Assignment on

Use of nucleic acid

probes and antibodies in
clinical diagnosis

Probes in clinical diagnosis

Recent advances in the area of recombinant DNA technology have led to the
possibility of using nucleic acid probes in the diagnosis of many diseases and the
progress in developing these genetic tools for use in the diagnostic laboratory has
generated a lot of excitement among clinical microbiologists.
The excitement stems from the likelihood that DNA probes can shorten the time
required to identify fastidious pathogens, detect organisms directly in clinical
specimens, and reduce the overall costs associated with processing specimens for the
isolation of infectious agents.
The nucleic acid probe approach to identification is unique because the focus of the
method is the nucleic acid content of the organism rather than the products that the
nucleic acid encodes. Since every properly classified species has some unique
nucleotide sequences that distinguish it from every other species, each
microorganism's genetic composition is in essence a fingerprint that can be used for
its identification.

1.1.

What is a Probe?

A probe is a pure or partially pure nucleic acid sequence which can be labeled and
used to search out and detect complementary sequences in the presence of a large
amount of non-complementary DNA. The probe may be a partially pure DNA,
mRNA, or a chemically synthesized oligonucleotide which could be labeled using
radioactive or non-radioactive substance. The denatured probe hybridises to the
complementary sequences and detection of such hybrids can be done either by
autoradiography, liquid scintillation counting, colourimetry, fluorometry or using
chemiluminescent assays.

1.2.

Designing a probe

Every organism, no matter how simple or complex, contain some unique sequences
of DNA or RNA within their genome that distinguish them from all other organisms.
The key to developing a nucleic acid probe (i.e., either a DNA or an RNA probe) is to
isolate those sequences, reproduce them in large quantities, and attach a reporter
molecule to them so that they can be incorporated into a hybridization reaction.
Sequences that might make effective probes can be isolated in a number of ways. For
example, the DNA from a pathogenic organism can be cut with a restriction
endonuclease and cloned into a plasmid vector. Recombinant plasmids are screened
with the genomic DNA from both pathogenic and nonpathogenic strains. Those
plasmids that contain sequences that hybridize only to the pathogenic strain form the

basis

for

species-specific,

and

even

strain-specific,

probes

(Fig).

1.3.

Probe hybridization and target detection

Nucleic acid hybridization is the basis for rapid and reliable assays. The physical
basis of these systems is precise nucleotide base pairing and hydrogen bonding
between one string of nucleotides and a complementary nucleotide sequence. A
general laboratory nucleic acid hybridization scheme is as follows.
1. Bind single-stranded DNA (the target) to a membrane support.
2. Add single-stranded labeled DNA (the probe) under appropriate conditions of
temperature and ionic strength to promote base pairing between the probe and the
target DNAs.
3. Wash the support to remove excess unbound labeled probe DNA.
4. Detect the hybrid sequences that form between the probe and target DNA.
A nucleic acid hybridization diagnostic test has three critical elements: probe DNA,
target DNA, and signal detection. This type of detection system can be both
extremely specific and highly sensitive.

1.4.

Nucleic acid probes in clinical diagnosis

1.4.1.

Diagnosis of malaria

An example of a diagnostic protocol that utilizes a DNA probe as a means of

detection is the procedure developed for the detection of Plasmodium
falciparum. This parasite causes malaria, a disease that affects about one-third of
the worlds population. The parasite infects and destroys red blood cells, leading
to fever and, in severe cases, damage to the brain, kidneys, and other organs.
Sensitive, simple, and inexpensive methods are required to identify the source(s)
of the parasite in various localities, to assess the progress of eradication
programs, and to facilitate early treatment.
Currently, malarial infections are diagnosed by either microscopic examination
of blood smears or immunological detection of parasite antigens, effective but
labor-intensive and time-consuming processes, especially given the large
numbers of samples that need to be examined. Although immunological
procedures for Plasmodium detection, such as ELISAs, are rapid and amenable
to automation, they do not always discriminate between current and past
infections, because they are designed simply to detect anti-Plasmodium
antibodies in the blood of affected individuals.
A DNA diagnostic procedure that selectively measures only current infections,
i.e., the presence of DNA-containing organisms, was developed by using highly
repeated DNA from P. falciparum. First, a genomic library of the parasite DNA
was screened with labeled whole-genome parasite DNA. The most intensely
labeled hybridizing colonies were selected because they were expected to
contain repetitive DNA. The DNA from each of the selected colonies was then
tested for its ability to hybridize with DNA from several other Plasmodium
species that do not cause malaria. The DNA sequence that was chosen as a
specific probe hybridized with P. falciparum but not with P. vivax, P. cynomolgi,
or human DNA, despite the fact that P. vivax causes a less severe form of

malaria. This probe can detect as little as 10 picograms of purified P. falciparum

DNA or 1 nanogram (ng) of P. falciparum DNA in blood.

1.4.2.

Diagnosis of diarrheal pathogens

One of the least cost-effective but most time-intensive procedures undertaken in

the clinical microbiology laboratory is stool culture. Moseley et al. first reported
the use of probes to detect organisms harboring the genes encoding LT
enterotoxin in ETEC in 1980. By using a modification of the colony
hybridization technique developed by Grunstein and Hogness, isolates of E. coli
were picked from stool cultures and inoculated onto nitrocellulose filters resting
on MacConkey agar plates and grown overnight. The filters were removed from
the agar, and the bacterial growth was lysed in situ by the addition of NaOH. As
the organisms lysed, the DNA released from the bacterial cells was denatured to
single strands by the NaOH. After neutralization of the base and a baking step to
attach the DNA to the filter, the single-stranded DNA was hybridized with a 32Plabeled DNA fragment from within the gene encoding the LT enterotoxin. After
overnight hybridization, the excess probe was removed by washing the filters in
buffer and those colonies containing the toxin gene were detected by
autoradiography. These investigators also demonstrated that the same method
could be used to identify ETEC directly in stool if the sample was spotted onto
filters and grown overnight in a fashion similar to that used for isolated colonies.
Probes also have been developed to Shigella species and enteroinvasive E. coli.
Probes to Salmonella serotypes, Y. enterocolitica, and enteroadherent and
enterohemorrhagic E. coli have all been described.
Probes to detect viral agents of diarrhea have also been developed and hold
promise for future diagnostic kits. For example, rotaviruses are among the most
common causes of diarrheal illness in children throughout the world. The rapid
recognition of these agents can prevent unnecessary antimicrobial therapy and
aid in epidemiologic studies of outbreaks of diarrheal disease in nurseries.
Dimitrov et al. have developed probes to detect rotavirus in stool.

1.4.3.

Diagnosis of viruses

Molecular cloning procedures have been especially useful for obtaining

abundant supplies of those viral nucleic acid probes which cannot be propagated
in tissue culture systems, such as hepatitis B virus (HBV) and human papilloma
virus (HPV). DNA probes also have been obtained from RNA viruses such as
rotavirus and influenza A by synthesizing a complementary DNA (cDNA) from
the viral RNA using the enzyme "reverse transeriptase". Rotbart and co-workers
and Hyypia et al. have explored the use of DNA probes for the detection of
enteroviruses, such as poliovirus and coxsackievirus, in cerebrospinal fluid.

1.4.4.

Diagnosis of STDs

Organisms causing sexually transmitted diseases include bacteria, viruses, fungi,

and protozoa. Each organism in this group presents a unique challenge to the

laboratory. While N. gonorrhoeae has been recognized as a sexually transmitted

disease agent for years, its propagation and identification in the clinical
laboratory are still problematic. A DNA probe to N. gonorrhoeae was reported
by Totten et al. In this study, the cryptic plasmid of the gonococcus, which was
labeled with 32P by nick translation, was used to probe male urethral exudates
which had been spotted on nitrocellulose filters.The sensitivity of the probe was
89%, while the specificity was 100%.

Antibodies in clinical diagnosis

Ever since the development of Hybridoma Technology in 1975 by Kohler and
Milstein, our vision for antibodies as tools for research for prevention, detection
and treatment of diseases, vaccine production, antigenic characterization of
pathogens and in the study of genetic regulation of immune responses and disease
susceptibility has been revolutionized.

2.1 Monoclonal antibodies

The monoclonal antibodies being directed against single epitopes are
homogeneous, highly specific and can be produced in unlimited quantities. In
animal disease diagnosis, they are very useful for identification and antigenic
characterization of pathogens. Monoclonal antibodies have tremendous
applications in the field of diagnostics, therapeutics and targeted drug delivery
systems, not only for infectious diseases caused by bacteria, viruses and protozoa
but also for cancer, metabolic and hormonal disorders. They are also used in the
diagnosis of lymphoid and myeloid malignancies, tissue typing, enzyme linked
immunosorbent assay, radio immunoassay, serotyping of microorganisms,
immunological intervention with passive antibody, antiidiotype inhibition, or
magic bullet therapy with cytotoxic agents coupled with anti mouse specific
antibody. Recombinant deoxyribonucleic acid technology through genetic
engineering has successfully led to the possibility of reconstruction of monoclonal
antibodies viz. chimeric antibodies, humanized antibodies and complementarily
determining region grafted antibodies and their enormous therapeutic use.

2.1.1

Diagnosis of prtozoans and helminthes

The immuno diagnosis of protozoal and parasitic diseases has significantly

been improved by MAb technology because the tests involving MAb as
diagnostic reagents overcome the limitations of polyclonal antibodies. MAbs
were found extremely useful in the rapid outbreak of East Coast Fever (ECF).
MAbs of diagnostic value have also been developed against Trichomonas
vaginalis, Leishmania donovani, Trypanosoma congolense, Babesia bovis.

2.1.2

Diagnosis of viruses

With improved sensitivity and specificity of the diagnostic test system, MAbs
have also been developed against a number of animal viruses viz., bovine
herpes virus, cervine herpes virus type I, pseudo rabies virus, calf strain RIT
4237 (sub-group I) and human strain 82-561 (sub group 3) of rotavirus.

2.2 ELISA
There are a number of different ways to determine whether an antibody has bound to
its target antigen. The enzyme-linked immunosorbent assay (ELISA) is one method,
and it is frequently used for diagnostic detection. The ELISA procedure may be either
indirect or direct.
A generalized indirect ELISA protocol has the following steps.
1. Bind the sample being tested for the presence of a specific molecule or organism to
a solid support, such as a plastic microtiter plate, which usually contains 96 sample
wells. Wash the support to remove unbound molecules.
2. Add a marker-specific antibody (primary antibody directed against the target
antigen) to the bound material, and then wash the support to remove unbound
primary antibody.
3. Add a second antibody (secondary antibody) that binds specifically to the primary
antibody and not to the target molecule. Bound (conjugated) to the secondary
antibody is an enzyme, such as alkaline phosphatase, peroxidase, or urease, that can
catalyze a reaction that converts a colorless substrate into a colored product. Wash
the mixture to remove any unbound secondary antibodyenzyme conjugate.
4. Add the colorless substrate.
5. Observe or measure the amount of colored product.
If the primary antibody does not bind to a target site in the sample, the second
washing step removes it. Consequently, the secondary antibody enzyme conjugate
has nothing to bind to and is removed during the third washing step, and the final
mixture remains colorless. Conversely, if the target site is present in the sample, then
the primary antibody binds to it, the secondary antibody binds to the primary
antibody, and the attached enzyme catalyzes the reaction to form an easily detected
colored product.
Since secondary antibodies that are complexed with an enzyme are available
commercially, each new diagnostic test requires only a unique primary antibody. In
addition, several secondary antibody molecules, each with several enzyme molecules
attached, bind to one primary antibody molecule, thereby amplifying the intensity of
the signal.
With a direct ELISA protocol, a monoclonal antibody specific for the target antigen
is first bound to the surface of the microtiter plate. To assess the amount of a
particular antigen in a sample, the sample is added to the well of the microtiter plate
and allowed to interact with the bound antibody. This is followed by a wash to
remove any unbound molecules.
Then, the primary antibody and the secondary antibody conjugated to an enzyme are
added, as described above, before the presence of bound antigen is visualized.

References:
1. Kulski. J.K, Norval Mary.Nucleic acid probes in
diagnosis of viral diseases of man.

2. Waldmann TA. Monoclonal antibodies in

diagnosis and therapy. Science. 1991;252:1657
62.
3. Pfaller MA. Diagnostic applications of DNA
probes. Infect Control Hosp Epidemiol. 1991
Feb;12(2):103-10.
4. F C Tenover. Diagnostic deoxyribonucleic acid
probes for infectious diseases. Clin. Microbiol.
Rev. 1988, 1(1):82. DOI: 10.1128/CMR.1.1.82.
5. David T. Kingsbury. DNA probes in the diagnosis
of genetic and infectious diseases.
6. Saleem Muhammad and Kamal Mustafa.
Monoclonal antibodies in clinical diagnosis: A
brief review application.
7. Gupta P.K. Biotechnology and Genomics.