JP7117824B2 - Monoclonal antibody, detection method, and detection device - Google Patents

Description

TECHNICAL FIELD The present invention relates to monoclonal antibodies used for detecting Mycoplasma pneumoniae and related techniques using the same.

Mycoplasma pneumoniae is one of the bacteria that cause pneumonia, and is characterized by severe coughing and long-term fever when infected.

The bacterium is isolated from the respiratory tract of patients for weeks to months, spreads mainly by droplet infection through contact and coughing, and tends to spread in closed spaces such as homes, schools, and facilities such as hospitals. . Inflammation occurs in the interstitium, a tissue located outside the bronchi and alveoli, so unlike other pneumonias, the noise heard with a stethoscope is less pronounced, and diagnosis is often delayed.

Typical symptoms include fever, general malaise, and dysphoria accompanied by headache for several days, followed by a dry cough, which later worsens to bloody phlegm. Cough persists for 3 to 4 weeks after fever subsides.

Mycoplasma pneumonia is an interstitial pneumonia, and although it is said that it rarely becomes severe, bronchitis and pneumonia may be accompanied by bronchitis and pneumonia, and bronchial asthma may occur if it is prolonged. In rare cases, severe pneumonia and pleural effusion may occur.

Some patients develop gastrointestinal symptoms such as nausea, vomiting, and diarrhea, and may also develop otitis media, sinusitis, myringitis, muscle pain, joint pain, and rash.

As complications, various cases such as abnormalities of the central nervous system (aseptic meningitis, encephalitis, Guillain-Barré syndrome), skin lesions, hepatitis, pancreatitis, hemolytic anemia, myocarditis, and arthritis have been reported. There are individual differences in these symptoms, and some people recover in a few days while others last more than a month.

Unlike pneumococcal pneumonia, which is specific to the elderly, Mycoplasma pneumoniae pneumonia affects a wide range of age groups, especially in childhood, school-age children, and adolescents. Since the incubation period is quite long, 2 to 3 weeks, it is necessary to pay attention to the spread of infection to the surrounding area during that period. It is generally said that the severity of the disease is rare, but because it is difficult to develop sufficient immunity, it is possible to be repeatedly infected.

Mycoplasma pneumoniae, unlike other bacteria, does not have a cell wall and therefore is not sensitive to beta-lactam (penicillin and cepham) antibiotics.

Therefore, as a therapeutic drug, macrolide antibiotics are the first choice, and erythromycin, licamycin, claris, myocamycin, josamycin, claricid, zithromax and the like are often used.

Recently, the number of resistant bacteria that are resistant to macrolide antibiotics has increased, and this has become a problem. For such resistant bacteria, tetracycline antibiotics such as minomycin and new quinolone antibiotics are used.

Diagnosis of Mycoplasma pneumoniae is difficult with these general blood tests, since white blood cells, indicators of general infections, and CRP (inflammatory response) are low. One of the methods of diagnosis is to use the antibody produced in the body by the infection with Mycoplasma pneumoniae, and to examine it by collecting blood.

An accurate test method is a method of comparing using a pair of sera, and blood is collected twice when symptoms begin to appear (acute phase) and after about two weeks after recovery (recovery phase). This is a method of diagnosis based on the degree of increase in antibody titer. Usually, Mycoplasma pneumoniae can be diagnosed if the antibody is elevated more than 4 times. A single blood sample in the acute phase is diagnosed with a high titer, such as 320 times or more.

However, this method has the drawback that accurate results cannot be obtained unless blood is collected at an appropriate time, and that it takes a long time to obtain the results.

More reliable diagnostic methods include isolate culture and genetic testing. Isolation culture is also a definitive diagnosis method, and judgment is made by seeding a throat swab specimen or sputum specimen in a dedicated PPLO medium and culturing it for a period of 2 to 4 weeks, but it may not grow well at a rate of several percent. .

As a genetic test, there are PCR (polymerase chain reaction) and LAMP (Loop-Mediated Isothermal Amplification) methods that can extract and amplify mycoplasma DNA and detect it (Patent Document 1: See Japanese Patent Application Laid-Open No. 2009-131174). ), which is known to correlate well with the culture method.

Although these methods are very useful as highly sensitive and highly specific methods, they require special equipment, require complicated sample pretreatments, and take several hours to obtain results. Furthermore, since the examination cost is high, it cannot be widely adopted for examinations by general practitioners and the like.

Attempts to detect Mycoplasma pneumoniae by immunoreaction have been made from early on. Detection by the method is disclosed (Patent Document 2: Japanese Patent Application Laid-Open No. 63-184064).

Furthermore, in 1993, preparation of a monoclonal antibody against the P1 protein highly specific to Mycoplasma pneumoniae and a detection method using this (Patent Document 3: JP-A-5-304990), and in 2001 Mycoplasma pneumoniae Also disclosed is a monoclonal antibody specific to the Ribosomal Protein L7/L12 protein and a detection method (Patent Document 4: International Publication No. 2001/57199).

And recently, as a simple and rapid test method for immunologically detecting Mycoplasma pneumoniae antigen, an immunochromatographic method using a monoclonal antibody against the P1 protein (Patent Document 5: JP-A-2013-72663) and a monoclonal antibody against Dnak have been used. An immunochromatographic method (Patent Document 6: International Publication No. 2011/068189) and an immunochromatographic method using a monoclonal antibody against P30 protein (Patent Document 7: International Publication No. 2015/025968) are also disclosed.

Although Patent Document 7 conceptually describes a monoclonal antibody against the P30 protein, it only vaguely states that "the epitope exists in the amino acid sequence AA74-274." Of course, of the 274 amino acid sequences, the range from 74 to 274 accounts for about 73% of the total, and it cannot be said that the epitope is specifically specified. Ultimately, even those skilled in the art cannot put Patent Document 7 into practical use.

Several reagents using these immunochromatographic methods are already on the market, and they appear within ten minutes or so with a simple operation of wiping the affected pharynx with a cotton swab, eluting it with a buffer solution, etc., and dropping it onto the test device. Since it is possible to know the presence or absence of infection based on the presence or absence of a line, it has come to be adopted in clinical sites where early diagnosis of infection is desired for the selection of antibiotics.

However, since the amount of antigen in Mycoplasma pneumoniae is relatively small, these methods have insufficient sensitivity, which poses a major problem of missing infected patients.

In addition, when Dnak is targeted, it cross-reacts with Mycoplasma genitalium, which is a related species (Patent Document 6), and even a commercial kit targeting L7/L12 protein shows cross-reactivity with Mycoplasma genitalium. Have been described. Thus, depending on the target sequence, there is a problem of cross-reactivity with closely related bacteria.

On the other hand, if amino acid substitution mutations or deletions occur in the target sequence, the reactivity will change and the specificity of the antibody will not match. There is also sex.

Here, the P1 protein and P30 protein, which are the target proteins of the immune reaction, are present at the tip of the cell-adsorbing organ, and play a very important role as adhesion proteins (adhesins) when they adhere to the respiratory epithelium and become infected. play.

Among them, the P30 protein generally consists of 274 amino acids, and sequence analysis of various mutant strains and comparative analysis of similar sequences with other related bacteria of the genus Mycoplasma (M. genitalium and M. gallisepticum) (Non-Patent Document 1: JOURNAL OF BACTERIOLOGY, APR.2011, p1726-1733).

In the sequence of the P30 protein, exactly the same sequence is present in P32, which is a similar adhesion protein present in the related species Mycoplasma genitalium, and the entirety of the 50 to 130 positions from the N-terminus. High homology is recognized.

The sequence from 73rd to 125th is very similar to the sequence from 68th to 120th of the P32 protein of Mycoplasma Genitalium as shown below, and the 81st C of P30 is the 76th of P32. In S, the 117th L of P30 is replaced with the 112th I of P32.

Mycoplasma pneumoniae P30 amino acid sequence 73-125
"WFIPTVAGCFGFSALAIILGLAIGLPIVKRKEKRLLEEKERQEQLAEQLQRIS"

Mycoplasma genitalium P32 amino acid sequence 68-120
"WFIPTVAGSFGFSALAIILGLAIGLPIVKRKEKRLLEEKERQEQIAEQLQRIS"

Thus, even if a monoclonal antibody against P30 has an epitope in a sequence that is highly homologous to other types of bacteria such as Mycoplasma genitalium, it is conceivable that the effect of cross-reactivity will be greater. .

In the sandwich reaction, if there is cross-reactivity with both the immobilized antibody and the labeled antibody, it will be a false-positive determination due to the presence of closely related bacteria even if it is not Mycoplasma pneumoniae, and only one of the antibodies is cross-reactive. In some cases, even if Mycoplasma pneumoniae is present, it is possible that the coexistence of closely related bacteria inhibits sandwich binding, resulting in false-negative determinations.

The P30 protein is known to have a characteristic repeat sequence in the 177th sequence from the N-terminus (C-terminal side), and a polyclonal antibody was also prepared by synthesizing the binding peptide of this repeat sequence and using it as an immunogen. It is

However, its unfavorable properties, such as immunological cross-reactivity with human tissue proteins keratin, myosin, and fibrinogen, have also been reported (Non-Patent Document 2: INFECTION AND IMMUNITY, July, 1996, p2595- 2601).
JP 2009-131174 A JP-A-63-184064 JP-A-5-304990 International Publication No. 2001/57199 JP 2013-72663 A International Publication No. 2011/068189 International Publication No. 2015/025968 JOURNAL OF BACTERIOLOGY, APR. 2011, p.1726-1733 INFECTION AND IMMUNITY, July, 1996, p2595-2601

An object of the present invention is to provide a highly reactive monoclonal antibody to Mycoplasma pneumoniae and a detection device using the same.

The monoclonal antibody according to the first invention specifically reacts with an amino acid sequence appearing multiple times on the C-terminal side from positions 177 to 274 from the N-terminus of the P30 protein of Mycoplasma pneumoniae, It does not react to the 176th from the N-terminus.

More specifically, it is a first monoclonal antibody reactive with the amino acid sequence PGMAPRP.

It is also a second monoclonal antibody having reactivity with another amino acid sequence PHPGMAP.

The present invention also provides an apparatus for immunologically detecting Mycoplasma pneumoniae by sandwich reaction using a first monoclonal antibody and a second monoclonal antibody.

Either the first monoclonal antibody or the second monoclonal antibody is the monoclonal antibody of the invention, and in a more preferred embodiment, the monoclonal antibody is combined with both the first monoclonal antibody and the second monoclonal antibody. use.

Preferably, the immunological detection method is an immunochromatographic method.

Provided is a method for recognizing Mycoplasma pneumoniae P30 protein and diagnosing Mycoplasma pneumoniae by detecting a plurality of amino acid sequences present on the C-terminal side after 177th position from the N-terminus of the P30 protein.

The presence of repeat sequences in the P30 protein is very useful because it is possible to increase the sensitivity corresponding to the number by creating a method for detecting multiple targets present in the same protein.

Furthermore, there is a great advantage that even if a partial deletion or partial mutation occurs in the target site, it can be detected without missing anything.

As described below, the present inventors have succeeded in producing monoclonal antibodies against multiple sequences present in the P30 protein, and have established a technique capable of efficiently detecting Mycoplasma pneumoniae.

By using the monoclonal antibody of the present invention, it is possible to provide an immunochromatographic detection device capable of detecting Mycoplasma pneumoniae simply and quickly. In addition, it can distinguish between the P30 protein of Mycoplasma pneumoniae and the P32 protein of Mycoplasma genitalium, and furthermore, it can detect partial deletion or partial mutation in the target protein. It can be detected and a correct diagnosis is possible.

(Development history)
First, the circumstances leading up to the completion of the present invention by the present inventors will be described.

Monoclonal antibodies were produced according to a conventional method using a lysate of Mycoplasma pneumoniae FH strain as an immunogen.

That is, a solubilized antigen of Mycoplasma pneumoniae FH strain was mixed with complete Freund's adjuvant to prepare an emulsion, which was intraperitoneally immunized to mice.

Two weeks after the immunization, the solubilized antigen of Mycoplasma pneumoniae FH strain and incomplete Freund's adjuvant were similarly mixed to prepare an emulsion, and the mice were boosted intraperitoneally three times.

Three days after the final immunization, the spleen was excised from the mouse, and the immune cells contained therein were taken out and fused with myeloma myeloma cells in the presence of polyethylene glycol to prepare hybridomas. 23 types of monoclonal antibody-producing strains that react to the antigen by confirming antibodies in the culture supernatant produced from each hybridoma by ELISA using an antigen-binding plate and an anti-mouse POD (horseradish peroxidase)-labeled polyclonal antibody. was selected.

The selected monoclonal antibodies were converted to ascites and then purified as IgG using a protein G affinity chromatography column.

That is, each monoclonal antibody was prepared at a concentration of 1 mg/ml in PBS buffer, and 1.3 μL/cm was coated on a nitrocellulose membrane (HF0135 high-flow membrane by Millipore) in a line to bind. After sufficiently drying this, it was cut into strips having a width of about 5 mm to prepare test strips.

On the other hand, each monoclonal antibody was bound to colloidal gold particles (average particle size: 60 µm) to prepare a label.

These were assembled and prepared as detection reagents for immunochromatographic antigen sandwiching, and their reactivity with Mycoplasma pneumoniae antigens was confirmed.

Here, the present inventors have selected two types of monoclonal antibodies that are reactive to the P30 protein and have particularly high detection sensitivity, respectively, anti-mycoplasma monoclonal antibody # 004 (hereinafter, antibody # 004), and anti- It was named Mycoplasma monoclonal antibody #006 (hereinafter, antibody #006).

A representative full sequence of the P30 protein is shown below.
[Full sequence of 274 amino acids of P30 protein (Wild Type: M129 strain)]
ＭＫＬＰＰＲＲＫＬＫＬＦＬＬＡＷＭＬＶＬＦＳＡＬＩＶＬＡＴＬＩＬＶＱＨＮＮＴＥＬＴＥＶＫＳＥＬＳＰＬＮＶＶＬＨＡＥＥＤＴＶＱＩＱＧＫＰＩＴＥＱＡＷＦＩＰＴＶＡＧＣＦＧＦＳＡＬＡＩＩＬＧＬＡＩＧＬＰＩＶＫＲＫＥＫＲＬＬＥＥＫＥＲＱＥＱＬＡＥＱＬＱＲＩＳＡＱＱＥＥＱＱＡＬＥＱＱＡＡＡＥＡＨＡＥＡＥＶＥＰＡＰＱＰＶＰＶＰＰＱＰＱＶＱＩＮＦＧＰＲＴＧＦＰＰＱＰＧＭＡＰＲＰＧＭＰＰＨＰＧＭＡＰＲＰＧＦＰＰＱＰＧＭＡＰＲＰＧＭＰＰＨＰＧＭＡＰＲＰＧＦＰＰＱＰＧＭＡＰＲＰＧＭＰＰＨＰＧＭＡＰＲＰＧＦＰＰＱＰＧＭＡＰＲＰＧＭＱＰＰＲＰＧＭＰＰＱＰＧＦＰＰＫＲ

(Analysis of epitope)
Antibody #004 and antibody #006 were bound to a ligand-immobilizing column to prepare an affinity chromatography column, and the P30 protein from the solubilized solution of Mycoplasma pneumoniae cells was purified. It was clarified from the results of amino acid analysis.

It was found that this is a sequence from the 56th A (alanine) of SEQ ID NO: 55 amino acids from the N-terminus of the total 274 amino acid sequences of P30 are deleted.

That is, the antibody #004 and the antibody #006 use any one of the 56th to 274th sequences of the P30 antigen (adhesin protein) known as Mycoplasma pneumoniae Adhesin Protein as an epitope. monoclonal antibody that recognizes

In order to examine the epitope in more detail, the following expression protein was prepared by fragmenting P30. These expressed proteins were produced by culturing Escherichia coli in which gene sequences corresponding to the respective sequences were incorporated into plasmid DNA by a general genetic recombination technique.

Western blotting was performed using these recombinant protein sequences, and the results confirmed reactivity with antibody #004 and antibody #006.

Fragmentation fragment name and amino acid sequence number of P30 SS-: SEQ ID NO: 52-274
No. 4: SEQ ID NO: 52-155
No. 5: SEQ ID NOS:81-175
No. 6: SEQ ID NOS: 101-195
No. 7: SEQ ID NOs: 121-215
No. 8: SEQ ID NO: 141-235
No. 9: SEQ ID NOs: 161-255
No. 10: SEQ ID NOs: 181-274
No. 5′: SEQ ID NO: 52-175
No. 6': SEQ ID NO: 52-195
No. 7′: SEQ ID NO: 52-215
No. 8': SEQ ID NO: 52-235
No. 9′: SEQ ID NO: 52-255

As a result, antibody #004 and antibody #006 were fragmented fragment no. 4, No. No response to No. 5; 6 to No. 10 and no. 6'-No. Reactivity was observed by 9'.

When these common sequences were confirmed, it was found that they recognized the amino acid sequence PRPGMPPHPGMAPRP at positions 181-195 of P30.

<Example 1>
[Measurement by immunochromatographic competitive method using synthetic peptide]
The following peptides were prepared from the P30 amino acid sequence PRPGMPPHPGMAPRP. Using these as specimens, measurement was performed by immunochromatographic competitive method using Mycoplasma pneumoniae antigen as a solid phase.

(synthetic peptide)
Peptide 1-1: PRPGMPPHPGMAPRP
Peptide 1-2: PGMPPHPGMAPRP
Peptides 1-3: GMPPHPGMAPRP
Peptides 1-4: MPPHPGMAPRP
Peptides 1-5: PHPGMAPRP
Peptides 1-6: PGMAPRP
Peptides 1-7: PGMAPR
Peptides 1-8: PGMAP
Peptides 1-9: MPPHPGM
Peptides 1-10: GMPPHPGMAP
Peptide 1-11: MPPHPGMAP
Peptide 1-12: GMPPHPGM
Peptides 1-13: PPHPGM

With reference to FIG. 1, the method of making the detection device will now be described. Here, FIG. 1(a) is a side view of a detection device according to an embodiment of the present invention, and FIGS. 1(b) and 1(b') are front views of the same detection device.

As shown in FIG. 1 , this detection device has a dropping section 11 , a labeling section 12 , a porous carrier 13 , a detection section 14 , a target section 15 and a liquid absorption section 16 .

In the figure, A is Mycoplasma pneumoniae, B is a labeled monoclonal antibody obtained by labeling the monoclonal antibody of the present invention, C is a complex, and X is a synthetic peptide.

This detection device is a device for confirming the reactivity of monoclonal antibody-labeled substances to various synthetic peptides using the immunochromatographic competitive method. , ordinarily used ones may be used. A more detailed description will be given below.

(Preparation of labeled component)
G. Gold colloid particles were prepared according to the method of Frens (Nature, 241, 20-22, 1973). 40 μg of antibody #004 was mixed with 10 mL of this gold colloid at room temperature to prepare gold colloid-labeled antibody #004. Gold colloid-labeled antibody #006 was also prepared in the same manner.

(Preparation of labeled area 12)
Colloidal gold-labeled antibody #004 was prepared so that OD520nm = 1.0, and colloidal gold-labeled rabbit gamma globulin was prepared so that OD520nm = 0.2 as a control for reaction confirmation. was made.

36 μL of this preparation solution was applied to each test on a glass fiber pad, and then freeze-dried to prepare a gold colloid-labeled antibody #004# application pad as the labeled area 12 . For antibody #006, a colloidal gold-labeled antibody #006 application pad was similarly prepared.

(Preparation of detection area 14)
Mycoplasma pneumoniae FH strain (ATCC#15531) was prepared with PBS buffer solution to 8.1×10 ⁸ CFU/mL, and placed on a nitrocellulose membrane (Millipore) as the porous carrier 13. A detection area 14 was formed by applying 0.73 μL in a line.

In addition, 0.73 μL of anti-rabbit antibody was applied per test to the downstream side of the detection zone 14 to form a control zone 15 for reaction confirmation. After drying, a Mycoplasma pneumoniae antigen-immobilized membrane (porous carrier 13 on which detection area 14 is arranged) was prepared.

(Fabrication of detection device)
Using ADVANTEC Grade 60 (trademark) filter paper as the dropping part 11, a gold colloid-labeled antibody #004 coating pad, a Mycoplasma pneumoniae antigen-immobilized membrane, and a filter paper as the liquid absorbing part 16 are superimposed on each other, and a mount paper with an adhesive is applied. We prepared a detection device using the immunochromatographic competitive method by attaching to Similarly, a detection device was also prepared using antibody #006 instead of antibody #004.

(Preparation of specimen sample solution)
A Tris-HCl buffer solution was used as an extract solution, and each peptide was adjusted to 1.0 mg/mL with this extract solution and used as a specimen sample solution. As a control, only the extract was used.

(reactivity test)
After 100 μL of the specimen sample liquid was dropped onto the two types of detection devices produced by a pipette, line color development in the detection area 14 was confirmed 15 minutes later. Here, the Mycoplasma pneumoniae antigen-immobilized membrane and each monoclonal antibody (antibody #004, antibody #006) immunoreact to form a reddish-purple color line of colloidal gold. When present, it is competitively inhibited and a colored line is not formed.

That is, the inhibition of color development confirms that the peptide contains the epitope of the monoclonal antibody.

(Results of examination of monoclonal antibody #004)
Color development was inhibited: Peptide 1-1, Peptide 1-2, Peptide 1-3, Peptide 1-4, Peptide 1-5, Peptide 1-6

Color developed: Peptide 1-7, Peptide 1-8, Peptide 1-9
From the above results, monoclonal antibody #004 was found to be "PGMAPRP (Pro-Gly-Met-Ala-Pro-Arg-Pro)".

(Results of examination of monoclonal antibody #006)
Color development was inhibited: Peptide 1-1, Peptide 1-2, Peptide 1-3, Peptide 1-4, Peptide 1-5, Peptide 1-10, Peptide 1-11

Color developed: Peptide 1-6, Peptide 1-9, Peptide 1-12, Peptide 1-13

From the above results, it was confirmed that monoclonal antibody #006 has reactivity with the peptide sequence of "PHPGMAP (Pro-His-Pro-Gly-Met-Ala-Pro)".

Furthermore, antibody #006 is also reactive with "P"R"PGMAP" in which the second amino acid of "PHPGMAP" is substituted with R, and "PHPGM"P"P" in which the sixth amino acid is substituted with P. confirmed to have

The positions of the amino acid sequences of these epitopes in the P30 protein were investigated. First, when the P30 protein of Mycoplasma pneumoniae was searched by Protein BLAST of NCBI (National Center for Biotechnology Information), 27 types of data were obtained. (As of December 10, 2015)

Here, AAD40198 is a very special mutant (class 2-3 mutant) and does not have the desired sequence, so it is excluded from the analysis target, and when the sequences of other strains are examined, the epitope is found after the 177th from the N-terminus. It can be seen that the corresponding sequence is included.

This sequence is well known to have a repeating sequence structure containing many P (prolines), and as described in Non-Patent Document 1, it mainly contains three types of repeating sequences. These 177th and subsequent sequences are grouped into the following five groups.

The Wild type M129 strain is classified into Group 1, the FH strain is classified into Group 2, and most of the strains belong to Group 1 or Group 2.

(Group 1)
AAD40199, P75330, WP_010874809, AGC04353, NP_110141, ALA30330, ALA35258, ALA34557, ALA33841, ALA33136, ALA32435, AAB96036, AAB36603

(Group 2)
BAL22031, WP_014325546, ALA37372, ALA39474, ALA38776, ALA38073, ALA36662, ALA35953, ALA30620, ABR09215

(Group 3)
WP_038534422

(Group 4)
CAB56613

(Group 5)
AAC45467

Then, according to Non-Patent Document 2, the repeating sequence PGMAPR (Pro-Gly-Met-Ala-Pro-Arg) consisting of 6 amino acids is the sequence A, the repeating sequence PGMPPH (Pro-Gly-Met-Pro-Pro-His ) is represented as sequence B, and the repeated sequence PGFPPQ (Pro-Gly-Phe-Pro-Pro-Gln) is represented as sequence C, each group is represented as follows.

Group 1
"Array A, array B, array A, array C, array A, array B, array A, array C, array A, array B, array A, array C, array A+PMGQPPRPMGPPQPGFPPKR"

Group 2
"Array A/Array B/Array A+SGFPPQ+Array A/Array B/Array A/Array C/Array A/Array B/Array A/Array C/Array A+PMGQPPRPMGPPQPGFPPKR"

Group 3
"Array A/Array B/Array A/Array C+PGMALRQGMPPH+Array A/Array C/Array A+PMGQPPRPMGPPQPGFPPKR"

Group 4
"Array A, array B, array A, array C, array A+PMGQPPRPMGPPQPGFPPKR"

Group 5
"Array A + PMGQPPRPMGPPQPGFPPKR"

Regarding the sequence PGMAPRP, which is considered to be the epitope of antibody #004, the sequence A is followed by sequence B, sequence C or P (Pro). It can be seen that there are four locations in Group 3, three locations in Group 4, and one location in Group 5.

Regarding the sequence PHPGMAP, which is considered to be the epitope of antibody #006, it is the part where sequence A follows sequence B or sequence A follows PH (Pro-His). 2 in group 3, 1 in group 4, and not in group 5.

Here, since antibody #006 also reacts with "P"R"PGMAP" and "PHPGM"P"P", the continuous portion of sequence A and sequence B, and the sequence on the C-terminal side of each sequence, " It is thought that it will also react to PRPGMPP.

Although this sequence may overlap with other epitopes, there are 4 in group 1, 4 in group 2, 2 in group 3, 1 in group 4, and 1 in group 5. I know there is. Therefore, even a special mutant such as group 5 can be detected.

Also, in group 2, there is a portion where array C "PGFPPQ" is converted to ""S"GFPPQ", and in group 3, array A "PGMAPR" is converted to "PGMA"L"R", array There is a part where "PGMPPH" which is B is converted to ""Q"GMPPH".

In this way, it is possible to detect other sequence parts by catching the repeat structure, which is very important for eliminating false negatives, in contrast to partial mutation depending on the strain.

In other words, recognizing such repetitive sequences not only leads to higher sensitivity because it can increase the chances of reaction, but also has the great advantage of being able to detect all kinds of mutations and deletions. Do you get it.

<Example 2>
Next, examples of the detection device will be described, but various immunoassay systems established using the monoclonal antibody, such as the ELISA method and the immunoagglutination method, are conceivable for the device, and are not limited to the immunochromatographic method described here. do not have.

As for the monoclonal antibodies used in the device, it is desirable to use a combination of two types of monoclonal antibodies of the present invention, but any one type can be used in combination with another antibody that reacts with the P30 protein. , which is also included in the present invention.

As for other antibodies, not only monoclonal antibodies but also polyclonal antibodies can be used.

[Measurement by immunochromatographic sandwich method]
FIG. 2(a) is a side view of a detection device according to an embodiment of the present invention, and FIG. 2(b) is a front view of the same detection device. In the figure, A is Mycoplasma pneumoniae, B is a monoclonal antibody-labeled product labeled with the monoclonal antibody of the present invention, C is a complex, D is the monoclonal antibody of the present invention, Y is Mycoplasma pneumoniae in the test object ( P30 protein). This detection device is characterized by a monoclonal antibody, but other elements may be those commonly used as detection devices using the immunochromatographic sandwich method. Here, it is preferable to use the first monoclonal antibody (PGMAPRP reactivity) obtained by removing the label from the labeled monoclonal antibody B and the second monoclonal antibody (PHPGMAP reactivity) as the monoclonal antibody D. On the other hand, although the reactivity is reduced, various changes such as using both as the first monoclonal antibody, both as the second monoclonal antibody, and replacing the above with the first and second are also possible. Such cases are also included in the protection scope of the present invention. A more detailed description will be given below. Here, a colloidal gold-labeled antibody #004 application pad was prepared in the same manner as in Example 1 (preparation of labeled component) and (preparation of labeled area 12).

(Preparation of detection area 14)
Antibody #006 of the present invention was prepared in a PBS buffer solution to a concentration of 1.0 mg/mL, and 0.73 μL per test was applied to a predetermined position of the porous carrier 13, a nitrocellulose membrane (Millipore). to form a detection area 14. Similarly, 0.73 μL of anti-rabbit antibody per test was applied downstream of the detection area 14 to form a control area 15 for reaction confirmation. After drying, an antibody #006-immobilized membrane (porous carrier 13 provided with detection area 14) was prepared.

(Fabrication of detection device)
Using ADVANTEC Grade 60 (trademark) filter paper as the dropping part 11, a gold colloid-labeled antibody #004 coating pad, an antibody #006 immobilized membrane, and a filter paper as the liquid absorbing part 16 are superimposed on each other. A detection device was prepared by attaching it to a mount and using the immunochromatographic sandwich method.

(Preparation of specimen sample solution)
The same extract as in Example 1 was used as the extract.

[Reactivity Confirmation Test]
The following Mycoplasma spp. were prepared with the extract solution to the concentration shown in the table and used as the specimen sample solution. Only the extract was used as a control.

i) Mycoplasma pneumoniae (each 1×10 ⁶ CFU/mL)
Mycoplasma pneumoniae FH (ATCC15531)
Mycoplasma pneumoniae M129-B7 (ATCC29342)
Mycoplasma pneumoniae M22 (ATCC 39505)
Mycoplasma pneumoniae M52 (ATCC 15293)
Mycoplasma pneumoniae Mac (ATCC 15492)
Mycoplasma pneumoniae PI 1428 (ATCC 29085)
Mycoplasma pneumoniae UTMB-10P (ATCC49894)
Mycoplasma pneumoniae Bru (ATCC 15377)

ii) related species bacteria Mycoplasma genitalium Mycoplasma genitalium (1×10 ⁶ CFU/mL)

(reactivity test)
After 100 μL of the specimen sample liquid was dropped onto the manufactured detection device with a pipette, line color development in the detection area 14 was confirmed 15 minutes later.

(Measurement result)
All eight Mycoplasma pneumoniae strains could be detected. On the other hand, it did not show reactivity with Mycoplasma genitalium, a closely related species.

This is because the monoclonal antibody has a useful recognition site that reacts with a wide variety of strains of Mycoplasma pneumoniae by detecting conserved sequences among strains of Mycoplasma pneumoniae, but does not cross-react with different closely related species. is.

(Cross-reactivity test)
Non-Patent Document 2 describes that the P30 repeat sequence exhibits strong immunological cross-reactivity with structural proteins such as keratin, fibrinogen, and myosin.

Therefore, a negative measurement sample containing no Mycoplasma pneumoniae and a positive measurement sample to which 0.2 μg/mL of Mycoplasma pneumoniae was added were prepared as specimens, and keratin and fibrinogen were added to each of these to 100 μg/mL to react. It was confirmed.

In this detection system, keratin and fibrinogen at a concentration of 100 µg/mL gave negative determinations, and there were no false positives due to cross-reaction. In addition, a Mycoplasma pneumoniae-positive sample (FH strain), which originally shows a positive reaction, was measured, and keratin and fibrinogen were added to confirm reactivity, but the positive determination was not inhibited and there was no effect on the reaction. .

Therefore, it was confirmed that the anti-Mycoplasma pneumoniae monoclonal antibodies #004 and #006 did not exhibit cross-reactivity with keratin and fibrinogen, and were not affected by both negative and positive reactions.

<Example 3>
[Comparison of measurement by immunochromatographic sandwich method and PCR method]
Pharyngeal swab specimens from 184 patients clinically suspected of having Mycoplasma pneumoniae infection were used for comparison between the immunochromatographic sandwich method and the PCR method.

(Measurement with a detection device using the immunochromatographic sandwich method)
A Mycoplasma pneumoniae detection device using the immunochromatographic sandwich method was prepared in the same manner as in Example 2 from (preparation of labeling component) to (preparation of detection device).

(Preparation of specimen sample solution)
The same extract as in Example 1 was used as the extract. The patient's pharynx was wiped with a sterilized pharyngeal swab, and the swab was extracted with an extract solution to obtain a specimen sample liquid.

(reactivity test)
After 100 μL of the specimen sample liquid was dropped onto the manufactured detection device with a pipette, line color development in the detection area 14 was confirmed 15 minutes later. Those in which line color development was observed were regarded as positive.

(Measurement by PCR method)
DNA extraction and real-time PCR measurement were performed using a portion of the specimen sample solution used in the immunochromatographic sandwich method. QIAamp DNA Mini Kit (trade name) manufactured by QIAGEN was used for DNA extraction.

Real-time PCR was performed according to the procedure of "Construction of a test system for rapid diagnosis of Mycoplasma pneumoniae pneumonia by Real-Time PCR method for outpatient care (Japanese Journal of Clinical Microbiology Vol.19, No.3, 2009)". rice field. Thermal Cycler Dice RealTime System TP800 manufactured by Takara Bio Inc. was used as a detection device, and SYBR Green 1 (SYBR Premix DimerEraser) (trade name) manufactured by Takara Bio Inc. was used as a detection reagent.

(As a result of the measurement)
The measurement results of each method are summarized as follows.

As described above, the positive concordance rate for the PCR method was 70.4% in this method.

The package insert of the commercially available immunochromatographic kit "Ribotest Mycoplasma" targeting ribosomal proteins L7/L12 states that the correlation with the PCR method is 57.6%.

In addition, the package insert of another mycoplasma antigen kit, "Prolast Myco," states that the correlation with the PCR method is 44.9%.

In contrast to these, it was confirmed that the present method can detect mycoplasma antigens with high sensitivity. On the other hand, for 103 negative specimens, no color development was observed in all of them, and the judgment was negative, and the specificity was also good.

(a) A side view of a detection device according to an embodiment of the present invention, (b) A front view of a detection device according to an embodiment of the present invention, (b') A front view of a detection device according to an embodiment of the present invention. figure FIG. 2(a) is a side view of a detection device according to an embodiment of the present invention, and FIG. 2(b) is a front view of the same detection device.

11 Dropping section 12 Labeling section 13 Porous carrier 14 Detection section 15 Target section 16 Absorbing section A Mycoplasma pneumoniae B Monoclonal antibody label C Complex D Monoclonal antibody X of the present invention Synthetic peptide Y Mycoplasma pneumoniae in the test object (P30 protein)

Claims (4)

It specifically reacts with an amino acid sequence that appears multiple times on the C-terminal side from the 177th to 274th positions from the N-terminus in the P30 protein of Mycoplasma pneumoniae, but does not react with the 176th position from the N-terminus. figure,
A monoclonal antibody in which the amino acid sequence is PGMAPRP (Pro-Gly-Met-Ala-Pro-Arg-Pro), and which does not react with the amino acid sequence PGMAPR and neither with keratin nor fibrinogen . It specifically reacts with an amino acid sequence that appears multiple times on the C-terminal side from the 177th to 274th positions from the N-terminus in the P30 protein of Mycoplasma pneumoniae, but does not react with the 176th position from the N-terminus. figure,
A monoclonal antibody in which the amino acid sequence is PHPGMAP (Pro-His-Pro-Gly-Met-Ala-Pro), which does not react with the amino acid sequence PGMAPR, nor with keratin and fibrinogen . 3. A detection method for specifically detecting Mycoplasma pneumoniae without showing reactivity to Mycoplasma Genitalium, using the monoclonal antibody according to claim 1 or 2. A detection device comprising a porous carrier and a detection region on which the monoclonal antibody according to claim 1 or 2 is immobilized, and performing immunological detection by immunochromatography.

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