JPH07108913B2 - Amyloid protein derived from dog serum, method for diagnosing canine infectious disease using the same, and method for quantifying the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an amyloid protein (hereinafter abbreviated as SAA) that is specifically increased in serum during canine disease.
And, its separation and purification method, and the detection of SAA using an antibody obtained by sensitizing a mammal with SAA as an antigen,
The present invention relates to a quantification method and a disease diagnosis method.

(Prior Art) Dogs have been favored as hunting animals and pets since they have coexisted with humans since ancient times, and their utility value has been increasing as an experimental animal in recent years, and the need to manage their health is desired. ing.

It has long been known that when humans and animals are infected with microorganisms, specific antibodies are produced in the living body. As research on these immune phenomena progresses, in various humans, C-reactive protein (serum specifically presenting a precipitation reaction with C polysaccharide contained in the cell wall of Pneumococcal pneumococcus in the presence of calcium ions) Protein) (hereinafter abbreviated as CRP) has been reported to increase.

It is known that in humans, CRP increases up to 500 to 1000 times as much as in normal conditions during acute inflammation, and disappears promptly as the symptoms recover. For this reason, it is used as an important index for disease activity, treatment progress, and prognosis judgment, and is used for diagnosis of disease state, but it is not so effective index for viral diseases.

In addition, the existence thereof in animals other than humans has not been known. In this regard, the present inventors have previously succeeded in separating and purifying CRP from dogs, and have proposed a method of using such diagnostic reagents (Japanese Patent Laid-Open No. 62-155299).

Although Eriksen et al. Reported the possibility of the existence of CRP-like proteins (J.Clin.Inves
t., 61: 390-394.1978) The substance has not been specified due to reasons such as the purification method.

(Problems to be solved by the invention) Attempts to sensitize a living body with CRP, which is commonly recognized to increase during various diseases, as an antigen, and to determine disease states by the obtained antiserum and therapeutic effect determination have been reported so far. However, there is a limit in industrial productivity such as purification purity and yield improvement of CRP, and it has been used only for human diagnosis, and has not been widely used for therapeutic diagnosis of livestock animals.

Further, human CRP and dog CRP have different antigenicities (although they have some common antigenicity), and it is hardly practical to use an antiserum against human CRP for detecting canine CRP.

Further, the previously proposed CRP for dogs by the present inventors (Japanese Patent Laid-Open No. 62-155299) is a highly accurate and simple method for determining the pathology of inflammatory diseases, but it is viral. With respect to diseases and the like, fluctuations in CRP had a problem in understanding the disease state.

Moreover, CRP appears in a relatively large amount during acute inflammation, but does not increase in viral diseases. Therefore, in order to improve the accuracy of diagnosing a disease state, it has been desired to find a factor in which the increase in serum than CRP varies more sensitively in proportion to the disease state and which can be observed for a long period of time.

The above-mentioned dog CRP by the present inventors is the SAA according to the present invention.
It has been confirmed by the present inventor that it has the following differences as compared with.

1. CRP does not increase with viral infections.

2.SAA increases faster than CRP and disappears (decreases) faster than CRP with recovery.

Therefore, SAA has the advantage of being able to know the pathological changes more accurately than CRP over all disease states including viral infections, and the significance of SAA measurement according to the present invention is extremely important in the sense of complementing CRP defects. The present invention discloses that there is.

(Means for Solving the Problem) From such circumstances, the present inventor tried development research of various test items for the purpose of diagnosing the comprehensive disease state of dogs and determining the therapeutic effect. As a result, Staphylococcus aureus SAA derived from dog serum with increased biological properties in dog serum sensitized to dogs
That is, the present invention has been completed and the present invention has been completed.

That is, according to the present invention, the following amyloid protein derived from dog serum, a method for diagnosing canine infectious disease using the same, and a method for quantifying the same are provided.

1. An amyloid protein (SAA) having a peak of an average molecular weight of 13700 by a column gel filtration method and an average molecular weight peak at 12000 by an SDS-polyacrylamide gel electrophoresis method, which was separated and purified from dog serum sensitized with Staphylococcus aureus.

2. A method for diagnosing a canine infectious disease, which comprises measuring an increase or decrease in SAA using an amyloidogenic protein-reactive antibody obtained by a method of sensitizing a rabbit with SAA as an antigen.

3. A method for diagnosing a canine infectious disease, which comprises measuring the increase / decrease in SAA using the above-mentioned amyloid protein-reactive antibody, measuring C-reactive protein (CRP), and diagnosing the pathological condition based on the correlation between the two.

4. Put a solution of sensitized amyloidogenic protein-reactive antibody on latex particles and sample serum in a capillary tube, centrifuge, measure the height of the latex particles that have sedimented, and measure the height of the latex particles with a known concentration of SAA. A method for quantifying the SAA concentration in the sample serum by comparing it with the sedimentation height.

SAA according to the present invention is an amyloid protein having the following physicochemical properties.

a) Average molecular weight 13,700 by column gel filtration b) Average molecular weight 12,000-15,000 by SDS-polyacrylamide gel electrophoresis c) High density lipoprotein (HDL ₃ ) bound to the serum.

d) Reactivity with specific substances: CRP binds to C-polysaccharide,
The SAA has not yet found anything like that.

It is clear that SAA and CRP are substances with different antigenicity because the anti-rabbit antiserum using SAA as an antigen does not react with CRP. It has been confirmed that the anti-rabbit antiserum using SAA as an antigen does not react with normal dog serum that does not contain CRP, but with dog serum that has an inflammatory disease that contains CRP. The degree also shows a tendency to be proportional,
Since it has a correlation with CRP, confirmation of both SAA and CRP can be used as a means to increase the probability of qualifying disease diagnosis.

SAA tends to increase faster with the onset of inflammation and decrease with recovery compared to CRP. Since SAA increases even with virus infection or the like in which CRP does not increase, it is possible to determine that it is a viral disease when CRP concentration and SAA concentration are simultaneously measured and only SAA concentration increases. The SAA measurement is extremely meaningful in the sense that it supplements the case where CRP cannot be used as an index.

(Example) Hereinafter, the present invention will be described in more detail with reference to examples.

Production Example 1 (a) Production and purification of SAA Dog SAA purification method.

Material: Staphylococcus aures (approx. 3 x 10 ¹¹ pcs / m
l) was intraperitoneally inoculated, and dog serum collected 1 to 2 days later, and dog serum collected 1 to 2 days after surgery. Method: Purification by Eriksen, et al. N.
and Benditt, EP) according to the following steps.

1) Add KBr to serum to adjust the specific gravity to 1.063 g / cm ³ .
While maintaining this at about 10 ° C, centrifuge for 20 hours at 50,000 rpm using an ultracentrifuge (Hitachi RP50T or Beckman 60Ti rotor).

2) Remove the upper 1/4 and collect the lower 3/4. This is KBr
Adjust to 1.125 g / cm ³ . While maintaining the temperature at 10 ° C, an ultracentrifuge (using Hitachi RP70T or Beckman 60Ti rotor) is used for centrifugation for 21 hours at 55,000 rpm.

3) Take the upper 1/4 and pool (as a HDL ₂ fraction).
Collect the lower 3/4 and dissolve the insoluble sediment well. KBr is added to this to give a specific gravity of 1.21 g / cm ³ . Using an ultracentrifuge (Hitachi RP70T or Beckman 60Ti rotor), this is centrifuged at 55,000 rpm for 22 hours.

4) Obtain the upper 1/4 as an HDL ₃ fraction. Save the lower 3/4 as a CRP-containing fraction. For HDL ₂ and HDL ₃ fractions, proceed with the following steps separately.

5) Put the HDL ₂ and HDL ₃ fractions into Spectropol No. 1 tubing and dialyze against DW for 5-6 days at 4 ° C. Change the DW of dialysate at least twice a day.

6) After dialysis, freeze-dry.

7) Freeze-dried HDL ₂ fraction and HDL ₃ fraction at 0 ° C or below,
Add ethanol / ether (3: 2vol / vol), stir, and centrifuge to degrease. Centrifuge at 5,000 rpm for 15 minutes and collect the sediment. This operation is repeated 3 times over 2 days. This melts the fat in the supernatant.

8) Add anhydrous ether to the defatted fraction and wash. This operation is repeated twice. After adding ether and stirring,
Centrifuge at 5,000 rpm for 15 minutes and collect the sediment.

9) Fraction washed with dry ether (HDL ₂ apoprotein and HDL ₃
Cover the centrifuge tube containing the apoprotein) with medicine wrapping paper, make a hole in it, put it in a desiccator, suck with a vacuum pump, and dry.

10) The dried HDL ₂ apoprotein and HDL ₃ apoprotein fractions were dissolved in 0.01 M HCOONa / HCOOH buffer containing 6 M Urea, pH 3.0.

11) Sephadex G100 swollen with the above format / Urea buffer is packed in a 2.5 × 90 cm column, and the HDL ₂ apoprotein and HDL ₃ apoprotein fractions are subjected to gel filtration with this column. Perform at room temperature.

As a result of gel filtration of the HDL ₃ apoprotein fraction on a Sephadex G-100 column, two large peaks and one small peak were obtained.

The small peak part almost coincided with the elution part of ribonuclease (MW13,700) used as a molecular weight marker. (See Fig. 1) Therefore, a small peak (the part that matched MW13,700)
Was collected as an apo SAA fraction and concentrated.

12) The apo SAA fraction was subjected to gel filtration again on a Sephadex G-75 column under the same conditions as for the Sephadex G-100 column to collect the main peak. Ericsen et al.
After 11 we are carrying out ion exchange chromatography with DEAE-Cellulogue (whatman DE52), but since the amount of protein is not sufficient here, we determined that the yield would be poor,
Instead, gel filtration with Sephadex G-75 was performed.

SDS-PAGE of the fractions obtained by gel filtration on Sephadex G-75
A band was observed at a site having a molecular weight of about 12,000 to 15,000. In addition to this, two weak bands were observed at the site of large molecular weight.

There was a distance of several cm between the band at the molecular weight of 12,000 and the other two bands.

The yield was about 80 mg of SAA fraction from dog serum 1.

Production Example 2 (b) Preparation of antiserum A major band recognized at a site having a molecular weight of about 12,000 to 15,000 was cut out by SDS-PAGE, crushed with a homogenizer, and then the protein was quantified using a Coomassie protein assay reagent.

Equivalent amount of Frozenate of gel containing about 600 μg / ml of protein was used.
It was mixed with und's complete adjuvant, and 1 ml of the mixture was immunized to 16 places on the toes of rabbits (primary immunization).

Two weeks after the first immunization, the protein prepared in the same manner was immunized at 10 intradermal sites in rabbits. After that, immunization was repeated in the same manner at 1-week intervals, and a total of 7 immunizations were performed.

Five days after the final immunization, whole blood was collected and antiserum was separated.

The obtained antiserum formed a single settling line with the canine acute phase protein by the Ouchterlony method.

This antiserum shows a marked increase in inflammation in dogs,
No reaction with CRP. Since this antiserum did not react with normal canine serum containing no CRP but only with acute-phase serum containing CRP, it was determined to be an antiserum against SAA (rabbit anti-dog SAA serum).

Single radial with this rabbit anti-dog SAA serum
Single radial immunodiff
As a result of quantification of SAA by usion), it is considered that this antiserum can be used for qualitative and quantification of SAA.

By immunoelectrophoresis, this antiserum was in the α position with the dog acute phase serum containing SAA, and in the β position with purified SAA.
One settling line was formed each.

Test Example 1 (c) Changes in SAA during inflammation Two 2-year-old Beagle dogs were subjected to ovaries and hysterectomy, and then one was given penicillin administration to the skin incision and intramuscularly. The infection was prevented, and one animal was not administered with penicillin to induce infection, and penicillin treatment was started at the time when suppuration was observed in the wound site (7th day).
Changes in SAA were quantified by collecting blood over time and measuring the SAA concentration in serum. The results are as shown in the table below, and it was revealed that SAA increased rapidly after the operation and showed a tendency to increase even after suppuration, and disappeared with healing.

Table SAA concentration (μg / ml) Wound infection control dogs Wound-free dogs Preoperative ND ND 6 hours postoperative ND ND 6 hours 63 74 1 day 624 895 2 days 586 920 3 days 463 945 5 days 109 952 10 days 36 781 15 Day 12 530 20 days ND 128 30 days ND ND 60 days ND ND ND Not measurable (not present) (d) SAA quantification method 1) Latex particles with a particle size of 0.32μ were used to obtain the anti-dog obtained in Example 2. Sensitize the antibody separated from the SAA serum, put the 2% liquid into a capillary with a length of 9 cm and an inner diameter of 1 mm up to 3 cm, add the same amount of sample serum to this, and mix one mouth of the capillary,
Cut 1.5 cm from the top to 7.5 cm and immediately centrifuge for 1 minute at a speed of 800 rpm to measure the height of the latex that has settled. On the other hand, using a known concentration of SAA, the SAA concentration in the sample was quantified from the calibration curve regarding the relationship with the height of the sedimented latex.

As a method for quantifying SAA, in addition to the above method, 2) reverse passive latex agglutination reaction (RPLA) was performed on a black glass plate using a latex reagent sensitized with antibody, and as a result, clear latex agglutination was obtained. Admitted. Therefore, RPLA was performed on the diluted test serum, and the semi-quantification of SAA was possible by the highest dilution factor of the serum showing the reaction.

3) In addition to the above, Single radial immunodiffusion (Mancini
Law). Enzyme-linked immunosorbent assay (ELISA),
The assay can also be performed by a quantification method using an enzyme inhibitory homogeneous immunoassay, and the present invention is not limited to the examples.

[Brief description of drawings]

FIG. 1 is a graph showing the SAA elution pattern on a Sephadex G-100 column. 1 ... Ribonucleade (molecular weight 13,70)
Elution site of 0), 2 ... No 78-96 fraction, elution site of SAA.

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Masanori Kurono 35 Higashi Sotobori-cho, Higashi-ku, Nagoya, Aichi Prefecture Sanwa Chemical Research Institute, Inc. Company Sanwa Chemical Research Institute (56) References J. Biol. Chem. Vol. 257, No. 17 (1982) P. 10510-10517 Comp. Biochem. Physi ol. Vol. 9413, No. 1 (1989) P.I. 175-183

Claims (4)

[Claims] 1. An amyloid protein (SAA) having a peak of an average molecular weight of 13700 by a column gel filtration method and an average molecular weight peak at 12000 by an SDS-polyacrylamide gel electrophoresis method, which is separated and purified from dog serum sensitized to Staphylococcus aureus. 2. A SA using an amyloidogenic protein-reactive antibody obtained by a method of sensitizing a rabbit with SAA as an antigen.
A method for diagnosing a canine infectious disease, which comprises measuring an increase or decrease in A. 3. An increase / decrease in SAA is measured using the amyloid protein-reactive antibody, while C-reactive protein (CRP) is used.
A method for diagnosing a canine infectious disease, which comprises measuring blood pressure and diagnosing a pathological condition by correlating the two. 4. A solution in which latex particles are sensitized with an amyloidogenic protein-reactive antibody and a sample serum are put in a capillary tube, and after centrifugation, the height of the precipitated latex particles is measured, and when SAA of a known concentration is used. A method for quantifying the SAA concentration in the serum of a sample by comparing it with the sedimentation height of latex particles.