Individual-specific antibody identification methods

An identification method, applicable to the identification of animals or inanimate objects, is described. The method takes advantage of a hithertofore unknown set of individual-specific, or IS antibodies, that are part of the unique antibody repertoire present in animals, by reacting an effective amount of IS antibodies with a particular panel, or n-dimensional array (where n is typically one or two) consisting of an effective amount of many different antigens (typically greater than one thousand), to give antibody-antigen complexes. The profile or pattern formed by the antigen-antibody complexes, termed an antibody fingerprint, when revealed by an effective amount of an appropriate detector molecule, is uniquely representative of a particular individual. The method can similarly by used to distinguish genetically, or otherwise similar individuals, or their body parts containing IS antibodies. Identification of inanimate objects, particularly security documents, is similarly affected by associating with the documents, an effective amount of a particular individual's IS antibodies, or conversely, a particular panel of antigens, and forming antibody-antigen complexes with a particular panel of antigens, or a particular individual's IS antibodies, respectively. One embodiment of the instant identification method, termed the blocked fingerprint assay, has applications in the area of allergy testing, autoimmune diagnostics and therapeutics, and the detection of environmental antigens such as pathogens, chemicals, and toxins.

BACKGROUND OF THE INVENTION 
The instant patent provides an identification method. It is premised on the 
formation of antigen-antibody reactivity profiles or antibody 
fingerprints, that can be used to uniquely identify animals and inanimate 
objects. The method is particularly valuable in the rapid identification 
of large numbers of individuals. One embodiment of the method, termed the 
blocked fingerprint assay, centers on detecting allergens, autoantigens, 
defined as "self antigens", and environmental agents such as pathogens, 
chemicals, or toxins, all of which substances possess the same epitopes, 
or antibody binding sites. Regarding the identification of inanimate 
objects, the method is particularly useful to identify security documents, 
and the like that have legal significance, as well as valuable art work. 
As applied to identifying animals, particularly humans, the instant method 
permits identification of individuals, and thus can be expected to be 
applied to the fields of forensic medicine, law enforcement, and 
immigration. 
SECURITY DOCUMENT IDENTIFICATION 
A new generation of photocopying, and photo offset machines has increased 
the potential for counterfeiting a wide variety of security documents. 
These machines are capable of producing near perfect replicas that are 
virtually indistinguishable from their authentic counterparts. 
Traditionally, security documents have been treated in generally one of 
two ways to discourage counterfeiters. The first method relies on the 
formation of a color associated with the original security document when 
attempts are made to undetermine its integrity. In involves impregnating 
into the document a small organic molecule which changes colors if the 
integrity of the document is tampered with. Examples of the latter are 
erasure marks, chemical treatment of the documents to remove valid 
signatures, etc. Chemicals that have been so employed are acid-base 
indicators such as phthaleins and sulphonephthaleins as described in U.S. 
Pat. No. 2,445,586 and in German Pat. No. 856,842. Unfortunately, however, 
these molecules are generally insoluble in water and, thus, are difficult 
to impregnate into documents without the use of organic solvents which in 
turn may adversely affect the document. Problems associated with the use 
of organic solvents lead to the development of water soluble indicators 
such as pyrene sulphonic acid as described in U.S. Pat. No. 4,136,229, 
which is soluble in aqueous solutions, and therefore, compatible with 
dispersion techniques used to impregnate a variety of security documents. 
A second approach often used to ensure the authenticity of security 
documents, is to incorporate into the document materials which visually 
distinguish copies made of the original. Perhaps the best example of this 
approach is the use of security threads presently employed by a number of 
countries in their currencies. Other techniques involve incorporating 
holographic images so that unique visual images are apparent when 
documents are inspected with either visible, or ultra-violet light. 
IDENTIFICATION OF INDIVIDUAL HUMANS 
The three most common non-visual means for identifying people are blood 
typing, fingerprinting, and voice exemplars. Other methods include retinal 
scans and dental X-rays. Blood typing is based on the existence of groups 
of antigens present on blood cells. For example, the ABO system refers to 
four different groups of blood cell antigens: A, B, AB and O. The letters 
designate antigens present on the surface of red blood cells. Type A 
individuals have the A antigen; Type B individuals have the B antigen; 
Type AB have both antigens; and Type O has neither antigen. Thus, by 
analyzing a sample of a person's blood it is possible to identify him as 
to a particular blood group. It is, of course, immediately apparent that 
while this method may be used to identify one individual out of a small 
group of individuals, the method is limited when identification of an 
individual out of thousands of individuals is the goal. To do this, 
testing for many more blood group antigens is required and each test is a 
separate assay. Some newer tests make use of different isozymes that are 
present in body fluids, and suffer from the same limitations as do the 
blood typing tests. These methods can exclude certain individuals but can 
not differentiate between members of the same blood group. 
Fingerprinting is perhaps a more accurate way of identifying an individual, 
and is widely used by virtually all law enforcement agencies around the 
world. It is based on the appearance of characteristic patterns of an 
individual's fingers, such as swirls, valleys and ridges. When the method 
is used, a statistical evaluation is given as to the degree of 
correspondence between known fingerprints obtained from the individual, 
and those fingerprints which are sought to be matched to the individual. 
The procedure is technically arduous, and often not definitive. For 
example, the way that fingerprints are catalogued allows room for 
ambiguities. Furthermore, in many instances of crime, fingerprints are not 
available. 
The third procedure for identifying individuals is to match a voice 
recording with a voice exemplar of the individual. It is, of course, 
apparent that this method has little value in most instances, as there is 
seldom access to a recording of an individual's voice prior to the time 
that the match is sought. 
A variety of immunological/biochemical tests based on genetics, are 
routinely employed in paternity testing, as well as for determining the 
compatibility of donors and recipients involved in transplant or 
transfusion procedures, and also sometimes as an aid in the identification 
of human and animals. While the instant method is of some use for 
paternity testing or organ or transfusion matching, it very importantly 
offers an identification method that is based on an alternate principle. 
Generally, the former procedures involve seriological testing for proteins 
encoded by the Human Leukocyte Antigen gene loci or, as it is more 
commonly known, HLA complex. Although a good deal of information is known 
concerning the genetic makeup of the HLA locus there are many drawbacks 
using HLA seriological typing as the means for identifying individuals in 
a large group. This is primarily because of the complexity of the serum 
used to do the testing, and the lack of widespread availability of 
standard serum necessary to conduct the test, especially when dealing with 
species other than mouse or man. However, the relatively recent advent of 
monoclonal antibody technology offers to bring considerable 
standardization to this field. Each of the HLA antigens must be tested for 
in a separate assay, and many such antigens must be identified in order to 
identify an individual, an arduous process when trying to identify one 
individual in a large group. 
In addition to serological and mixed lymphocyte testing for the products of 
the HLA loci, more recent studies have identified DNA restriction fragment 
length polymorphisms (RFLP'S) indicative of different individuals, and 
these have been used in paternity testing, and transplant and transfusion 
compatibility testing. For example, Erlich, U.S. Pat. No. 4,582,788, has 
described a method for typing the HLA system based on the HLA DNA RFLP's. 
Further, Jeffreys et al. Nature, Volume 314, Page 67 (1985), have 
developed a powerful new identification system based on the analysis of 
repetitive DNA sequences (called "hypervariable minisatellite" regions) in 
human DNA. This method can also be applied to animals (see Wetton et al. 
Nature, Volume 327, Page 147 (1987)). 
It will be appreciated by those skilled in the art that while the above 
methods are useful for identifying security documents, individuals, as 
well to perform paternity tests, and transplant and transfusion 
compatibility testing, that these methods are presently technically 
arduous, when used for the purpose of identifying individuals, are also 
time-consuming, and often necessitate the use of expensive laboratory 
equipment. Thus, it would be a significant contribution in the field if a 
single test could be developed for identification purposes, that is at 
least as powerful as those presently used, and that does not have many of 
their shortcomings, and that preferably is based on an entirely different 
principle. The systems that are based on DNA RFLP, or DNA "hypervariable 
minisatellite" regions, do not discriminate between genetically identical 
animals such as twins, and depend on the particular DNA probe used to 
discriminate between individuals that are closely related. The instant 
method, while influenced by the genetics of the individual in a general 
way, resembles most closely the forensic fingerprinting method in that it 
is the product of normal developmental processes that are unique for each 
individual, namely, that the immune system of each individual is highly 
variable when producing antibodies in response to antigens, and, in 
addition, the antibody genes are known to undergo a high degree of 
"somatic mutations" acting effectively as a "random number generator" 
leading to further diversity of the antibody repertoire (including the IS 
antibody repertoire). The instant method shares with the DNA 
fingerprinting method, the ability to discriminate between closely related 
individuals, and matching up newborns and mothers. It has the advantages 
of being able to discriminate between identical twins and is simple and 
rapid, especially when used with saliva or body fluids other than blood. 
SUMMARY OF THE INVENTION 
The instant invention, termed antibody fingerprinting, presents a general 
identification method whereby animate and inanimate objects can be 
identified. It is premised on the hithertofore unrealized discovery that 
humans, as well as animals generally have present in their body, a 
heretofore unknown set of individual-specific, or IS antibodies. When an 
individual's body fluid (or solids) containing IS antibodies, is screened 
against a panel (an n-dimensional array where n is typically 1 or 2), of 
multiple antigens (typically greater than 10,000 different antigens), 
distinct antigen-antibody complexes are formed. The antigen-antibody 
complexes are detected using an appropriate antibody-binding detector 
molecule, typically radioactive or enzymes that give a colored product on 
reaction with substrate. The antigen-antibody reactivity profile, or 
antibody fingerprint, can be used to identify individual humans or 
animals. 
It is therefore an object of the instant invention to describe a simple 
immunological identification method that is applicable to people, and 
animals generally. The method is premised on the generation of an antibody 
mediated immune response. Saliva, tears, blood, serum, semen, urine, 
perspiration, lung washings, or other bodily fluid, or bodily solids such 
as tissue or feces, containing an organism's antibody repertoire is 
screened against a panel consisting of multiple different antigens, 
resulting in the formation of antibody-antigen complexes highly distinct 
of the organism from which the body fluid was obtained. All IS antibody 
isotypes are represented, thus allowing for the use of a wide range of 
detector molecules. The detector molecules used to detect the 
antigen-antibody complexes are widely available and include antibody 
binding proteins such as Staphylococcus aureus Protein A., or antibodies 
such as goat anti-human antibody or rheumatoid factor, or even cells with 
receptors for antibodies, such as lymphocytes. The detector molecules are 
appropriately labeled with tracer molecules, examples of such being 
enzymes, radioactive isotopes, magnetizable metals or photosensitive 
chemicals. The signals generated by the detector molecules on binding to 
the antigen-antibody complexes are then analyzed visually, or with 
appropriate instruments such as optical scanners or gamma radiation 
scanners. The profiles can be computer analyzed and stored for comparison 
at a later date with profiles from samples of unknown origin. 
In addition to presenting a method for identifying people and animals, 
another object of the instant invention is to provide a method whereby 
severed bodily parts from people or animals can be identified. This 
situation might arise, for example, as a result of a catastrophic event 
such as a plane crash wherein bodily parts may be identified using IS 
specific antibodies. 
It is a further object of the instant invention to describe a method 
whereby security documents can be identified by incorporating into, or 
associating with the document, IS antibodies obtained from one or more 
known individuals, that when tested with one or more panels of antigens, 
gives particular profiles of antigen-antibody reactivity, or antibody 
fingerprints. Security documents can also be identified by incorporating 
into, or associating with the document, a panel of multiple different 
antigens, that when screened with an effective amount of one or more 
particular sera containing IS antibodies, forms distinct antigen-antibody 
complexes, that when detected, generates an antigen-antibody reactivity 
profile, or antibody fingerprint. 
It is yet another object of the instant invention to present an embodiment 
of the antibody fingerprint method, termed the blocked fingerprint assay. 
The method relies on competition by an effective concentration of IS 
antibody for epitopes present in a panel of multiple or primary antigens, 
with similar epitopes present in other antigenic or secondary molecules, 
usually in solution, and leads to inhibition or blocking of the formation 
of the antigen-antibody complexes on the panel on which is formed the 
antibody fingerprint, thus reducing the number of elements in the 
fingerprint. The blocking assay can be used, in a single assay, to detect 
many different secondary antigenic molecules with the same epitopes as are 
present on the primary antigens in the panel such as allergens, 
autoantigens, or environmental antigens such as infectious agents, 
chemicals, toxins, or synthetic peptides, as appropriate. This method also 
allows the identification of IS antibodies with anti-pathogen, 
anti-allergen, or autoantibody function. The IS antibodies thusly 
identified are useful in their own right in the construction of 
diagnostics for pathogens or allergens, therapeutics or diagnostics for 
autoimmune diseases. 
An additional object of the instant invention is to provide kits that can 
be used to identify animals or security documents that consist of 
individual specific antibodies and accompanying reagents.

DETAILED DESCRIPTION OF THE INVENTION 
The instant invention, antibody fingerprinting, provides a generally 
applicable identification method that is rapid and simple. It is premised 
on the finding that animals with an immune system react to the presence of 
foreign substances, or antigens, by mounting an immune response which 
involves the production of antibody molecules by lymphocyte cells. The 
antibody response is maintained over a multiyear period. I have found that 
during the early development (aged newborn to two years old) of each 
individual's immune system, an effective individual-specific antibody 
response (termed IS antibody response) is obtained, involving a large 
number of different antibody molecules. Consequently, IS antibodies 
present in the body fluids or solids of humans, or animals, can act 
effectively as an individual-specific "fingerprint" of that individual 
when screened against a suitable panel (n dimension array where n is 
usually one or two) containing multiple antigens. The effective number of 
antigens or epitopes (antibody-binding sites) that are present in the 
antigenic array is less than 10.sup.20 and typically greater greater than 
one thousand, depending on the level of statistical certainty desired in 
matching or discriminating between a few or a large number (greater than 
100 ) of individual IS antibody "fingerprints." The antigen-antibody 
reactivity profile, when detected with an appropriate detector molecule, 
provides an individual-specific antibody fingerprint. 
The instant identification method can be used to identify humans, or any 
organism that produces IS antibodies. Moreover, the method can be applied 
to distinguishing between or identifying inanimate objects such as blood 
transfusions, body parts, body excretions, or security documents. Further, 
it will be appreciated by those skilled in the art that elements of the 
antibody fingerprint may reveal much useful information about the immune 
status of an individual. 
A unique feature of the instant identification method is that it is 
sensitive to persistent environmental antigens. In the blocked fingerprint 
assay, one embodiment of the instant method described at length below, the 
ability of secondary antigens is described such as environmental antigens, 
that compete with an effective amount of primary antigens in the panel for 
limited IS antibodies. Successful competition leads to a loss of 
antigen-antibody reactions with primary antigens in the panel, and 
subsequent loss of elements of the antibody fingerprint. Antibodies 
(including IS antibodies) that effectively cross-react with primary 
antigens in the panel, and with different secondary antigens can be used 
to identify immunologically similar primary and secondary antigens, such 
as environmental allergens, infectious agents, chemicals and toxins. The 
blocked fingerprint assay can also be used to detect persistent antigens 
that are natural or synthetic "autoantigens" that possess similar epitopes 
as do the primary antigens in the panel (autoantigens are defined as 
"self" antigens). Conversely, when the primary antigens in the panel 
consist of "self" or "autoantigens," or pathogens, the blocked fingerprint 
assay can be used to identify environmental antigens with similar epitopes 
as the autoantigens or pathogens, respectively. Some IS antibodies 
probably correspond to "autoantibodies" and on purification, may be useful 
in and of themselves, in the construction of diagnostics and therapeutics 
for autoimmune diseases. 
Similarly, if the primary antigens correspond to pathogens in the 
environment, then the blocked fingerprint assay can be used to identify IS 
antibodies with anti-pathogen function. These IS antibodies may also be 
useful in the construction of anti-pathogen diagnostics or therapeutics, 
such as vaccines. 
Thus it is readily apparent that this application of the method described 
herein has applications in the area of diagnostics and therapeutics for 
autoimmune diseases of man and animals. 
The immunological method also may be used in conjunction with other 
identification methods based on different principles, such as traditional 
fingerprinting, or genetic tests such as HLA testing, DNA RFLP assays, or 
DNA fingerprint assays, and the several tests taken together, can be 
expected to provide a particularly accurate and powerful identification 
method, especially in situations involving a large number (more than a 
hundred) of individuals. The instant method has advantages of rapidity, 
simplicity, and cost effectiveness, especially when saliva is used, thus 
avoiding the problems of blood letting. 
The basic immunological method, as applied to the identification of 
individuals includes: 
(a) Obtaining an effective concentration of IS antibodies, preferably, in 
solution, from an individual's body fluid, such as urine, plasma, serum, 
saliva, perspiration, semen, or lung washings, or from an individual's 
body solids such as tissue or feces. The IS antibodies may be obtained in 
the dry state and as in the case of the body solids, resuspended into 
solution using a suitable buffer, such as isotonic saline. The effective 
concentration of each IS antibody specificity is less than 1 g/ml, 
preferably about 10 ng/ml-10 mg/ml. 
(b) Reacting the antibody solution obtained in (a) with an effective panel 
of antigens where panel refers to an n-dimensional array of antigens, 
where n is typically one or two, to form antigen-antibody complexes. The 
panel of antigens contains multiple different antigens typically, but not 
necessarily, greater than ten thousand different antigens or epitopes and 
may be composed of natural antigens prepared from tissue cultured cells, 
or fresh animal tissue obtained from many different sources, or may 
consist of such extracts treated with enzymes or in other ways to generate 
fusion proteins or degradation fragments of different size, or may consist 
of purely synthetic polypeptides, or may consist of mixtures or 
subfractions of any of the above, so long as the antigens in the panel 
contain epitopes recognized by IS antibodies, and so long as the antigens 
are not contaminated with IS antibodies. The number of antigen-antibody 
complexes may be effectively increased or decreased by mixing the 
different kinds of antigens used in the panel. The effective concentration 
of each antigen in the panel is less than 1 g/mm.sup.2, preferably 0.5 
nanogram to 1 mg per square millimeter. The panel of antigens is formed by 
separating the antigens in n dimensions which is typically one or two, and 
can be effected by electrophoresis, isoelectric focusing, or other means; 
(c) detecting the antigen-antibody complexes with an effective amount of an 
appropriate detector molecule. 
The identity of an individual is established by comparing the resulting 
antigen-antibody reactivity profile, or antibody fingerprint, with that of 
an earlier obtained profile known to be characteristic of the individual. 
Similarly, individuals or inanimate objects, such as blood transfusions or 
tissues obtained after an air crash, may be distinguished by comparing 
antibody fingerprints obtained from each. 
The method as applied to the identification of security documents includes: 
(a) as above; 
(b) associating an effective amount of characterized IS antibodies obtained 
from one or more individuals in (a) on or with the security document; 
(c) reacting the IS antibodies associated on or with the security document 
with a particular panel of antigens; 
(d) detecting the amount of antigen-antibody complexes with an effective 
amount of detector molecules. 
The identity of the security document is realized by comparing the antibody 
fingerprint associated on or with the document with that of an earlier 
obtained fingerprint known to be characteristic of the antibodies. 
Similarly, an authentic document may be distinguished from a fraudulent 
document by comparing the antibody fingerprints associated with each. It 
should be appreciated that the method as applied to the identification of 
security documents can be the compliment of the above, that is: 
(a) associating a particular panel of antigens on or with the security 
document; 
(b) reacting the panel of antigens with an effective concentration of one 
or more particular individuals' characterized IS antibodies obtained from 
body fluids or solids of that individual(s); 
(c) detecting antigen-antibody complexes with an effective concentration of 
an appropriate detector molecule. 
A wide variety of assay techniques are available for detecting 
antigen-antibody complexes, commonly referred to as "immune complexes." It 
will be appreciated by those skilled in the art that the instant invention 
does not rely on an understanding of the molecular interaction of each 
particular antibody and antigen. All that is necessary is that a method be 
employed for detecting the immune complexes formed as a result of the 
individual-specific antibodies (IS antibodies) reacting with multiple 
antigens present in a panel, or n dimension array, where n is typically 
one or two. 
A variety of immune complex assays are described in the Methods of 
Enzymology, Volume 73, Part B, Editor J. J. Langone and H. V. Vunakis, 
Academic Press. Immune complex assays described therein generally fall 
into two broad classes. In the first class are assays termed analyte 
excess/labeled antigen assays. Although these assays can be applied to 
realize the instant invention, they are tedious and time-consuming in that 
they are generally used to determine the presence of a single antigen. 
Since the instant method relies on a determination of the reactivity 
profile of multiple antibodies with multiple antigens, it will be 
appreciated that to obtain this information using analyte excess/labeled 
antigen assays would require considerable time wherein multiple different 
assays are performed for each antigen. 
The second broad class of immune complex assays can be considered 
antigen/labeled antibody excess assays. Here, the antibody composition of 
normal sera is ascertained by screening the sera against a mixture of 
antigens generally bound to, and separated on, a solid surface. The 
antibody present in sera is in excess, and hence the phrase "excess 
antibody assay". The antigen mixture can be attached to a wide variety of 
solid surfaces, and then assayed by applying the sera. A second labeled 
antibody or antibody-binding molecule is applied which reveals the 
presence of antibody bound to the antigen present in the sera. 
Typically, the antigenic material is separated in some way, most often by 
electrophoresis, to allow ready visualization of the reactivity profile of 
antibodies in the sera with multiple antigens. A wide variety materials 
has been used to construct solid support matrices that can be employed in 
these types of immune complex assays. Representative materials include 
polyacrylamide, and agarose. Additional, other materials including 
polystyrenes, polyvinylchloride, polyethylene, cellulose, and other 
natural or synthetic polymers may be employed. 
A modification of the above-mentioned technique is the so-called 
immunoblotting procedure, also called Western blotting, described by 
Towbin et al. in Proceedings of the National Academy of Science, USA, 
Volume 76, Page 4350 (1979). This consists of separating an antigen 
mixture in one or two dimensions on a polyacrylamide gel, and then 
transferring the antigens from the gels onto a suitable surface, for 
example, nitrocellulose paper. The procedure generates a panel of antigens 
arrayed in one or two dimensions. The panel of antigens is then incubated 
with a blocking agent, for example a solution of bovine serum albumin, and 
detergents, such as Tween-20, to block or bind to sites on the panel that 
are not occupied by antigen. The panel is subsequently incubated with 
primary antibodies, washed to remove non-specifically bound antibodies, 
and then incubated with a detector molecule that recognizes the 
antigen-antibody complexes, typically through the antibody portion. The 
immunoblotting technique is advantageous in that it generally has lower 
backgrounds than other techniques, and further, it lends itself to a 
dipstick assay. The instant method is different from those previously 
described for the formation or detection of immune complexes because it 
involves individual-specific or IS antibodies. 
The method of detecting antibody bound to antigen using a tracer-labeled 
antibody-binding molecule will differ depending on the nature of the 
substrate material to which the antigen is attached, the nature of the 
antigens themselves, and the nature of the antibodies involved. IS 
antibodies react in a typical fashion. Traditional methods utilize S. 
aureus Protein A or a labeled second antibody directed against the first 
antibody, wherein the label is most often radioactive, or an enzyme 
capable of hydrolyzing a substrate thereby producing detectable color. The 
color can be associated with fluid surrounding the matrix, or can be 
associated with the matrix itself. It will be appreciated by those versed 
in the art, that the second antibody itself will give an antibody 
fingerprint in the instant method, and is first preferably incubated 
extensively with the panel of antigens to adsorb out second 
antibody-specific IS antibodies present. The adsorbed second antibody may 
then be used as a detector molecule when combined with a suitable reporter 
molecule well known to those skilled in the art. By adsorbing the second 
anti-body to remove IS antibodies present, clear IS antibody fingerprint 
profiles are realized. Immunoblotting enables antibody binding to antigen 
to be detected by visualizing colored particulate precipitates and avoids 
the use of radioactive compounds. An effective concentration of antibodies 
present in immune complexes is typically 1 ng to 10 ug per complex per 
mm.sup.2. 
A number of enzymes can be coupled to protein A or second antibody, that 
form precipitates on a solid surface in the presence of a suitable 
substrate. A partial list includes horseradish peroxidase, glucose 
oxidase, and alkaline phosphatase. 
In addition to enzymatically revealing the antigen-antibody complexes 
formed on a solid matrix, there exists non-enzymatic means of revealing 
the immuno complex. Many of these are well known to those skilled in the 
art, but perhaps particularly useful is the colloidal gold technique. The 
manner in which it is constructed and used with antibodies is described by 
De Mey et al in Prot. Biol Fluids (Editor Pepters), Paragon Press, 
Oxford, page 943 (1981), and Ami in Immunochemistry: Applications and 
Pathology and Biology (Editor Polak and Van Noorden) and Wright and Sons 
Ltd., London, page 83 (1983). 
The preferred embodiment of the instant invention involves constructing a 
panel of one to ten thousand different antigens, using the immunoblotting 
procedure described by Towbin et al. Proceedings of the National Academy 
of Sciences, Volume 76, Page 4350 (1979). The particular antigens used in 
the panel may vary, depending on the number of antigen-antibody reactions 
desired. For example, human HeLa cells contain approximately ten thousand 
different antigens, and can be prepared and separated electrophoretically 
according to molecular mass, using denaturing polyacrylamide gel 
electrophoresis systems, and subsequently electrophoretically transferred 
onto a matrix, such as nylon or nitrocellulose paper. The antigens are 
prepared according to appropriate protocols, for example, see Francoeur et 
al. Journal of Immunology, Volume 136, Page 1648 (1986). Unlike previously 
documented use of the immunoblotting technology, the instant invention is 
based on the formation and detection of immune complexes formed with 
individual-specific antibodies. A different individual-specific antibody 
fingerprint is obtained if a single individual's IS antibodies are reacted 
with total HeLa cell antigens, or partially degraded HeLa cell antigens. 
In both cases, the IS antibodies are likely the same, but the HeLa 
antigens are different in molecular mass and size. Thus, the antigens are 
located in different areas of the panel. 
Individual specific antibodies are obtained from an individual's body 
fluids or solids, and reacted with the panel of antigens for a time 
sufficient for IS antibodies to bind antigen, typically for 30 minutes to 
an hour, but not more than three days. The IS antibodies are incubated 
neat (undiluted) or in an appropriate solution (e.g., isotonic saline) at 
a dilution of about less than one in 10.sup.7, preferably in the range of 
1:10 or 1:20. The effective concentration of each antigen in the panel is 
less than 1 g/mm.sup.2, preferably 0.5 nanogram to 1 mg per square 
millimeter. The nonspecifically bound antibodies are removed by washing, 
and the immune complexes detected by further incubation with an effective 
concentration of a labeled detector molecule such as .sup.125 I-Protein A, 
or with a labeled second antibody such as alkaline phosphatase-conjugated 
goat anti-human IgG, previously adsorbed to remove second 
antibody-specific IS antibodies reactive with the panel of antigens. The 
panel of antigens is washed to remove nonspecifically bound detector 
molecules, and an effective concentration of substrate material added if 
the second antibody carries an enzyme, resulting in the development of a 
colored product. If the detector molecule carries a radioactive tracer, 
X-ray film or other techniques can be used to detect the presence of the 
detector molecules by methods well known to those skilled in the art. 
It will be appreciated by those skilled in the art that the IS antibodies 
present in a particular individual sera or other body fluids or solids, 
can be employed to identify or distinguish between inanimate objects, such 
as blood or body parts, or security documents. For the latter, a 
particular panel of antigens can be employed for this purpose as well, to 
be used to generate an antibody fingerprint with a particular individuals' 
IS antibodies. Such are generically referred to as security documents, by 
which is primarily meant any negotiable instrument which is convertible to 
cash, particularly currencies, checks, travelers checks, postal order, 
lottery tickets, trading checks, bearer bonds, and other financial 
documents. In addition, other documents which cover a different array of 
valuable rights are intended to come within the scope of the term security 
documents. Such would be passports, admission tickets, travel tickets and 
the like. Also, the instant method is applicable to determining the 
authenticity of labels attached to clothing, such as those which exhibit 
the trademark of a particular manufacturer. 
The instant method may also be employed to ensure the authenticity of works 
of art, and the like. As applied to security documents the instant method 
involves associating with the item whose authenticity is sought to be 
determined, a record of the antibody-antigen reactivity profile. The 
preferred method is to associate with the security document an effective 
concentration of a particular individual's IS antibodies, for example, in 
the form of dried plasma on a filter paper inside of a sealed tube, and 
then, when the authenticity of the document is sought to be proved, react 
the antibodies with a particular panel of antigens and after detection of 
the immune complexes, observing the antigen-antibody reactivity profile, 
or antibody fingerprint, in comparison to a previously obtained 
fingerprint using the same IS antibodies. Alternatively, the particular 
panel of antigens may be associated with the documents and the fingerprint 
obtained with particular IS antibodies. In those instances where the 
antigens are small molecular weight molecules, this can be accomplished by 
impregnating the antigens directly into the document using the techniques 
as described in U.S. Pat. No. 4,037,007 and 4,136,226. 
An additional approach is to chemically modify either the security 
document, the IS antibodies, the antigens, or both, so as to affect 
binding between the two, yet preserve the function of the molecules 
involved. One method for binding the panel of antigens whereby this can be 
accomplished is to chemically activate the security document, assuming the 
latter is composed at least partially of paper, or other similar 
chemically reactive substrate, by cyanogen bromide (CNBr) treatment. When 
CNBr activated paper is combined with proteinaceous antigens, the antigen 
binds irreversibly to the paper. The procedures for carrying out this 
reaction are well known in the art and are described by Eska in Methods of 
Enzymology, Volume 73, page 646, Editors Langone and Vunakis (1981). 
Additional reactions are available, and are well known to those skilled in 
the art. 
When the identity of the security document is questioned, the IS 
antibodies, or the panel of antigens associated with it are reacted to 
form the immune complexes with the appropriate antibodies or panel of 
antigens respectively, and the immune complexes are detected with suitable 
detector molecules. In addition to the detector molecules described 
previously, additional labels attached to the antibody-binding molecule 
(i.e., protein A or adsorbed second antibody) can include fluorescent, 
bioluminescent, or chemiluminescent molecules as described in U.S. Pat. 
No. 4,478,817, or magnetizable, or radiation sensitive materials. 
The following examples illustrate various aspects of the invention, but it 
will be obvious to those skilled in the art that various changes and 
modifications may be made therein without departing from the scope of the 
invention. For example, total antibodies, including the IS antibodies, 
from a particular individual may be labeled directly by reaction with 
detector molecules, and the detector-IS antibody complexes subsequently 
incubated with the panel of antigens, thus generating an antibody 
fingerprint. Another example, perhaps more properly called an antigen 
fingerprint, includes first separating the total antibodies, including IS 
antibodies (eg. by isoelectric focusing), and then reacting the antibodies 
with a mixture of labeled or unlabeled antigens, or a particular antigen 
detector molecule, and the immune complexes formed detected directly, or 
after reaction with a labeled detector molecule. 
EXAMPLE I 
"Antibody Fingerprinting" of Humans 
An extract of human HeLa cells was prepared and used to form an antigenic 
panel as described in Francoeur et al. Journal of Immunology, Volume 136, 
Page 1648 (1986). HeLa cells were grown in standard laboratory tissue 
culture medium, isolated, lysed with a detergent, and centrifuged to 
remove any insoluble debris. The extract contained approximately ten 
thousand immunogenically different antigens, the bulk of which have not 
yet been antigenically defined. The material was subjected to 
electrophoresis on a sodium dodecyl sulfate polyacrylmide gel to separate 
the mixture according to molecular mass. The separated antigens present in 
the polyacrylamide gel were electrophoretically transferred onto Immobilon 
paper (obtained from Millipore Corporation) and the sites on the paper 
with no antigens bound were then blocked by incubation with a solution 
containing 4% instant powdered dry milk, using standard techniques. The 
paper was cut into strips approximately two millimeters in width. Each 
strip consists of a panel of some ten thousand different antigens arrayed 
in one dimension according to molecular mass. Separate panels of antigens 
were incubated with individual sera (containing IS antibodies), diluted 
1:20 in the appropriate buffer, obtained from different individuals at 
different times, in this case, for three hours at room temperature, with 
constant agitation. The panels of antigens were washed, to remove unbound 
or nonspecifically bound antibodies, and incubated with .sup.125 I-Protein 
A for two hours. Following subsequent washing to remove unbound detector, 
the panels of antigens were dried and exposed to X-ray film for 4 hours at 
-70.degree. C. with an enhancer screen, and the film developed, according 
to established protocols. 
FIG. 1 shows two different exposures of the film; a long (3-day) and short 
(8-hour) exposure. The fingerprints are stable over a period of 4 years, 2 
months for one individual (lanes 2-4); for 6 years, 7 months for a second 
individual (lanes 5 and 6); for 7 years for a third individual (lanes 7 
and 8); for 7 years, 4 months for a fourth individual (lanes 9-11), and 
for 8 years, 3 months for a fifth individual (lanes 12-14). It is apparent 
that the fingerprints are virtually identical at early and late times, and 
are constant over a multiyear period. The individual whose fingerprints 
are shown in lanes 9 and 10 became gravely ill (vasculitis), and his 
fingerprint is obtained during this condition (lane 11). It is apparent 
that during the illness, the amount of antibody-antigen complexes was 
increased and thus the intensity of the fingerprint was stronger than when 
he was well. Minor changes in the fingerprint were detected. The second 
individual (lanes 5 and 6) is the daughter of the third individual (lanes 
7 and 8). While the fingerprint of the mother and daughter are unique to 
those individuals, certain elements within the fingerprint appear to be 
common to both, suggesting that they share a common environment and/or 
genetic background that influences the immune response similarly. The 
fourth individual (lanes 9-11), is the son of the first individual (lanes 
12-14), and again, while the fingerprints of the son and mother are unique 
to each individual, certain elements within the fingerprints are common to 
both. Lastly, lanes 1 and 15-17 show fingerprints of unrelated individuals 
while lane 18 shows the control fingerprint used for quality control. The 
molecular mass (Mr) of the antigens used in the panel is shown in 
kilodaltons (Kd). 
EXAMPLE II 
Antibody-antigen Reactivity Profiles, or Antibody Fingerprints of Old and 
Young Normal Humans 
The method of the instant invention is applicable to both young and old 
humans. FIG. 2 shows the antibody fingerprints obtained from sera of 
normal individuals aged newborn to 90 years old. The panel of antigens and 
detector molecules used were the same as described in Example I. 
Approximately equal numbers of males and females are represented. An 
individual-specific profile was obtained in all cases. Some or all of the 
IS antibodies in newborns are likely transferred from the mother to the 
fetus as maternal antibodies in general are known to be passed to the 
fetus via the placenta, and are replaced by the child's own antibodies by 
approximately the sixth month after birth. The individual-specific (IS) 
antibodies appear to be fixed by approximately two years of age (see also 
examples IV and V). Lanes 1-7, 80-90 years; lane 8, 20 years; lanes 9-18, 
70-79 years; lanes 19-28, 60-69 years; lanes 29-38, 50-59 years; lanes 
39-42, 40-49 years; lanes 43-45, 30-39 years; lanes 46-49, Newborns. Lane 
50 is a quality control fingerprint. The molecular mass (Mr) of the 
antigens in the panel is indicated. 
EXAMPLE III 
Antibody Fingerprints of a Single Normal Human Serum Obtained by Screening 
Against Different Panels of Antigens 
Normal human sera, or other body fluids containing IS antibodies, or 
similar fluids from other animals, can be screened against different 
panels of antigens prepared from complex antigenic mixtures. FIG. 3 
presents antibody fingerprints obtained with a single normal human serum 
and reacted against panels of antigens prepared from human HeLa cells, a 
line of human cervical carcinomal cells that grow in the laboratory (lane 
1); WIL-2 cells, a line of human B lymphocytes (lane 2); CV-1 monkey cells 
(lane 3); MDBK-cow cells (lane 4); dog muscle cells (lane 5); chicken 
embryo cells (lane 6); a mixture of different mouse cells lines (lane 7); 
frog oocytes (lane 8); Drosophila cells (lane 9); Bakers yeast (lane 10); 
RY1090 bacteria (lane 11). The detector molecule used was .sup.125 
I-Protein A. It is apparent that the sera yields a different fingerprint 
with each antigenic panel tested. It is important to point out that this 
finding permits a large number of antigens to be employed, and, moreover, 
permits a cross check of the identity of the individual. 
EXAMPLE IV 
Antibody Fingerprints of Various Children, Aged Two Days to One Year 
Sera were obtained from various male or female children, both normal and 
ill. The panel of antigens and detector molecule used were as described in 
Example I. Lanes 1-26: female, 12 months, normal; male, 10 days, normal; 
female, 12 months, ill; female, 8 months, ill; female, 7 months, ill; 
female, 1 month, normal; female, 10 months, normal; male, 12 months, 
normal; male, 4 months old, ill; male, 10 months, ill; male, 9 months, 
ill; male, 10 months, ill; male 3 months, ill; female 2 months, normal; 
male 9 months, normal; female, 2 days, normal; female, 5 months, ill; male 
11 months, ill; male, 3 months, normal; male, 1 months, ill; male, 2 days, 
normal; male, 1 month, normal; female, 4 months, ill; male, 5 months, ill; 
male, 9 months, ill; female, 2 months, normal. It can be seen that the 
fingerprints of children less than a year old are generally simpler than 
older individuals, presumably because the immune system is in its early 
developing stages, and the full complement of IS antibodies is not yet 
established. 
EXAMPLE V 
Antibody Fingerprints of Various Children, Aged One Year to Six Year Old 
FIG. 5 shows that both normal and ill children of ages one to six years 
have IS antibodies that can be used to generate antibody fingerprints, 
similar to those of older individuals. The panel of antigens and detector 
molecules were as described in Example I. Lanes 1-12; female, 3 years, 
ill; male, 1 year, normal male, 2 years, ill; male, 4 years, ill; female, 
5 years, ill; female, 6 years, ill; female 2 years, ill; male, 2 years, 
ill, female, 5 years, ill; male, 3 years, ill; female, 5 years, ill; male, 
3 years, ill; male, 1 year, well; male, 1 year, ill. While it is clear 
from Example I that the antibody fingerprints are stable over a multiyear 
period, there is some indication that the fingerprints are altered by 
disease. The correlation with disease that we have found so far is that in 
some instances there is an increase in the amount of total antibody which 
presents a more intense fingerprint (see FIG. 1, lane 11, FIG. 5, lane 7. 
Lane 13, quality control fingerprint. The molecular mass of the antigens 
in the panel is indicated in kilodaltons. 
EXAMPLE VI 
Antibody Fingerprints of Animals 
FIG. 6 shows antibody fingerprints of different animals. Sera was obtained 
from animals and tested against a panel of HeLa cell antigens. The 
detector molecule and antigens were as described in Example I. Lanes 1-3, 
6, 15, 16, 19, 20, are sera from individual cats; lanes 4, 5, 7, 10-14, 
17, 18, 21, are sera from individual dogs; lanes 8 and 9 are sera from 
individual monkeys; lane 22 is a horse serum; lane 23 is a quality control 
fingerprint. The various animals ranged from 2 months old to 18 years old 
and were approximately equally represented by both sexes. Most of the 
animals were well or had mild conditions, except for the cat and dog shown 
in lanes 5 and 6, which were gravely ill. FIG. 6 shows that antibody 
fingerprints may be obtained from different animals as well as from 
humans. The molecular mass (Mr) of the antigens in the panel is indicated 
in kilodaltons (Kd). 
EXAMPLE VII 
Antibody Fingerprints of Normal Human Families 
FIG. 7 shows antibody fingerprints of several normal human families. The 
children were all aged 12 years or older. The panel of antigens and 
detector molecules used were as described in Example I. Despite the 
genetic relatedness of the individuals in the families, each individual 
has a unique antibody fingerprint. In some cases, common "motifs" within 
the fingerprints can be discerned, which appear to be shared by members of 
a family, suggesting a common response to probably common environmental 
antigens. The fingerprints are marked off in sets of families with members 
of the families designated as F (father), M (mother), C (child). Quality 
control fingerprints are designated "M." The molecular mass of the 
antigens in the panel is indicated in kilodaltons. 
EXAMPLE VIII 
The Blocked Fingerprint Assay as Used to Detect Environmental Antigens That 
Can Compete With Antigens in the Panel for IS Antibody Binding 
FIG. 8 shows the antibody fingerprints of a normal human individual, with 
or without the presence of competing secondary antigens. The X-ray film 
was purposely overexposed to show the fine details of the fingerprints. 
The panel of antigens consisted of separated human HeLa cell antigens, and 
the detector molecule was .sup.125 I-Protein A, as described in Example I. 
Different mixtures of antigens were incubated simultaneously with the IS 
antibodies and the panel of antigens at the same time for two hours. All 
fingerprints should be identical if no blocking occurs. Lane 1 shows no 
blocking agent, and it is apparent that there is no blocking of the 
fingerprint; lane 2, shows the results when human HeLa cell extract is 
used as the blocking agent. Two elements of the fingerprint are blocked 
arrows, bands 3 and 4), suggesting that one or more antigens present in 
the HeLa cell extract compete effectively with two HeLa antigens present 
in the panel for limiting IS antibodies; lanes 3 and 6 show the effect of 
blocking with bacterial extracts. Three elements of the fingerprint are 
blocked each instance (bands 2, 3 and 4. Lane 4 shows the results when 
yeast extract is used as the blocking agent. One element of the 
fingerprint is blocked (band 3). Lane 5 presents a control for the 
detector molecule in which the serum containing the IS antibodies was 
omitted. The control is blank indicating that the fingerprint results from 
binding of IS antibodies to antigens, and not as a result of nonantibody 
substances binding to the antigens in the panel. 
There will be many modifications of the methods and compositions described 
above that are obvious to those of ordinary skill in the fields of 
immunology, immunochemistry, and genetic engineerng. Such modifications 
are intended to come within the scope of the invention.