Method of inhibiting binding activity of immunoglobulins

Details Method of inhibiting binding activity of immunoglobulins Method of inhibiting binding activity of immunoglobulins

1997-02-13

2002-02-19

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Chemical stimulation of growth or activity by addition of...

C435S253600, C435S252900, C435S068100

Reexamination Certificate

active

06348346

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to the field of biotechnology and, particularly, to a method for inhibiting the binding activity of immunoglobulins with immunogens, a method for inhibiting culture agglutination and the actual inhibitors.
BACKGROUND OF THE INVENTION
Many forms of immune proteins exist in mammals including humans, and other classes of animals having immune systems. It is generally believed that the immune proteins IgM and IgG are the most important proteins that provide protection against microbial invasion of mammals. IgM is similar to IgG but is composed of five IgG-like molecules in a pentamer arrangement. The immunoglobulins IgM and IgG are circulated in the blood to combat invasion by foreign matter, mainly bacteria and are transferred to milk to establish immunity in nursing offspring.
Scientific literature suggests that the immune proteins IgM and IgG interact when a binding site on the variable regions of the light and heavy chains (see
FIG. 1
) attaches to an antigenic site or to a complimentary binding site on the tail of the heavy chain (Fc fraction) of another IgM or IgG molecule. Many researchers believe that IgM and IgG lose their interactive ability when the heavy chains are removed from the Fab fractions. Our work in the development of the present invention, however, indicates that the carbohydrate moieties or constant regions on both the light and heavy chains of immune proteins are capable of interacting with antigenic sites and/or bridging compounds thereby causing immune reactions and agglutination. Thus, it should be appreciated, that simple cleavage of the Fab fraction is not effective to inhibit immune reactions. Accordingly, a new method must be developed to achieve this end.
SUMMARY OF THE INVENTION
Accordingly, it is a primary object of the present invention to provide a method for inhibiting the binding activity of immunoglobulins with immunogens. Such a method has a number of beneficial applications including, but not limited to, inhibiting culture agglutination in a fermentation medium and blocking selected antigenic sites so as to prevent problematic immune reactions in the health care field.
Yet another object of the present invention is to provide a method for inhibiting culture agglutination in a fermentation medium wherein agglutination of the microorganisms responsible for fermentation is substantially prevented. Such a method allows, for example, more efficient production of a more consistent and higher quality fermentation product (e.g. cheddar cheese, cottage cheese).
Still another object of the present invention is to provide a method for inhibiting the binding activity of mammalian immunoglobulins and in particular IgA, IgG and IgM in a biologically safe and effective manner. Such an approach may be utilized to limit or prevent immune reactions and prevent infection and, accordingly, are beneficial for a number of uses in the care and treatment of patients suffering from a number maladies.
Yet another object of the present invention is to provide inhibitors of the binding activity of immunoglobulins including mammalian immunoglobulins and particularly IgA, IgG and IgM. This is accomplished through the utilization of corresponding immunoglobulins hydrolyzed with plant proteases to provide peptides having a molecular weight of less than 10,000 Daltons and preferably of about 1,000 Daltons.
Additional objects, advantages and other novel features of the invention will be set forth in part in the description that follows and in part will become apparent to those skilled in the art upon examination of the following or may be learned with the practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention as described herein, an improved method is provided for inhibiting the binding activity of immunoglobulins with immunogens. The method is particularly adapted for utilization in inhibiting the binding activity of mammalian immunogens including, specifically, IgA, IgG and IgM immune proteins. The method broadly includes the initial step of treating a source of immunoglobulins with an enzyme that hydrolizes the immunoglobulins into immunoreactive peptides having a molecular weight of less than 10,000 Daltons. Preferably, the enzyme is a plant protease that cleaves the immunoglobulins, IgA, IgG and IgM, above the second disulfide bond (see FIG.
1
). This results in the liberation of two Fab fractions. The enzymes papain, ficin and bromelain are particularly useful for this purpose.
In addition to cleaving the immunogens IgA, IgG and IgM above the second disulfide bond, the hydrolysis is continued for an additional period of time to clip and remove the carbohydrate moiety or constant regions from the variable regions so as to provide a peptide fraction having a molecular weight of less than 10,000 Daltons. This may be accomplished through ultrafiltering the source of immunoglobulins as they are hydrolized with the enzyme. A diafiltration technique may be utilized so that the smaller peptide fractions produced as a result of hydrolysis (i.e. those under 10,000 Daltons) pass as permeate through a polysulfone or other membrane having a molecular weight cut off of 10,000 Daltons. Specifically, to achieve this end, hydrolysis preferably is completed at a temperature between 30-50° C. for a period of at least two hours. As permeate is removed from the source of immunoglobulins undergoing hydrolysis a buffer of equivalent volume may be added.
The permeate, consisting of peptides of a molecular weight of less than 10,000 Daltons and preferably approximately 1,000 Daltons is then collected. Following collecting is the reacting of immunogens with the peptides so as to bind and block antigenic sites on the immunogens. Then follows the introducing of the peptide bound and blocked immunogens into the mammal. Advantageously, the bound and blocked antigenic sites on the immunogens make the immunogens substantially “invisible” to the immune system of the mammal thereby preventing undesired and problematic immune reactions from occurring.
Such an approach has a number of applications including, specific use in the artificial insemination of mammals including humans and other animals. Specifically, the sperm cells to be inseminated are reacted with the peptide so as to bind and block the antigenic sites or determinants on the sperm cells. When subsequently inseminated into a recipient, the immune reaction of the recipient is reduced and/or substantially eliminated, and accordingly, the aggregation of sperm cells is virtually prevented. Thus, the sperm cells remain free to reach the ovum for purposes of fertilization.
In accordance with yet another aspect of the present invention, a method for inhibiting culture agglutination in a fermentation medium including immunogens is provided. As before, this method includes an initial step of treating a source of immunoglobulins with an enzyme such as a plant protease to hydrolize the immunoglobulins into peptides having a molecular weight of less than 10,000 Daltons and preferably, approximately 1,000 Daltons. Specifically, as previously described these peptides are produced utilizing ultrafiltration and diafiltration techniques. Preferably, papain, ficin and/or bromelain is used as the enzyme.
The immunoglobulins may be obtained from a number of sources including, but not limited to, raw whey, dry whey, whey protein isolates, non-fat-dried milk, blood serum protein isolates, purified immunogen preparations and mixtures thereof.
Following hydrolysis is the collecting of the peptides produced as a result of the treatment of the immunoglobulins with papain, ficin and/or bromelain and in accordance with the ultrafiltration and diafiltration techniques. Specifically, the resulting peptides present in the permeate may be concentrated utilizing reverse

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