Chloroform

–Water dilution Chloroform: Larsen (US 12/299670 and WO2007/079755) discloses an alternative protocol for IgY isolation from egg yolk. First separate egg white and yolk, add 100 mM sodium phosphate buffer pH 7.6 to volume 60 ml and mix, add 40 ml chloroform and mis to a semi solid phase, centrifuge, isolate the supernatant. PEG-6000 is added to a final concentration of 12% w/v, mix until PEG dissolved, centrifuge for 10 min, dissolve pell in 100 mM sodium phosphate buffer pH 7.6. sodium azide is added to 0.05%. 

Salting Out Agents

Ammonium sulfate precipitation: See outline

Sodium Sulfate precipitation:

Jensenius “J Immunol Methods, 46, 63-8, 1981) disclsoes two methods of separation of IgG from egg yolk. In the first methods, egg yolk is combined with TBS, then the supernatant from this mixed with dextran sulphate and CaCl2, the sediment from this discarded and then the supertants mixed with Na2SO4. In the second method, yolk is mixed with water and NaoH ot pH 7.0 and then the IgG precipitated with Na2SO4. Ammonium sulfate may be used instead of sodium sulphate.

–Water dilution – CEX: Aqueous extract-Cation exchange-sodium sulfate preciptation: 

Charter, “A new process for the separation and purification of egg yolk antibodies” Thesis 1987) disclose a three stage process for the separation of an antibody (IgY) form chicken egg yolk which invloves aqueous extraction of the water soluble fraction (WSF) from the yolk by dilution with distilled water and pH adjustment, spearation of IgY from the WSF using a cation exchange column and then precipitation with sodium sulfphate.

Sodium Chloride

–Water dilution – NaCL: Larsen (US 12/299670 and WO2007/079755) discloses an alternative protocol for IgY isolation from egg yolk. First separate egg white and yolk, wash the yolkd with 2 volumes water, puncture the yolk and dissolve it in 1:10 weight/volume water, freeze and dry the solution, add 133.25 g solid NaCl to 25% saturation, incubate at room temp for 20 min, centrifuge for 30 min, transfer supernatant to new glass and add solid NaCl to 40% saturation, incubate at room temp for 20 min, centrifuge. The pellet is redissolved. 

Lithium Sulfate

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.

PEG:

–PEG-PEG:

Larsen (US 12/299670 and WO2007/079755) discloses a protocl for islation of IgY from egg yolk using PEG.

Polson, A., M.B. von Wechmar and M.H. van Regenmortel, “Isolation of Viral IgY antibodies from yolks of immunized hens,” Immunological Communicaitons 9:475-493 (1980) disclose isolation of antibodies from the yolks of hens by PEG at 3.5% to casue the lipids and vitellin to separate and then precipitation of IgY with 12% PEG.  

Polyethylene glycol (PEG): by a 25% cold ethanol precipitation to displace the immunoglobulin fraction from chicken egg yolks (Polson “Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunol. Invest. 14: 323-327 (1985). The mechanism is not clear but Poson speculated that the immunoglobulin fraction in the PEG extraction precipitates by means of displacement.

Polson (US4,550,019) discloses a method of separation of IgY which comprises collecting yolks form a number of eggs, washing them with distilled water to remove all the albumen, dropping them into a large glass funnel  and adding buffer equivalent to two volumes of yolk and adding PEG to a final concentraiton of 3.5% by weight of polymer to volume of diluted yolk. The mixutre was centrigued, causing the seapration of three phases in the centrifuge tubes; a yellow fatty layer on the surface, a clear supernatnat layer occupying the largest volume and a semi solid pliable layer of the builk of the yolk and caseinous protein “pellet” representing about 1/3 of the total volume. The supernatant fluid with the fatty layer was decanted into a funnel contianing an adsorbent cotton plug in the neck of the funnel. This plug filtered off the lipid layer that was decanted with the supernatant fluid. The volume of the clear fitlrate was measured and pulverised PEG was added which caused complete displacement of the IgY.

Mostov (US6,340,743) also discoses extracting chicen antibody IgY from egg yolks by a series of PEG precipitations followed by a series of ammonium sulfate precipitations accoridng to the method of Poison. 

Other Agents

Caprylic (octanoic) acid:

–caprylic acid –ammonium sulfate

Mclaren (J Immunol Methods, 1994, 177(1-2): 175-84) discloses collegcting eggs from immunised hens, harvesting the egg yolk, diluting them with phosphate-buffered saline (PBS), pH 7.5, adjusting pH to 4.6 with acetic acid and caprylic acid, stirring at room temeprature for 2 hour, centrifuging, the resulting supernatant, contining the immunoglobulin fraction, was then collected, the pH adjusted to 7.5 with Tris and centrifuged. The prepration was thenn cooled to 4C and ammonium sulfate was added, stirred for 1 hr and centfiuged for 20. The sueprnatnat discard, the plellet washed by resuspending in ammonium sulphate and recentrifuged. The washed pellet was then dissolved in PBS pH 7.5.

–Caprylic acid -HCIC

Tong (“A new purificaiton process for goose immunoglobuilin IgY (dltaFc) wit6h hydrophobic charge-induction chromatography) Biochemical Engineering J. 56 (2011) 205-211) discloses pretreating goose plasma with caprylic acid to precipitate some impurities and loading the supernatant onto a HCIC column for IgY(deltaFc) separation.