Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
ANTIBODY FOR DETECTING MYCOPLASMA PNEUMONIAE
Field of the Invention
The present invention relates to antibody useful to a detection of
microorganism that
belongs to Mycoplasma pneumoniae, that is -the cause microorganism of common
pneumonia, a detection method of the microorganism, a reagent kit for the
detection of the
microorganism, and a method for preparing the antibody useful to a detection
of the
microorganism.
The invention is important to pharmaceutical industry specifically to the
diagnosis of
atypical pneumonia caused by Mycoplasma pneumoniae, and has industrial
applicability in
the art.
The invention can be useful for detecting the species Mycoplasma pneumoniae in
test samples, such as from throat swabs, tissue samples, body fluids,
experimental solutions
and cultures.
Background Art
Diagnosis of microbial infections can be confirmed either by detection of the
causative pathogen from the infection site or by detection of antibodies to
the disease
contributing microorganisms in serum and body fluids. The diagnosis, i.e. the
detection of
the causative pathogen, is particularly important in the sense that it makes
possible quick
treatment available to the patient.
Detection of the causative pathogen of infections can be generally classified
as
cultivation and identification methods, that the causative pathogen is
selectively. cultivated
and then identified based on its physiological, biochemical or structural
properties; genetic
diagnosis, that the causative pathogen is amplified by PCR or target specific
nucleic acid
hybridization etc., thus the causative pathogen is detected; or immunological
methods, that
the causative pathogen is detected using a specific reaction of antibody with
antigen marker
of the pathogen.
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However, it takes time to obtain results by cultivation and identification
methods or
genetic diagnosis methods. Therefore, diagnosis by immunological methods is
commonly
used because the pathogen can be detected within a short time with high
sensitivity thus the
patient can be quickly and appropriately treated.
Depending on the species of microorganism, a combination of a variety of
marker
antigens and antibody or antibodies can be used for detecting the causative
pathogen of
infections by conventional immunological methods.
Mycoplasma pneumoniae is the common causative pathogen of pneumonia,
tracheobronchitis and pharygitis. It is small Gram-negative bacteria that
invade selectively
human host and cause diseases. It causes approximately 15-20% of all community-
acquired
pneumonias in general populations and up to 50% of pneumonias in certain
confmed groups
(Ragnar Norrby 1999).
The microorganism is very small in size, fastidious in culture and forms very
small
colony on enriched media. Growth of Mycoplasma pneumoniae on enriched agar
plates are
very slow, and might take at least 21 days or more for the identification
(Granato, Poe et al.
1980). Several Mycoplasma species of human origin can produce similar
biochemical
reactions. On the other hand, lacks of cell wall add further difficulties to
recognize them
under light microscope (Knudson and MacLeod 1979). Therefore, Gram staining
and
culture methods are not practiced for detecting this pathogen quickly.
DNA hybridization assay probes directed to genomic sequences for detecting
Mycoplasma pneumoniae are mentioned by Hyman et al., Buck et al., and Bernet
et al.
(Hyman, Yogev et al. 1987; Bernet, Garret et al. 1993; Buck, O'Hara et al.
1993). Probes
directed to ribosomal RNA (rRNA) sequences of Mycoplasma pneumoniae are
mentioned
by Tilton et al. (Tilton, Dias et al. 1980), Yogev et al. (Yogev, Halachmi et
al. 1988), Gobel
et al. (Gobel, Geiser et al. 1987), Zivin and Monahan (EPO 305145, and Gobel
and
Stanbridge (EPO 250662). Kai et al. (Kai, Kamiya et al. 1987) and Jensen et
al. (Jensen,
Sondergard-Andersen et al. 1993) describe primers directed to 16S rRNA
sequences of
M_ycoplasma pneumoniae. All mention probes are designed to DNA of Mycoplasma
pneumoniae or DNA of Mycoplasma pneumoniae and Mycoplasma genitalium. As these
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probes are relatively insensitive, the more recently available amplified
techniques e.g.
PCR have largely captured them. PCR is highly sensitive and can be done within
shorter
time than hybridization. However, problems of false positive often rise among
asymptomatic carriers who are negative to cultured Mycoplasma pneumoniae, or
persons
who indicate positive after their disease.
Japanese Patent Application Laid-open No. 63(1988)-298 discusses an immuno-
detection method based on the western blotting method, that uses a monoclonal
antibody
to an approximately 43 kilo Dalton membrane protein antigen of Mycoplasma
pneumonia.
However, there are problems with the said antibody and detection methods based
on that
antibody in that species specificity to Mycoplasma pneumoniae is insufficient
for proper
diagnosis due to low specificity. Moreover, the diagnostic method takes at
least five hours
to complete (Madsen et al. 1988).
In Chest. 1999 Apr;115(4):1188-94, Chan ED et al. concluded that in adults,
Mycoplasma-associated bronchiolitis without pneumonia is rarely reported, but
in
hospitalized patients, it may be more common than expected and may be
associated with
severe physiologic disturbances. Also, in Pediatr Rev. 1998 Oct;19(10):327-31,
Cimolai N.
discussed about Mycoplasma pneumoniae respiratory infections.
Disclosure of the Invention
The present invention has been achieved to solve the above problems.
Specifically,
an object of the present invention is to provide a method for specifically,
high- sensitively
and rapidly detecting a microorganism that belongs to Mycoplasma pneumoniae, a
detection antibody using for the detection and a reagent kit for the
detection. Furthermore,
another object of the present invention is to provide a method for
manufacturing the
detection antibody using for the detection.
The inventors have identified a useful protein antigen that is conserved same
function in all bacteria. Generally, the structural change of said protein is
expected to be
very low. Surprisingly, it has been found that the antibody to the protein is
specific to
bacterial species or genus, has a protean property enable to use for
discrimination specific
to bacterial species or genus, and object microorganisms may be detected all
serotypes
thereof. In more detail, said proteins are useful proteins exhibiting species-
specific
property that the structures, through the change of the amino acid sequence,
are
completely same in same species, but structural changes are accompanied in the
case
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species are different.
The inventors focused on intracellular molecules that are present as molecules
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having same function in all microorganism cells and somewhat differ between
microorganisms in terms of it's amino acid sequence, particularly Ribosomal
Protein
L7/L12 that is one kind of ribosomal protein. Ribosomal Protein L7/L12 is a
protein with
a molecular weight of approximately 13 kilo Daltons and is known to exist as
an essential
ribosomal protein in protein synthesis. Progress has been made in
understanding the
complete amino acid sequence of Ribosomal Protein L7/L12 in several
microorganisms
including Mycoplasma pneumoniae.
The inventors focused on the fact that even though there are similarities
between
different microorganisms in terms of said molecule, this molecule also has a
structural
segment that is unique to each microorganism and discovered that, it is
possible to detect
various microorganisms with species specificity and to detect all serotypes
within the
same species by using antibody to said protein.
The inventors completed the present invention upon discovering that antibody
specific to the protein of Mycoplasma pneumoniae can be obtained and species-
specific
15 detection of Mycoplasma pneumoniae is possible using said antibody.
In accordance with the present invention a monoclonal antibody specific to
Ribosomal Protein L7/L12 of Mycoplasma pneumoniae has been discovered and
developed. The antibody is novel and different than those previously described
and has
property of reacting specifically to the said protein.
The present invention relates to an antibody which is an antibody to ribosomal
protein L7/L12 comprising SEQ ID NO:2 of microorganism belonging to Mycoplasma
pneumoniae and which reacts specifically with ribosomal protein L7/L12
comprising SEQ
ID NO:2 of said microorganism.
Sequences No. I and No. 2 in the Sequence List are the DNA sequence of the
Ribosomal Protein L7/L12 gene of Mycoplasma pneumoniae and the corresponding
amino acid sequence (NCBI database accession# NC_000912). The left terminal
and right
terminal of the amino acid sequences entered in the Sequence List are amino
group
(referred to below as the N terminal) and carboxyl group terminals (referred
to below as
the C terminal), respectively, and the left terminal and right terminal of the
base sequence
is the 5' terminal and the 3' terminal, respectively. Amino acid in the
sequence of closest
match test is expressed by one letter notation. The notation "+" in closest
match test
indicates that it is different amino acid but amino acid with similar
properties such as
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hydrophobic. The notation (blank) indicates that it is entirely different
amino acid
including properties
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thereof. Moreover, the series of bio-molecular experiments of gene preparation
mentioned
in this text can be performed by methods entered in standard experimental
manuals.
"Molecular cloning: A laboratory manual", Cold Spring Harbor Laboratory Press,
Sambrook, J. et al. (1989), is given as an example of the well-known standard
experimental
manual.
Table I
Closest Match Test:
Mp: 1 MAKLDKNQLIESLKEMTIMEIDEIIKAVEEAFGVSATPVVAAGAVGGTQEAASEVTVKVT 60
1 M KLDK QLIESLKEMTI+EIDEIIKAVEEAFGV+ATP+VAAGA G TQEAASEV+VKVT
Mg: 1 MGKLDKKQLIESLKEMTIVEIDEIIKAVEEAFGVTATPIVAAGAAGATQEAASEVSVKVT 60
Mp: 61 GYTDNAKLAVLKLYREIAGVGLMEAKTAVEKLPCVVKQDIKPEEAEELKKRFVEVGATVE 120
GY DNAKLAVLKLYREI GVGLMEAKTAVEKLPCVVKQDIKPEEAEELKKRFVEVGATVE
Mg: 61 GYADNAKLAVLKLYREITGVGLMEAKTAVEKLPCVVKQDIKPEEAEELKKRFVEVGATVE 120
Mp: 121 IK 122
+K
Mg: 121 VK 122
In the present invention, the term "microorganism", means Mycoplasma
pneumoniae, specifically, indicates microorganism having a pathogenic property
in
respiratory organ and high significance in diagnosis as a causative pathogen
of Mycoplasma
infections. In the present invention, the term "antibody specifically reacting
with
microorganism" indicates an antibody that can specifically react with species
or genus of
microorganism, and antibody specifically reacting with species is especially
useful in
diagnosis of bacterial infections.
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The term "antibody" in the present invention means a polyclonal antibody or
monoclonal antibody that can be made using the entire length or only a partial
peptide of said
Ribosomal Protein L7/L12. Although there are no special restrictions to the
peptide length
for making the antibody, in the case the antibody corresponds to Ribosomal
Protein L7/L12,
the segment may be of the length characterizing the Ribosomal Protein L7/L12,
and a
peptide of 5 amino acids or longer, particularly 8 amino acids or longer, is
preferred.
Antiserum containing antibody (polyclonal antibody) that identifies Ribosomal
Protein L7/L12 can be obtained by inoculating laboratory animals with adjuvant
and a
peptide or the full length protein, as is or, when necessary, after being
cross-linked with a
carrier protein such as KLH (keyhole-limpet hemocyanin) and BSA (bovine serum
albumin)
and recovering the serum. Moreover, the antibody can be used after it has been
purified
from the antiserum. The laboratory animals that are inoculated include sheep,
horses, goats,
rabbits, mice, rats, etc., and sheep, rabbits, etc., are particularly
preferred for preparation of
monoclonal antibody. Moreover, monoclonal antibody can also be obtained by
conventional methods of making hybridoma cells, but mice are preferred in this
case.
The entire length of said protein, or its amino acid sequence of 5 or more,
preferably
8 or more, residues that has been fused with glutathione S-transferase (GST),
etc., can be
purified and used as antigen, or it can be used as antigen without being
purified. The
antibody can also be produced from the genetic recombination antibody
expressed in
cultured cell using immunoglobulin genes that have been separated by a variety
of methods
in published documents ("Antibodies: A Laboratory manual," E. Harlow et al.,
Cold Spring
Harbor Laboratory), cloning methods, etc.
Antibody to Ribosomal Protein L7/L12 that can be employed as the marker
antigen
of the present invention can be obtained by the following methods, and other
similar
methods as well, though not to be limited within these methods:
a) The desired antibody can be acquired by synthesizing a peptide fragment, in
the
case microorganism has a known Ribosomal Protein L7/L12 genetic sequence and
amino
acid sequence, using the region least similar to the amino acid sequence of
said protein of
another microorcanism and making polyclonal antibody, or monoclonal antibody,
using this
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peptide fragment as the immune source.
Moreover, it is possible to acquire the entire sequence of said gene by using
a
conventional genetic procedure, such as gene amplification by PCR using the
DNA
sequence at both terminals of said laiown genetic sequence as the probe, or
hybridization
using the sequence of a homologous segment as the template probe.
Then, a fused gene with another protein gene is constructed and said fused
gene is
inserted into the host by conventional gene insertion methods using
Escherichia coli, etc., as
the host and expressed in large quantities. The desired protein antigen can
then be acquired
by purifying the expressed protein by affmity column methods with antibody to
the protein
that was used as the fusion protein. In such a case, even if antibody to the
amino acid
segment retained within the microorganisms is acquired, it does not coincide
with the
purpose of the present invention because the full length of Ribosomal Protein
L7/L12
becomes the antigen. Consequently, hybridoma that produces monoclonal antibody
to the
antigen that has been obtained by this method is acquired by conventional
methods and the
desired antibody can be obtained by selecting a clone, which produces antibody
that will
react only with the desired microorganism.
b) For microorganisms that the amino acid sequence of the Ribosomal Protein
L71L12 is unknown, as the amino acid sequence of the Ribosomal Protein L7/L12
has 50 -
60 % of homology between microorganisms, the protein gene can easily obtained
by using a
conventional genetic procedure, such as gene amplification of specific
sequence moiety by
PCR based on the sequence of a homologous segment of the amino acid sequence,
or
hybridization using the sequence of a homologous segment as the template
probe. Then, a
fused ;ene with another protein gene is constructed and said fused gene is
inserted into the
host such as Escherichia coli, and the like by conventional gene insertion
methods, and
expressed in large quantities. The desired protein antigen can then be
acquired by purifying
the expressed protein by affmity column methods with antibody to the protein
that was used
as the fusion protein. In such a case, even if antibody to the amino acid
segment retained
within the microorcyanisms is acquired, it does not coincide with the purpose
of the present
invention because the full lenQth of Ribosomal Protein L7/L12 becomes the
antigen.
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Consequently, hybridoma that produces monoclonal antibody to the antigen that
has been
obtained by this method is acquired by conventional methods and the desired
antibody can
be obtained by selecting a clone, which produces antibody that will react only
with the
desired microorganism.
c) Altematively, as another method that is suitable for the case where the
amino acid
sequence of the Ribosomal Protein L7/L12 is unknown, a peptide of 5 to 30
amino acids
corresponding to the common sequence segment retained in the microorganisms is
synthesized from the known amino acid sequence of the Ribosomal Protein
L7/L12, and
polyclonal antibody or monoclonal antibody to this peptide sequence is made by
conventional methods. Then Ribosomal Protein L7/L12 highly purified can be
obtained by
purifying the disrupted liquid of bacterial cells through affinity column
chromatography
using said antibody. If purity of the protein is insufficient, it can be
purified by conventional
methods, such as ion exchange chromatography, hydrophobic chromatography, gel
filtration, etc., after which the eluted fraction of Ribosomal Protein L7/L12
is identified by
method such as westem blotting using antibody that was made, and purified
protein can be
obtained. The desired antibody can be obtained by acquiring hybridoma by
conventional
methods using the purified Ribosomal Protein L7/L12 antigen that has been
obtained, and
selecting hybridoma reactable specifically with the desired microorganism.
The antibody of the present invention specific to various microorganisms that
has
been obtained by the methods in a), b) and c) can be used in a variety of
immunoassay
methods to provide various diagnostic reagents and kits specific to the
desired
microorganism. For example, this antibody can be used in aggregation
reactions, that is one
of known measuring method and where antibody is adsorbed on polystyrene latex
particles,
ELISA, which is a conventional technology performed in a microtiter plate,
conventional
immunochromatography methods, and sandwich assay, whereby said antibody
labeled with
colored particles or particles having coloring capability, or with enzyme or
fluorescence
substances, and magnetic micro-particles coated with capture antibody, etc.,
are used, etc.
The term "microorganism diagnosis methods using antibody" means diagnostics
methods using any known conventional immunoassay, such as aggregation whereby
said
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antibody is adsorbed on polystyrene latex particles, ELISA, which is a
conventional
method performed in a microtiter plate, conventional immunochromatography
methods,
or sandwich assay, whereby said antibody labeled with colored particles or
particles
having coloring capability, or with enzyme or fluorescence substances, and
magnetic
micro-particles coated with capture antibody, etc., are used.
Moreover, the optical immunoassay (OIA) technology described in Japanese (via
Intemational) Patent Application Laid-open No. 07-509565, in which
microorganisms are
detected by the principle of an optical interference induced by an antibody
reaction on the
optical thin film which is formed by silicone, silicon nitride or the like, is
a useful as a
high sensible diagnostic method, especially as a diagnostic method of
microorganisms
using an antibody.
Moreover, as a method for extracting intracellular marker antigen from the
desired
microorganism in aforementioned diagnostic method, reagent treatment using an
extraction reagent(s) comprising various surfactants, such as typically Triton
X-100TM and
Tween-20TM, enzyme treatment using an appropriate enzyme, such as protease,
etc., and
physical treatment using known cell structure crushing methods, typically cell-
crushing of
microorganism, can be used. It is preferred that the most suitable conditions
for extracting
with reagent are set to each kind of microorganism using a proper combination
of
surfactants, etc.
Moreover, in the present invention the term "reagent kit for diagnosis of
microorganisms using antibody" means a diagnostic kit that uses the above-
mentioned
diagnostic method.
The amino acid and DNA sequence of Ribosomal Protein L7/L12 of Mycoplasma
pneumoniae are shown in Sequence List. Consequently, in the case of this
microorganism,
it is possible to compare the amino acid sequence of Ribosomal Protein L7/L12
with the
same protein of closely related microorganisms. Which is shown in Sequence
List under
the heading "Closest Match". Synthesizing a peptide with the segment of low
homology
and making polyclonal or monoclonal antibody to that could short cut the
selection of
those having specificity to the microorganism.
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Especially in the case of a polyclonal antibody, it is preferred that IgG
fraction be
obtained by purification of the antiserum of immunized laboratory animals with
a protein A
column, etc., and affinity purification be performed with the synthetic
peptide used in
immunization of the laboratory animals.
5 Moreover, PCR primers are formed based on the sequences of N-terminal and
C-terminal from the DNA sequence of Ribosomal Protein L7/L12 of the
microorganism.
Utilizing homology of the PCR primers, DNA fragments are amplified by the PCR
method using genomic DNA and extracted, the fragments of Mvcoplasma pneumoniae
can
be thus acquired according to a conventional method. The entire length of the
gene for
10 Ribosomal Protein L7/L12 of Mycoplasma pneumoniae can be acquired through
the analysis
of the DNA sequence information of these fragments.
The Ribosomal Protein L7/L12 gene of Mycoplasma pneumoniae thus acquired
forms a fusion protein gene with, for example, GST, etc., and an expression
vector is built
using an appropriate expression plasmid, Escherichia coli is transformed and a
large
quan-tity of said protein can be expressed. A suitable amount of the
transformed Escherichia
coli is cultivated and disrupted bacterial fluid is subjected to purification
by an affinity
column using GST to obtain the GST fusion Ribosomal Protein L7/L12 of
Mycoplasma
pneumoniae.
It is also possible to acquire the target specific monoclonal antibody by
establishing a
multiple clone of hybridomas using said protein as is or GST moiety deleted
protein as an
antigen protein, and selecting the antibody which exhibits a specific response
to
Mycoplasma pneumoniae bacteria, a homogenized fluid of the bacteria, or
Ribosomal
Protein L7/L12 of Mycoplasma pneumoniae.
Antibody made based on the present invention can be used in all known types of
immunoassay, such as known aggregation reaction whereby said antibody is
adsorbed on
polystyrene latex particles, ELISA, which is a conventional technology
performed in a
microtiter plate, conventional immunochromatography, and sandwich assay,
whereby said
antibody labeled with colored particles or particles that have coloring
capability, or enzymes
or fluorescence substances, and magnetic particles coated with capture
antibody are used,
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etc.
Moreover, antibody that is made based on the present invention can
simultaneously
function as a so-called capture antibody that captures said antigen protein in
solid or liquid
phase and as a detecting antibody that is a so-called enzyme-labeled antibody
by
modificating an enzyme, such as peroxidase and alkali phosphatase, etc., by
conventional
methods in any of these immunoassay procedure.
Preferred Embodiment of the Invention
The following examples are given to explain specifically the present
invention; the
present invention the principle of is not being restricted to these examples.
Example 1
Cloning of Ribosomal Protein L7/L12 genes from Mycoplasma pneumoniae
After inoculating an appropriate amount of Mycoplasma pneumoniae.(ATCC15531,
distributed and purchased from ATCC) on PPLO agar (DIFCO: 0412-17-3)
supplemented
with Mycoplasma Supplement (DIFCO: 0836-68-9), the niicroorganism was
cultivated for 5
hours in a CO2 incubator under conditions of 37 C and 5% CO2. The grown
colonies were
suspended in a TE buffer (Wako Pure Chemical Industries, Ltd.) to a fmal
concentration of
approximately 5 x 109 CFU/ml. Approximately 1.5 ml of this suspension was
transferred to
a microcentrifuge tube and centrifuged for 2 minutes at 10,000 rpm. The
supematant was
discarded. The sediment was resuspended in 567 l of TE buffer. Then 30 l of
10% of
SDS and 3 l of 20 mg/mi proteinase K solution were added and thoroughly
mixed, followed
by incubating for 1 hour at 37 C. The suspension was incubated for further 1
hour at 56 C.
After 80 1 of 10% acetyl trimethyl ammonium bromide/0.7 M NaCI solution were
added
and mixed, it was incubated for 10 minutes at 65 C. Equal volume of chloroform-
isoamyl
alcohol mixed solution at a volume ratio of 24:1 was added and stirred well.
The solution was centrifuged for 5 minutes at 4 C and 12,000 rpm with a
microcentrifuge and the aqueous fraction was transferred to a new
microcentrifuge tube.
Isopropanol was added to the fraction at 0.6-times its volume and the tube was
vigorously
shaken to form sediment of the DNA. The white DNA sediment was scooped with a
glass
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rod and transferred to a different microcentrifugation tube containing I ml of
70% ethanol
(cooled to -20 ). The tube was centrifuged for 5 minutes at 10,000 rpm and the
supernatant was gently removed. Then another 1 ml of 70% ethanol was added and
the
mixture was centrifuged for 5 more minutes.
Once the supernatant had been removed, the sediment was dissolved in 100 1 of
TE buffer to obtain the DNA solution. The concentration of the genomic DNA
solution
was determined quantitatively in accordance with E5: Spectrophotometric
determination
of the Amount of DNA or RNA, "Molecular cloning: A laboratory manual", Cold
Spring
Harbor Laboratory Press, Sambrook, J. et al. (1989).
PCR (polymerase chain reaction) was performed using lOng of this genomic
DNA. Taq polymerase (Takara Co., Ltd., code ROOIA) was used for PCR Five 1
of
buffer attached to the enzyme, 4 l of a dNTP mixture attached to the enzyme,
and 200
pmol of each oligonucleotide (shown in Sequence No. 3 and 4 of the Sequence
List) were
added to the enzyme. Purified water was added to bring the final volume to 50
l.
The mixture was performed for 5 cycles using TaKaRa PCR Thermal Cycler 480,
where one cycle was consisted of treatments for 1 minute at 95 C, 2 minutes at
50 C, and
3 minutes at 72 C. Then, 25 cycles of treatments for 1 minute at 95 C, 2
minutes at 60 C,
and 3 minutes at 72 C per one cycle were carried out. Electrophoresis was
performed in
1.5% agarose gel using a part of the PCR product. This product was then
stained with
ethidium bromide (Nippon Gene Co., Ltd.) and observed under ultraviolet rays
to confirm
amplification of approximately 400 by cDNA. After fragmentation treatment with
restriction endonucleases BamHI and Xhol, electrophoresis was performed in
1.5%
agarose gel and staining with ethidium bromide was carried out. An
approximately 400 by
band was cut out from the gel. This band was purified with Suprecol (Takara
Co., Ltd.)
and then inserted into pGEX-6P-lTM (Pharmacia), which is a common vector. The
vector
can function as an expression vector for the desired molecule, which can
express fused
protein with GS.T protein, by insertion of the desired gene fragment into the
appropriate
restriction endonuclease site.
Actually, vector pGEX-6P-1 TM and the previous DNA were mixed together at a
molar
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ratio of 1:3 and DNA was inserted into the vector with DNA lipase (Invitrogen
Co.).
Vector pGEX-6P-1TM into which DNA had been inserted was genetically introduced
to
Escherichia coli one-shot competent cells and then inoculated in a plate of
LBL-broth agar
(Takara Co., ltd.) that was a semi-sold culture plate containing 50 g/ml
ampicillin
(Sigma). The plate
was then incubated at 37 C for 12 hours and the grown colonies were selected
at random
and inoculated into L-Broth liquid culture medium containing the same
concentration of
ampicillin. Shake cultivation was performed at 37 C for 8 hours and the
bacteria were
recovered and the plasmid was separated using Wizard MiniprepTM in accordance
with the
attached literature. The plasmid was cleaved with restriction endonuclease
BamHUXhoI.
Insertion of said PCR product was confirmed by cutting out approximately 370
by DNA.
The DNA base sequence of the DNA that had been inserted was determined using
said
clone.
Determination of the base sequence of the inserted DNA fragment was performed
using the Fluorescence Sequencer of Applied Biosystems.
The sequence sample was prepared using PRISMTM, Ready Reaction Dye
Terminator Cycle Sequencing Kit (Applied Biosystems). First, 9.5 ) l of
reaction stock
solution, 4.0 l of 0.8 pmol/pl T7 promoter primer (Gibco BRL) and 6.5 I of
0.16
i_tg/ l template DNA were added to a microtube of 0.5 ml and mixed. After
covering the
mixture with a double layer of 100 gl of mineral oil, PCR amplification was
performed for
cycles, where one cycle was consisted of treatments for 30 seconds at 96 C, 15
seconds at 55 C, and 4 minutes at 60 C. The resulting product was then kept at
4 C for 5
minutes. After the reaction was completed, 80 gl of sterilized pure water was
added and
stirred. The product was centrifuged and the aqueous layer was extracted 3
times with
25 phenol-chloroform mixed solution. Ten ill of 3M-sodium acetate with pH 5.2
and 300 1
of ethanol were added to 100 1 aqueous layer and stirred. The product was
then
centrifuged for 15 minutes at room temperature and 14,000 rpm and the sediment
was
recovered. Once the sediment was washed with 75% ethanol, it was dried under a
vacuum
for 2 minutes to obtain the sequencing sample. The sequencing sample was
dissolved in
formamide containing 4 gI of 10 mM EDTA and denatured for 2 minutes at 90 C.
This
was then cooled in ice and
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submitted to sequencing.
Two out of the 5 randomly selected clones had homology of the sequence with
the
probe used for PCR. In addition, a DNA sequence was evidently identical to the
gene
sequence of Ribosomal Protein L7/L12. The entire base sequence and the
corresponding
amino acid sequence of the structural gene moiety are as shown in Sequence No.
1 and No. 2
of the Sequence List. This gene fragment clearly codes for the gene of
Mycoplasma
pneumoniae Ribosomal Protein L7/L12.
Example 2
Mass expression in Escherichia coil and purification of Ribosomal Protein
L7/L12 from
Mycoplasma pneumoniae
Escherichia coli into which expression vector had been inserted was cultivated
overnight in 50 ml of LB medium at 37 C. Then 500 ml of 2-times concentrated
YT medium
was heated at 37 C for 1 hour. Fifty milliliters of the Escherichia coli
solution that had been
cultivated overnight were introduced to 500 ml of the aforementioned medium.
One hour
later, 550 l of 100 mM isopropyl-(3-D(-)-thiogalactopyranoside (IPTG) were
introduced
and cultivated for 4 hours. The product was then recovered and introduced to
250 ml
centrifugation tubes and centrifuged for 10 minutes at 7,000 rpm. The
supernatant was
discarded and dissolved in 25 ml each of Lysis buffer containing 25% sucrose
in 50 mM Tris
buffer, pH7.4. The product was then added with 1.25m1 of 10% NP-40 and 125 1
of 1M
MgCI2, and transferred to a plastic tube. The product.was submitted to 5 times
of ultrasonic
treatment for 1 minute. After that, the product was centrifuged for 15 minutes
at 12.000 rpm,
and the supernatant was recovered.
Next, the aforementioned supernatant was adsorbed on a glutathione agarose
column
conditioned with PBS. Then the column was washed using 2 bed volume of a
washing
solution containing 4.2 mM MgCI2 and 1 mM dithiothreitol (DTT) in 20 mM Tris
buffer,
pH7.4. Elution was performed with 50 mM Tris buffer, pH 9.6, containing 5 mM
glutathione. The protein content in the eluted fraction was determined by the
pijment
bonding method (Bradford method; BioRad Co.) and the main fraction was
acquired.
Purity of the purified GST fusion Ribosomal Protein L7/L12 that was obtained
was
CA 02398469 2002-07-23
confirmed by electrophoresis to be approximately 75%, showing that purity
satisfactory for
an immunogen.
Example 3
Preparation of monoclonal antibody to Ribosomal Protein L7/L12 of Mycoplasma
5 pneumoniae
First, regarding to immunization of mice, 100 g of the GST fusion Ribosomal
Protein L7/L12 antigen of Mycoplasma pneumoniae were dissolved in 200 l of
PBS and
then 200 l of Freund's complete adjuvant were added and mixed and
emulsification was
performed. Two hundred microliters of the emulsion were injected
intraperitoneally to
10 immunize mice. Then the same emulsion antigen was intraperitoneally
injected after 2
weeks, after 4 weeks, and after 6 weeks. Two-fold the concentration of antigen
emulsion
was injected intraperitoneally after 10 weeks and after 14 weeks. The spleen
was excised on
3 days after the final immunization and submitted to cell fusion.
After thoroughly mixing 2 x 107 myeloma cells per 108 spleen cells from mice,
which
15 had been taken out aseptically, in a glass tube, the mixture was
centrifuged for 5 minutes at
1,500 rpm and the supernatant was discarded. The cells were thoroughly mixed.
The myeloma cells used for cell fusion were obtained by cultivation of cell
strain
NS-1 in an RPMI 1640 culture medium containing 10% bovine fetal serum,
cultivating this
product beginning 2 weeks before cell fusion using an RPMI 1640 medium
containing 0.13
mM azaguanine, 0.5 g/ml MC-210, and 10% bovine fetal serum for 1 weeks, and
then
further cultivating the cell strain for 1 week in an RPMI 1640 medium
containing 10%
bovine fetal serum.
Fifty ml of RPMI 1640 culture medium that had been kept at 37 C were added to
the
mixed cell sample and centrifuged at 1,500 rpm. After removing the
supernatant, 1 ml of
50% polyethylene glycol that had been kept at 37 C was added and stirred for 1
minute. Ten
ml of RPMI 1640 medium kept at 37 were added and the solution was vigorously
mixed for
approximately 5 minutes while it was suctioned and evacuated from a sterile
pipette.
After centrifugation for 5 minutes at 1,000 rpm and removal of the
supernatant, 30
ml of HAT culture medium were added to bring the cell concentration to 5 x 10
cells/ml.
CA 02398469 2002-07-23
16
This mixture was stirred till uniform and then poured, 0.1 ml per each well,
into a 96-well
culture plate and cultivated at 37 C in and under condition of 7% carbon
dioxide gas. HAT
culture was added, 0.1 ml at a time, on the first day and at after first week
and after second
week. Then ELISA screened the cells that had produced the desired antibody.
GST fusion Ribosomal Protein L7/L12 and GST of were dissolved in PBS
containing 0.05% sodium azide and diluted to 10 g/ml, and the diluted
solution was poured,
100 l each, into separate 96-well plates and adsorbed overnight at 4 C.
After removing the supernatant, 200 .l 1% of bovine serum albumin solution in
PBS
were added and the mixture was reacted and blocked for 1 hour at room
temperature. After
removing the supernatant, the product was washed with washing solution
containing 0.02%
Tween 20, in PBS. One hundred milliliters of culture solution of fused cells
were added to
this and the mixture was reacted for 2 hours at room temperature. The
supernatant was
removed and sediment was washed with washing solution. Next, 100 l of 50
ng/ml
peroxidase labeled goat anti-mouse IgG antibody solution were added and the
solution was
reacted for 1 hour at room temperature. The supernatant was removed and the
product was
again washed with washing solution. Then TMB solution (KPL Inc.) was added,
100 l at a
time, and the mixture was reacted for 20 minutes at room temperature. After
coloration, 100
l of 1 N sulfuric acid were added to stop the reaction and absorbance at 450
nm was
determined.
As a result, positive wells that only reacted with GST Ribosomal Protein
L7/L12, but
did not react with GST were detected, and it was concluded that antibody to
Ribosomal
~
Protein L7/L12 is produced.
Therefore, the cells in the positive wells were recovered and cultivated with
HAT
medium in a 24-well plastic plate.
The fused medium that had been cultivated was diluted with HT medium to a cell
count of approximately 20 cells/ml and then 50 1 of the diluted product was
mixed with 106
thymus cells of six-week-old mouse suspended in HT culture medium, in a 96-
well culture
plate. The mixture was then cultivated for 2 weeks at 37 C under condition of
7% carbon
dioxide gas.
CA 02398469 2008-04-14
17
Antibody activity in the culture supernatant was similarly determined by the
aforementioned ELISA method and the cells that showed positive reaction with
Ribosomal Protein L7/L12 were recovered. Furthen.nore, the same dilution and
cloning
procedure was repeated to obtain a total of 5 clones of hybridoma MPRB-1 - 5.
Example 4
Selection of monoclonal antibody that detect Ribosomal Protein L7/L12 of
Mycoplasma
pneumoniae.
Monoclonal antibody was produced and recovered in accordance with standard
methods using the positive hybridoma cells obtained as previously described.
Specifically, 5 x 106 cells in PBS that had been subcultured using RPMI 1640
culture medium containing 10% FCS were intraperitoneally injected into Balb/C
mice that
had been intraperitoneally injected with 0.5 ml of PristaneTM before 2 weeks
previously.
After 3 weeks, ascites was recovered and the centrifugation supernatant was
obtained.
The solution containing antibody that was obtained was adsorbed in a Protein A
column (5 ml bed, Pharmacia) and rinsed with PBS at 3-times of the bed volume.
Then
elution with citrate buffer, pH 3 was performed. The antibody fraction was
recovered and
the monoclonal antibody that produced by each hybridoma was obtained. The
monoclonal
antibody derived from these 5 strains of hybridoma was evaluated using ELISA
method.
The sandwich assay method was used to assess the monoclonal antibody. The
monoclonal antibody that was prepared was used as antibody for detection by
being
chemically bound to peroxidase.
That is, enzyme labeling was performed using horseradish peroxidase (Sigma
Grade VI) in accordance with the method described in "Analytical Biochemistry
132
(1983), 68-73" with the reagent S-acetylthioacetic acid N-hydroxysuccinimide
for
binding. In the ELISA reaction, a solution of commercial anti-Mycoplasma
pneumoniae
polyclonal antibody (Biodesign, rabbit) diluted to a concentration of 10 pg/ml
was
poured, 100 l each, into a separate 96-well plate and adsorbed overnight at 4
C.
After removing the supematant, 200 l of 1% bovine serum albumin solution in
PBS were added and the mixture was reacted and blocked for 1 hour at room
temperature.
The
CA 02398469 2002-07-23
18
supematant was removed and the product was washed with washing solution
containing
0.02% Tween 20, in PBS. One hundred microliters of antigen solution, which had
been
obtained by adding Triton X=100 to culture solutions of each species of
microorganism in an
amount to a concentration of 0.3% and then extracting the solution for 5
minutes at room
temperature, were added to this and the mixture was reacted for 2 hours at
room temperature.
The supematant was removed and the product was further washed with washing
solution.
Then 100 l of 5 g/ml peroxidase-labeled anti-Ribosomal Protein L7/L12
antibody
solution were added and the mixture was reacted for 1 hour at room
temperature. The
supernatant was removed and the product was washed again with washing
solution. TMB
solution (KPL Inc.) was added, 100 l each, and the mixture was reacted for 20
minutes at
room temperature. After coloration, 100 l of 1 N sulfuric acid were added to
stop the
reaction. Absorbance at 450 nm was determined.
It is clearly evident that when monoclonal antibody derived from hybridoma
MPRB-1 was used as the enzyme-labeled antibody, all strains of Mycoplasma
pneumoniae
tested were detected at a sensitivity of 106 cells/mi, while reactivity of
other microorganisms
e.g., Haemophilus influenzae, Klebsiella pneumoniae, Chlamydia pneumoniae and
Neisseria meningitides could not exhibit reactivity, even at high
concentrations of 10g
cells/ml and therefore, antibody with specific reactivity to Mycoplasma
pneumoniae can be
obtained by using monoclonal antibody to Ribosomal Protein L7/L12. The
antibody was
named as AMMP-1. Table 2 shows only those results with AMMP-1. Results with
other
antibodies, which cross-reacted with other microorganisms are not mentioned
here.
CA 02398469 2002-07-23
19
Table 2
Result of Detection (10 cells/mi)
M. pneumoniae +
Results of Detection (10 cells/ml)
N. meningitides -
N. lactamica -
N. mucosa -
N. sicca -
H. influenzae -
B. catarrharis -
N. gonorrhoeae -
E. coli -
K. pneumoniae -
(+: Positive, -: Negative)
Example 5
Acquisition of a polyclonal antibody, which specifically reacts with Ribosomal
Protein
L7/L12 of Mycoplasma pneumoniae usinj a Ribosomal Protein L7/L12-immobilized
affmity column
The Ribosomal Protein L7/Ll2 of Mycoplasma pneumoniae, which was acquired by
the method described in Examples 1, or the supernatant of Mycoplasma
pneumoniae bacteria
treated with Triton X-100 was used as an antioen. About 1.2 ml of a
physiological saline
solution containina 100 g of antigen was emulsified with the addition of 1.5
ml of Freund's
adjuvant. The emulsion was subcutaneously injected into SPF Japanese White
Rabbit to
immunize the animal. The rabbit was immunized 5 to 6 times, once every two
weeks, and
the antibody titer was confirmed.
The antibody titer was confirmed by the ELISA method. Solutions of Ribosomal
Protein L7/L12 of Mycoplasma pneumoniae dissolved in PBS containing 0.05%
sodium
azide diluted to 10 910,1/ml were poured, 100 l each, into 96-well plates and
adsorbed
overnight at 4 C. After removing the supematant, 200 l of 1% bovine serum
albumin
solution in PBS were added and the mixture was reacted and blocked for 1 hour
at room
temperature. The supernatant was removed and the product was washed with a
washino,
solution containinc, 0.02% Tween 20, in PBS. One hundred l of a solution
obtained by
CA 02398469 2002-07-23
diluting normal rabbit serum and immunized rabbit antiserum was added and the
mixture
was reacted for two hours at room temperature. The supernatant was removed and
the
product was further washed with a washin~ solution. Then, 100 l of 50 ng/ml
peroxidase-labeled goat anti-rabbit IgG antibody solution was added and the
mixture was
5 reacted for one hour at room temperature. The supernatant was removed and
the product
was washed again with a washing solution. OPD solution (Sigma Co.) was added,
100 l
each, and the mixture was reacted for 20 minutes at room temperature. After
coloration, 100
l of 1 N sulfuric acid was added to stop the reaction. Absorbance at 492 nm
was
determined.
10 After confirming that the antibody titer had increased, a large quantity of
blood was
collected. Blood was collected in a glass centrifuge tube from the ear artery,
allowed to
stand for one hour at 37 C, and then overnight at 4 C. The mixture was
centrifuged at 3000
rpm for 5 minutes and the supematant was recovered. The resulting anti-serum
was
preserved at 4 C.
15 An affmity column immobilized Ribosomal Protein L7/L12 of Mycoplasma
pneumoniae was prepared. HiTrap NHS-activated column (1 ml, manufactured by
Pharmacia) was used. Immediately after replacing the column with 1 mM HCI, a
PBS
solution of Ribosomal Protein L7/L12 (1 mg/ml) was charged. The column was
allowed to
stand for 30 minutes and a blocking reagent was charged, followed by
equilibration with
20 PBS.
Using the affmity column immobilized Ribosomal Protein L7/L12 of Mycoplasma
pneumoniae, the polyclonal antibody in the resulting anti-serum obtained as an
antigen from
the supematant of Triton X- 100 treated bacteria of Mycoplasma pneumoniae as
an antigen
was purified. This antiserum was diluted with PBS to a volume of 5 times,
passed through a
0.45 m filter, then adsorbed in the column immobilized with Ribosomal Protein
L7/L12 of
Mycoplas aa pneumoniae at a flow rate of 0.5 ml/min. After elution from the
colum.n with
0.1 M glycine buffer, pH 2.1 and immediately neutralizing with 1 M Tris
buffer, pH 9.0,
eluted fractions of the target antibody were recovered by the ELISA method,
the same as the
antibody titer measuring method.
CA 02398469 2002-07-23
21
The polyclonal antibody obtained in this manner was evaluated by the same OIA
method as described in Japanese Patent Application Laid Open No.07-509565.
The purified antibody was used as a capture antibody for the OIA method.
Moreover, AMMP-1 monoclonal antibody described in Example 4 was labeled with
peroxidase and used as the detect antibody. The enzyme labeling was performed
using
horseradish peroxidase (Sigma Grade VI) and the reagent S-acetylthioacetic
acid
N-hydroxysuccinimide for binding in accordance with the method in "Analytical
Bio-chemistry 132 (1983), 68-73".
In the OIA reaction, the purified polyclonal antibody in PBS containing 0.05%
sodium azide was diluted with 0.1 M HEPES buffer, pH 8.0, to a concentration
of 10 g/ml
and the diluted solution was added onto a silicon wafer, 50 l at a time, to
react for 30
minutes at room temperature, followed by washing with distilled water and
coated with a
coating solution including sucrose and alkali-treated casein.
Fifteen l of antigen solution, which had been obtained by adding Triton X-100
to
culture solutions of various species of microorganism to a concentration of
0.5% and then
extracting for 5 minutes at room temperature, was added onto the above-
described silicon
wafer and reacted for 10 minutes at room temperature. Then, 15 l of 20 g/ml
peroxidase-labeled monoclonal antibody was added and the mixture was reacted
for 10
minutes. After washing with distilled water, TMB solution (KPL Inc.) was
added, 15 l at a
time, and the niixture was reacted for 5 minutes at room temperature. The
product was
washed with distilled water and observed the blue colored as a result of
enzyme reaction.
As shown in Table 3 as a result, it is clear that when the purified polyclonal
antibody
of APMP-1 is used as the capture antibody, Mycoplasma pneumoniae can be
detected at a
sensitivity of 108 cells/ml, while reactivity of other microorganisms cannot
be detected.
Thus, it was confirmed that an affmity column immobilized with the Ribosomal
Protein
L7/L12 of Mycoplasma pneumoniae obtained the polyclonal antibody with specific
reactivity to Mycoplasma pneumoniae.
CA 02398469 2002-07-23
22
Table 3
Results of Detection (10 cells/ml)
M. pneumoniae +
H. influenzae ATCC10211 _
E. coli ATCC25922
E. faecalis ATCC19433 _
K. pneumoniae ATCC13883 -
N. gonorrhoeae IID821 _
N. lactamica ATCC23970 -
N. meningitidis ATCC13090 -
P. aeruginosa ATCC27853 -
Group B Streptococcus ATCC12386 -
S. aureus ATCC25923 -
S. pneumoniae ATCC27336 -
S. pyogenes ATCC19615 -
(+: Positive, -: Negative)
Industrial Applicability
According to the present invention, not only microorganisms can be detected
species
specifically, but also microorganisms of all serotypes in the same species can
be detected at
a high precision, by using antibodies to the evolutionary and functionally
conserved
intracellular molecules.
By using antibodies to Ribosomal Proteins L7/L12 of microorganisms as such
antibodies, Mycoplasma pneumoniae can be detected precisely.
Moreover, detection of microorganisms can be performed with higher precision
and
wider applicability by using the reagent kit for detecting microorganisms
comprising such
an antibody.
References Cited:
Patent Documents
United States Patent 5,552,279 Weisburg, et al. September 3, 1996. Nucleic
acid
probes for the detection of Mycoplasma pneumoniae and Mvcoplasma genitalium.
EPO 305145, Application No. 88307793.5 Zivin and Monahan. March 1,1989.
CA 02398469 2002-07-23
23
Methods and probes for detecting nucleic acid.
EPO 250662, Application No. 86304919.3. Gobel and Stanbridge. January 7, 1988.
Detection of Mycoplasma by DNA hybridization.
Japanese Patent Application Laid Open No. 63(1988)-298
Other Documents
Bernet, C., M. Garret, et al. (1993). "Detection of Mycoplasma pneumoniae by
using
the polymerase chain reaction." J Clin Microbiol 31(4): 1013-5.
Buck, G. E., L. C. O'Hara, et al. (1993). "Rapid, sensitive detection of
Mycoplasma
pneumoniae in simulated clinical specimens by DNA amplification." J Clin
Microbiol
31(4): 1013-5.
Gobel, U. B., A. Geiser, et al. (1987). "Oligonucleotide probes complementary
to
variable regions of ribosomal RNA discriminate between Mycoplasma species".
Granato, P. A., L. Poe, et al.(1980). "Use of modified New York City medium
for
growth of Mycoplasma pneumoniae. A preliminary report." Zh Mikrobiol Epidemiol
Immunobiol(8): 50-3.
Hyman, H. C., D. Yogev, et al.(1987). "DNA probes for detection and
identification
of Mycoplasma pneumoniae and Mycoplasma genitalium" .
Jensen, J. S., J. Sondergard-Andersen, et al. (1993). "Detection of Mycoplasma
pneumoniae in simulated clinical samples by polymerase chain reaction. Brief
report." J
Med Microbio138(3): 166-70.
Kai, M., S. Kamiya, et al. (1987). "Rapid detection of Mycoplasma pneumoniae
in
clinical samples by the polymerase chain reaction." J Gen Microbiol 133(Pt 7):
1969-74.
Knudson, D. L. and R. MacLeod (1979). "Mycoplasma pneumoniae and
Mycoplasma salivarium: electron microscopy of colony growth in agar." Sci Rep
Res Inst
Tohoku Univ [Med] 26(3-4): 71-91.
Madsen RD, Weiner LB, McMillan JA, Saeed FA, North JA, Coates SR. (1988).
Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a
monoclonal
antibody immunoblot assay. Am J Clin Pathol 89(1):95-9
CA 02398469 2002-07-23
24
Ragnar Norrby, S. (1999). "Atypical pneumonia in the Nordic countries:
aetiology
and clinical results of a trial comparing fleroxacin and doxycycline. Nordic
Atypical
Pneumonia Study Group." J Med Microbiol 48(12): 1115-22.
Tilton, R. C., F. Dias, et al. (1980). "DNA probe versus culture for detection
of
Mycoplasma pneumoniae in clinical specimens." J Clin Microbiol 12(6): 748-52.
Yogev, D., D. Halachmi, et al. (1988). "Distinction of species and strains of
mycoplasmas (mollicutes) by genomic DNA fmgerprints- with an rRNA gene probe."
J Clin
Microbiol 26(11): 2266-9.
Chan ED, Kalayanamit T, Lynch DA, Tuder R, Arndt P, Winn R, Schwarz MI.
"Mycoplasma pneumoniae-associated bronchiolitis causing severe restrictive
lung disease in
adults: report of three cases and literature review". Chest. 1999;115(4): 1188-
94.
NCBI database accession #NC#000912., Himmelreich,R., Hilbert, H., Plagens, H.,
Pirkl, E., Li, B. C. and Herrmann, R.
Cimolai N. Mycoplasma pneumoniae respiratory infection. Pediatr Rev. 1998;
19(10): 327-31.
Harlow, E., and D. Lane (1988). "Antibodies: A laboratory manual." New York.
Cold Spring Harbor Laboratory Press.
Shambrook, J., E. F. Fritsch, and T. Maniatis. (1989). "Molecular Cloning: A
laboratory Manual (2 d ed.)." Cold Spring Harbor Laboratory Press.
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1/4
SEQUENCE LISTING
<110> ASAHI KASEI KABUSHIKI KAISHA
<120> ANTIBOY FOR DETECTING MYCOPLASMA PNEUMONIAE
<130> ASAHI-8
<150> JP 2000 062729
<151> 2000-01-31
<160> 4
<210> 1
<211> 369
<212> DNA
<213> Mycoplasma pneumoniae
<400> 1
atg gca aaa cta gat aaa aac caa tta att gaa tcg ttg aag gaa atg 48
Met Ala Lys Leu Asp Lys Asn Gln Leu Ile Glu Ser Leu Lys Glu Met
1 5 10 15
acc atc atg gaa atc gat gaa atc att aag gct gta gaa gaa gct ttt 96
Thr Ile Met Glu Ile Asp Glu Ile Ile Lys Ala Val Glu Glu Ala Phe
20 25 30
gga gta tcg gca aca cct gta gta gct gct ggt gct gtt ggt ggt aca 144
Gly Val Ser Ala Thr Pro Val Val Ala Ala Gly Ala Val Gly Gly Thr
CA 02398469 2002-07-23
2/4
35 40 45
caa gaa gct gct agc gaa gtg act gtg aaa gtt act ggt tac act gac 192
Gln Glu Ala Ala Ser Glu Val Thr Val Lys Val Thr Gly Tyr Thr Asp
50 55 60
aac gct aaa tta gct gtg tta aag ctt tac cgc gaa att gct ggt gtt 240
Asn Ala Lys Leu Ala Val Leu Lys Leu Tyr Arg Glu Ile Ala Gly Val
65 70 75 80
ggt tta atg gaa gct aaa act gct gtg gaa aaa ctt cct tgt gtt gtt 288
Gly Leu Met Glu Ala Lys Thr Ala Val Glu Lys Leu Pro Cys Val Val
85 90 95
aag caa gac atc aaa cct gaa gaa gct gaa gaa ctt aaa aag cgt ttc 336
Lys Gln Asp Ile Lys Pro Glu Glu Ala Glu Glu Leu Lys Lys Arg Phe
100 105 110
gtt gaa gtt gga gca act gtt gaa atc aaa taa 369
Val Glu Val Gly Ala Thr Val Glu Ile Lys
115 120
<210> 2
<211> 122
<212> PRT
<213> Mycoplasma pneumoniae
<400> 2
Met Ala Lys Leu Asp Lys Asn Gln Leu Ile Glu Ser Leu Lys Glu Met
CA 02398469 2002-07-23
= 3/4
1 5 10 15
Thr Ile Met Glu Ile Asp Glu Ile Ile Lys Ala Val Glu Glu Ala Phe
20 25 30
Gly Val Ser Ala Thr Pro Val Val Ala Ala Gly Ala Val Gly Gly Thr
35 40 45
Gln Glu Ala Ala Ser Glu Val Thr Val Lys Val Thr Gly Tyr Thr Asp
50 55 60
Asn Ala Lys Leu Ala Val Leu Lys Leu Tyr Arg Glu Ile Ala Gly Val
65 70 75 80
Gly Leu Met Glu Ala Lys Thr Ala Val Glu Lys Leu Pro Cys Val Val
85 90 95
Lys Gln Asp Ile Lys Pro Glu Glu Ala Glu Glu Leu Lys Lys Arg Phe
100 105 110
Val Glu Val Gly Ala Thr Val Glu Ile Lys
115 120
<210> 3
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer DNA used for obtaining Ribosomal Protein L7/L12 gene from
Hycoplasma pneumoniae <400> 3
aatggatcca tggcaaaact agataaaaa 29
<210> 4
CA 02398469 2002-07-23
4/4
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR primer DNA used for obtaining Ribosomal Protein L7/L12 gene from
Mycoplasma pneumoniae
<400> 4
tgactcgagt tatttgattt caacagttgc 30
