060223986, US 6,680,376 and US2003/0009014) discloses a method of obtaining IgY(deltaFc) antibody isoform absorbing yolk antibodies in a water miscible fration obtained from egg yold with a water insoluble non-charged active constituent in the stationary phase such as silicate, carbon, cellulose and synthetic fiber, flowing the stationary phase with a buffer to obtain an aqueous fraction containing the yolk antibodies and then salting out this aqueous fraction with (NH4)SO4 at a first concentraiton ranging from about 15-24 (w/v) and then salting out the aqueous fraction with (NH4)2SO4 at a second concentration of 25-40% (w/v).