KR100427513B1 - IMMUNOASSAY, A DIAGNOSTIC REAGENT AND A DIAGNOSTIC METHOD FOR MALARIA BY DETECTION OF MALARIA-SPECIFIC IgM - Google Patents

Abstract

The present invention relates to an immunological measurement method, a diagnostic reagent and a diagnostic method for diagnosing malaria serologically, and more particularly, an immunological measurement method, a diagnostic reagent, and a diagnostic method for detecting IgM against malaria protozoan antigens. It is about. The immunological measurement method, diagnostic reagent and diagnostic method of malaria of the present invention are useful for early diagnosis of malaria by detecting malaria-specific IgM in the blood, and distinguish malaria from current malaria patients and past medical personnel. It can be useful for diagnosis.

Description

IMMUNOASSAY, A DIAGNOSTIC REAGENT AND A DIAGNOSTIC METHOD FOR MALARIA BY DETECTION OF MALARIA-SPECIFIC IgM}

The present invention relates to a serological measurement method, a diagnostic reagent and a diagnostic method of malaria, and more particularly, to an immunological measurement method, a malaria diagnostic reagent, and a diagnostic method for detecting IgM against malaria protozoan antigen in plasma or serum. will be.

Malaria is a mosquito-borne malaria parasite that infects human erythrocytes. One billion people worldwide live in malaria-risk areas. More than 500 million people are infected with malaria each year, and more than 2 million people die. It is one of the diseases to lose. Malaria has a global distribution, but in some areas it has ceased or decreased since the 1960s due to effective control. However, due to the recent increase in abnormal temperature and drug resistance strains such as El Niño, increasing resistance to insecticides, etc., the trend is spreading again worldwide.

Malaria is largely divided into African, Latin American, and Asian, mainly Indian and Southeast Asia, each of which differs not only in geographical distribution but also in the species characteristics and genetic inclinations of protozoa. .

The life cycle of malaria is divided into Schizogony, a life history in human hosts, and Sporogyny, a life history in mosquito hosts. After moving to the liver proliferation in the liver cells or exist in a dormant state, after a certain period of time to come out into the blood will be a full-fledged growth. Malaria sporeworms are present in the blood erythrocytes as nutrients in the form of cyclic to proliferative forms, and the cleavage formed through the cleavage process releases dozens of cleavage bodies, which enter into each erythrocyte and repeat the same reproductive cycle. do. Some of them exist in the form of germ cells, and when a mosquito sucks a person in this state, the germ cells are infected with the mosquito and multiply, and then again in the stomach and salivary gland through the growth process of the mosquitoes, the human is repeatedly infected. do.

Antibiotics, such as chloroquine and primaquine, have been used for the treatment of malaria, but the treatment of malaria has recently been difficult due to the emergence of malaria resistant to these antibiotics.

Blood smears are most commonly used to diagnose malaria, a classic method of staining malaria-infected blood cells under a microscope. However, this method has the disadvantage of being time-consuming and requiring professional training of the inspector. Although reagents for testing malaria antigens are currently commercially available, they are recommended for simultaneous use with blood smearing because of their low sensitivity. Another method of testing the malaria gene is a polymerase chain reaction (PCR) method, which is highly sensitive but still in the laboratory.

Malaria has long been known in Korea as a disease called malaria, and malaria has rarely been reported in the 1970s due to the malaria eradication project that began in the 1960s. However, in recent years, the number of patients with malaria has been reported to have spread rapidly since the outbreak of the patient near the armistice line in 1993.

Four types of malaria sporeworms are known : Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale . Among them, malaria that occurs in Korea is caused by Plasmodium vivax , which is characterized by a long incubation period and a low number of protozoa present in the blood, making it difficult to diagnose. Currently, malaria diagnostic reagents imported from abroad detect antigens such as malaria dehydrogenase (Lactate Dehydrogenase), the sensitivity of which remains at 50% level, and thus has a long incubation period and antigens present in blood and There is a difficulty in diagnosing Korean malaria with fewer protozoa.

It is an object of the present invention to provide an immunological measurement method for malaria by detecting malaria specific IgM present in blood using antigens of malaria protozoa.

Another object of the present invention is to provide an IgM capture immunoassay for detecting malaria specific IgM.

It is another object of the present invention to provide a malaria diagnostic reagent having improved sensitivity and specificity, including an antigen of malaria protozoa.

It is another object of the present invention to provide a method for diagnosing malaria patients comprising detecting malaria specific IgM present in blood using antigens of malaria protozoa.

1 is a result of diagnosing 216 malaria positive samples and 353 negative samples by an IgM capture enzyme immunoassay.

FIG. 2 compares the results of diagnosing 75 malaria positive samples and 92 negative samples by an IgM capture enzyme immunoassay and an indirect enzyme immunoassay.

FIG. 3 compares the results of diagnosing 129 malaria negative samples by age by an IgM capture enzyme immunoassay and an antigen sandwich enzyme immunoassay.

In order to achieve the above object, the present inventors have developed an immunological measurement method for diagnosing malaria by detecting only IgM against malaria among the specific antibodies against malaria protozoa present in the blood of malaria patients. The present inventors have recently invented and applied a method for diagnosing malaria, including triplet malaria, as a method for detecting malaria antibodies. This method detects malaria antibodies using malaria antigen, which can detect malaria patients or past medical personnel with higher sensitivity than the prior art. However, since IgG in malaria antibodies can last for many years or decades after malaria experience, the method of IgG-incorporation in the malaria antibody test in a long-term malaria epidemic can cause a lot of positive reactions and cause confusion. As a result of experiments by the antigen sandwich immunological measurement method, the present inventors showed that there was almost no positive malaria antibody at 10 to 30 years of age in non-malarial patients, but more than 5% of positive reactions were in the 40s. This is thought to indicate a relatively high IgG positive rate at the age of 40, since it was likely exposed to malaria mosquitoes before the 1960s, when malaria was prevalent. Similarly, malaria-specific IgG will remain in the body of a malaria-infected malaria, resulting in a relatively high positive rate for malaria antibody testing, and less effective for detecting malaria. . Therefore, to diagnose malaria in the age of 40 years or older or malaria epidemic area, IgM may be more effective in screening the titer that disappears after a certain period of time than IgG, which still has titer after malaria treatment.

The present invention provides an immunological measurement method for malaria by detecting malaria-specific IgM present in blood using antigens of malaria protozoa. In one embodiment, the present invention provides an IgM capture immunoassay using an anti-human IgM antibody in a fixture. Malaria measurement method of the present invention has a high sensitivity compared to the method of measuring the specific antigen of the prior art, it is possible to early diagnosis of malaria patients, and also has the characteristics that can distinguish the normal person who has been infected with malaria in the past from the patient . Unlike the immunological measurement method and diagnostic reagent of malaria, which detects malaria-specific antibodies present in the blood by using the surface antigens of malaria protozoa, which have been tested positive for all past history who have had malaria in the long-term malaria epidemic area, the present invention According to the method and the diagnostic reagents of malaria patients and the past history can be distinguished, the present invention was completed by confirming significantly improved specificity and sensitivity than the antibody test method by indirect enzyme immunological measurement described in Comparative Example 1 below.

The present invention provides an immunological measurement method for malaria by detecting malaria-specific IgM present in blood using antigens of malaria protozoa. Preferably, the present invention provides an IgM capture immunoassay for detecting malaria specific IgM. Preferably, the present invention provides a method for measuring IgM capture enzyme immunology. Preferably, the present invention provides an immunological measurement method of malaria by using an anti-human IgM antibody to detect malaria specific IgM present in the blood.

IgM capture immunoassay (IgM capture immunoassay) for the detection of malaria specific IgM of the present invention,

Attaching the anti-human IgM antibody to the fixture;

Binding the IgM by treating serum or plasma samples to the antibody-coated fixture;

Adding a labeled antigen conjugate to the above to react with IgM bound to the antibody attached to the fixture; And

Analysis using a label of a label antigen conjugate that binds to IgM.

The present invention provides a malaria diagnostic reagent comprising an antigen of malaria protozoa and detecting malaria specific IgM present in blood. Preferably, a fixture coated with an anti-human IgM antibody; Marker antigen conjugates; And it provides a malaria diagnostic reagent comprising a substrate solution containing a coloring agent. A preferred example of the labeled antigen conjugate is an enzyme marker attached to a malaria surface antigen produced from a recombinant transformant.

In another aspect, the present invention provides a method for diagnosing malaria patients comprising detecting malaria specific IgM present in the blood using the antigen of malaria protozoa.

Hereinafter, the present invention will be described in detail.

The present invention provides an immunological measurement method for malaria by detecting malaria-specific IgM present in blood using antigens of malaria protozoa. In this method, the surface antigen of the malaria protozoa is preferably used as the antigen of the malaria protozoa. In the above method, a recombinant antigen is preferably used as the antigen of the malaria protozoa.

Antigen-order using the antibody response detecting malaria-specific IgM present in blood, the present invention booties Zh surface protein (Merozoit surface protein of preferably Plastic Sumo rhodium ratio box (Plasmodium vivax) as the Plasmodium antigen: Mero tightening of provide in whole or in method immunological measurement of malaria detecting malaria-specific IgM present in blood by using a portion, more preferably from Plastic Sumo rhodium ratio box (Plasmodium vivax) of abbreviated below MSP) agent Provided is an immunological measurement method of malaria that detects malaria specific IgM present in blood using the C-terminal portion of surface protein (MSP). The C-terminal region comprises an amino acid sequence commonly present among subspecies of the trigeminal malaria protozoa, in particular 108 which are described as SEQ ID NO: 1 and which exhibit 100% homology among subspecies of the trigeminal malaria protozoa Polypeptides consisting of amino acids (hereinafter abbreviated as " PV200C ").

The present invention preferably provides a method for immunological measurement of malaria by detecting malaria-specific IgM present in the blood using all or part of the Merzozoit surface protein of Plasmodium vivax. More preferably, the method provides an immunological measurement method for malaria, which detects malaria-specific IgM present in blood using the C-terminal portion of the mesozoite surface protein (MSP) of plasmodium bibox. In another aspect, the present invention preferably malaria using all or a portion of PV200C polypeptide present in the C- terminal region of Plastic Sumo rhodium booties Zh surface of a non-box protein (MSP) for detecting malaria-specific IgM present in blood It provides an immunological measurement method of, and more preferably provides an immunological measurement method of malaria detecting the malaria specific IgM present in the blood using the amino acid sequence of the PV200C polypeptide amino acid sequence of SEQ ID NO: 1 do. The protein can be obtained by mass expression from recombinant transformants, and the present inventors manufactured yeast transformants and E. coli transformants expressing PV200C polypeptides and deposited them at the Biotechnology Research Institute Gene Bank on December 18, 1999. (Accession No .: KCTC 8977P, KCTC 8976P). The present invention preferably provides a method and diagnostic reagent for diagnosing malaria by separating malaria surface antigen PV200C isolated and purified from the recombinant transformant with high purity of 99% or more.

The present invention provides an immunological measurement method for malaria by detecting malaria-specific IgM present in blood using antigens of malaria protozoa. In the above method, an IgM capture immunoassay is preferably provided as an immunological measurement method for detecting the IgM, and more preferably, an IgM capture enzyme immunoassay is provided. to provide. In the above method, the present invention preferably provides an immunological measurement method of malaria by using an anti-human IgM antibody to detect malaria specific IgM present in the blood.

To this end, the present invention provides a 96-well plate coated with an anti-human IgM antibody, preferably from goats, as a fixture to be used for the diagnosis of malaria. In addition, the present invention provides a label antigen conjugate in which the antigen and the label of the malaria protozoa are combined to detect malaria specific IgM. The labeled antigen conjugate of the present invention is composed of an antigen and a label, and the antigen is preferably a recombinant malaria surface antigen purified from transformed yeast or E. coli to detect malaria specific IgM. Examples of the marker of the labeled antigen conjugate include enzymes such as horseradish peroxidase (hereinafter abbreviated as "HRP") and basic phosphatase, colloidal gold, and fluorescein. And dyes (dye) and the like, but is not limited thereto, preferably enzymes, more preferably horseradish peroxidase (HRP).

Preferred examples of IgM capture immunoassay for the detection of malaria specific IgM of the present invention include (i) attaching an anti-human IgM antibody to the fixture; (ii) binding the IgM by treating a serum or plasma sample to the antibody-coated fixture; (iii) adding a labeled antigen conjugate to the above to react with IgM bound to the antibody attached to the fixture; And (iv) analyzing the label of the labeled antigen conjugate bound to IgM. More preferably, the surface antigen of the malaria protozoa is used as the antigen of the labeled antigen conjugate of step (iii) in the IgM capture immunological assay. Even more preferably C- terminal region of booties Zh surface protein (Merozoit surface protein) of Plastic Sumo rhodium ratio box (Plasmodium vivax) as an antigen of the labeled antigen conjugate of the above step (iii) in the IgM capture immunological measurement method Use all or part of

More preferred examples of IgM capture immunoassay for the detection of malaria specific IgM,

(1) reacting by adding serum or plasma samples to each well of a plate coated with anti-human IgM antibody derived from goats;

(2) washing the well plate;

(3) reacting with an IgM attached to the anti-human IgM antibody with a labeled antigen conjugate consisting of recombinant surface antigen and HRP;

(4) washing the well plate;

(5) coloring by adding a substrate solution consisting of a coloring agent and a buffer solution; And

(6) measuring absorbance using a 96 well plate reader.

In a preferred embodiment, the immunological measurement method of malaria of the present invention provides a fixture coated with a goat-derived anti-human IgM antibody, and inserted into a expression vector prepared by inserting a gene encoding an antigenic protein of a triplet malaria protozoan. An antigen-labeled antigen conjugate conjugated with HRP by the method described in Example 1 below, wherein the antigenic protein expressed and purified from recombinant yeast or recombinant E. coli transformed is provided. When serum or plasma of a person wanting to know whether malaria is infected is added to the goat-derived anti-human IgM antibody-coated fixture, human IgM present in serum or plasma is bound to the anti-human IgM antibody coated on the well. do. Adding a conjugate of malaria surface antigen to HRP, the conjugate recognizes and binds the IgM of the malaria patient among the IgMs bound to the well-coated anti-human IgM antibody. The HRP of the conjugate thus decomposed the colorant by dissolving the colorant in the substrate solution and indirectly detecting the presence of IgM and the amount of antibody in the sample plasma to measure the absorbance to diagnose malaria infection. As a result of diagnosing malaria in the plasma of malaria patients and plasma of normal persons using the measurement method of the present invention, it showed a sensitivity of 98% or more and a specificity of 99% or more. Plasma from normal humans over 40 estimated to have had all negative treatments. That is, the immunological measurement method of malaria of the present invention showed an improved specificity than the antigen sandwich enzyme immunological measurement method described in Comparative Example 2 below, compared to the indirect enzyme immunological measurement method described in Comparative Example 1 below. Sensitivity and specificity are greatly improved.

The present invention also provides a malaria diagnostic reagent comprising an antigen of malaria protozoa and detecting malaria specific IgM present in blood.

Examples of anti-human IgM antibodies used in the malaria diagnostic reagent of the present invention include, but are not limited to, anti-human IgM antibodies derived from goats. The anti-human IgM antibody is preferably specific for the mu chain of IgM and is purified by the affinity of the antigen-antibody and uses no cross reaction with IgG or other immunoglobulins.

The present invention provides a malaria diagnostic reagent comprising an antigen of malaria protozoa and detecting malaria specific IgM present in blood. Preferably the present invention provides a malaria diagnostic reagent comprising the surface antigen of the malaria protozoa and detecting malaria specific IgM present in the blood. Using all or a portion of booties Zh surface protein (Merozoit surface protein) of preferably Plastic Sumo rhodium ratio box (Plasmodium vivax) as an antigen of the malaria parasite in the malaria diagnostic reagent of the present invention further preferably Plastic Sumo It uses the C- terminal region of the non-rhodium box Mero Zh surface protein. In addition, using all or a portion of PV200C polypeptide present in the C- terminal region of the surface protein of booties Zh preferably Plastic boxes Sumo rhodium ratio as an antigen of the Plasmodium malaria in the diagnostic reagent of the present invention, more preferably Preferably, the amino acid sequence of the PV200C polypeptide is an amino acid sequence as set forth in SEQ ID NO: 1. In another preferred embodiment, the present invention provides an anti-human IgM antibody coated fixture; Marker antigen conjugates; And it provides a malaria diagnostic reagent comprising a substrate solution containing a coloring agent. In the malaria diagnostic reagent, all or part of the mesozoite surface protein of the malaria protoplasma plasmodium bibox is preferably used as an antigen of the labeled antigen conjugate, and more preferably the mesozoite of the malaria protoplasmic plasmodium bibox. All or part of the C-terminal portion of the surface protein is used.

Examples of the label of the label antigen conjugate used in the malaria diagnostic reagent of the present invention include horseradish peroxidase (HRP), basic phosphatase (colloidal gold), fluorescent (fluorescein) and pigment ( dyes) and the like, but are not limited thereto. Horseradish peroxidase (HRP) is used as a preferred label in the present invention.

In addition, the malaria diagnostic reagent of the present invention includes a substrate solution that induces a color reaction by reacting with a label of a label antigen conjugate, preferably a colorant and a buffer solution that cause color development by reacting with a label of a label antigen conjugate. Use a substrate solution consisting of. Examples of the coloring agent include tetramethylbenzidine and the like. A coloring agent such as tetramethylbenzidine in the substrate solution was decomposed by horseradish peroxidase (HRP), which is used as a preferred labeling agent for labeling antigen conjugates to induce a color reaction, and to measure the color development by absorbance. Detect the quantity.

In another aspect, the present invention provides a method for diagnosing a malaria patient comprising detecting malaria specific IgM present in the blood using the antigen of malaria protozoa. In the above diagnostic method, a method for diagnosing malaria is preferably provided for detecting malaria-specific IgM present in the blood using an anti-human IgM antibody. In the diagnosis method, used in part or in whole, preferably as a Plasmodium antigen, and using the surface antigen of Plasmodium, more preferably booties Zh surface protein of Plastic Sumo rhodium ratio box (Merozoit surface protein). In addition, in the above diagnostic methods, a rhodium ratio preferably Plastic Sumo box booties Zh surface using the C- terminal part of the protein, and more preferably from Plastic Sumo rhodium ratio box (Plasmodium vivax) as an antigen of the malaria parasite Using all or part of the PV200C polypeptide present in the C-terminal portion of the Merzozoit surface protein, even more preferably the amino acid sequence of the PV200C polypeptide is the amino acid sequence of SEQ ID NO: 1 Use it.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Preparation Example 1 Expression and Purification of Malaria Protozoan Surface Antigen from Yeast Transformants

(Step 1) Preparation of Yeast Transformant and Expression of Malaria Protozoan Surface Antigen

Malaria surface antigen PV200C polypeptide and pYES-2 vector obtained by amplifying cDNA synthesized by reverse transcription from RNA purified in malaria positive blood with pYES-2 vector were treated with restriction enzymes HindIII and XhoI, respectively, followed by T4 ligase (T4 ligase, Promega) to produce a recombinant expression vector pYLJ-MSP. The recombinant expression vector pYLJ-MSP was transformed into yeast Saccharomyces cerevisiae INVSC1 ( Sacharomyces cerevisiae INVSC1) by the method of Hinnen ( Proc. Natl. Acad. Sci. , USA 75. 1929-1933, 1978). The transformant pYLJ-MSP / S. cerevisiae INVSC1 was obtained, and the yeast transformant was deposited on December 18, 1999 to the Bank of Biotechnology (Accession Number: KCTC 8977P).

After incubating the recombinant yeast pYLJ-MSP / S. Cerevisiae INVSC1 (KCTC 8977P) expressing the surface antigen PV200C polypeptide of the malaria protozoa prepared as described above in 37 ° C. in YEPD medium containing 2% glucose, the medium was 1 By incubating with YEP medium containing% glucose and 1% galactose, glucose was depleted to induce the expression of surface antigen from recombinant yeast. In this case, the surface protein PV200C polypeptide expressed from recombinant yeast is bound to the N-terminus. Factor leader peptides ( -factor leader paptide) was secreted out of the cell and bound to the N-terminus after the surface protein was secreted. The factor leader peptide was removed by peptidase present in the cell membrane, so that only the protein of interest was secreted into the culture.

(Step 2) Purification of Surface Antigen Expressed from Yeast Transformant

In order to obtain the surface protein PV200C polypeptide secreted into the culture as in step 1, the culture medium incubated for 2 days was centrifuged to remove the cells, and only the culture solution was taken and concentrated by an ultrafilter (available from Amicon, USA). In order to simply purify the concentrated culture broth, the histidine-bound surface protein, an anion nickel, was used as six an histidine residue attached to the C-terminus of the surface protein PV200C polypeptide expressed from the yeast transformant. The attached Probond (Invitrogen) column was adsorbed. Impurities not adsorbed on the column were removed with phosphate buffer in which 10 mM imidazol was dissolved, and surface proteins adsorbed on the column were separated by desorption with phosphate buffer in 500 mM imidazole. In the above process, the surface protein was purified almost 90% or more, and the separation solution was concentrated to perform Sephacryl S-200 gel filtration chromatography. .

In an embodiment of the present invention, the surface antigen of the malaria fission body was isolated and purified from the yeast transformant in high purity in the same manner as described above, and used in the immunological measurement method, diagnostic reagent, and diagnostic method of malaria of the present invention.

Preparation Example 2 Expression and Purification of Malaria Protozoan Surface Antigen from an E. Coli Transformant

(Step 1) Preparation of E. coli transformants and expression of malaria protozoan surface antigen

The DNA fragment of MSP PV200C obtained in the same manner as in Preparation Example 1 was purified with a phenol / chloroform mixture solution, and then the malaria surface antigen PV200C polypeptide and the pET-19b (Novagen) vector were combined with restriction enzyme NdeI and After treatment with BamHI, T4 ligase was linked to obtain a recombinant expression vector pELK-MSP. The produced expression vector pELK-MSP was transformed into E. coli BL21 (DE3) lysS by the calcium chloride method to secure the E. coli transformant pELK-MSP / BL21, and the E. coli transformant was transferred to the Biotechnology Research Institute Gene Bank in December 1999. Deposited 18 days (Accession No. KCTC 8976P).

The E. coli transformant pELK-MSP / BL21 (KCTC 8976P) prepared as above was incubated for 12 hours in LB medium to which ampicillin (100 µg / ml) and chloramphenicol (50 µg / ml) were added. 50 ml of the culture solution was inoculated again in 1 L LB medium and incubated at 37 ° C. for 2 hours, and when the absorbance (0D600) reached 0.3, IPTG (Isopropyl β-D-Thiogalactopyranoside) was added so that the final concentration became 0.2 mM. And incubated for another 7 hours.

(Step 2) Purification of surface antigen expressed from E. coli transformants

Centrifugation of the culture medium as in step 1 to separate the cells only, the separated cells were suspended in 30 ml of phosphate buffer and crushed using an ultrasonic crusher (Sonicator; Sonifier 450, Branson) and then centrifuged at 5000 rpm Separated. Some of the MSP proteins expressed from E. coli transformants are present in the supernatant and the remainder in the centrifuged precipitate, respectively.The supernatant and the precipitate were dissolved in 4M urea buffer (a buffer solution in which 4M urea was dissolved in phosphate buffer, pH 7.5). ) And completely suspended, and centrifuged again to collect only the supernatant.

In order to purify the surface protein expressed from E. coli transformants, 10 histidine residues labeled at the N-terminus of the protein were adsorbed onto a Probond column (Invitrogen). Impurities not adsorbed on the column were removed with phosphate buffer in which 10 mM imidazol was dissolved, and surface proteins adsorbed on the column were separated by desorption with phosphate buffer in 500 mM imidazole. In the above process, the surface protein was almost purely separated by more than 90%, and the separation solution was concentrated to perform Sephacryl (Sephacryl) S-200 gel filtration chromatography, and the surface protein was separated with high purity of almost 99%.

In the embodiment of the present invention, the surface antigen of the malaria cleavage body was isolated and purified from the E. coli transformant in high purity, and used in the immunological measurement method, diagnostic reagent, and diagnostic method of malaria of the present invention.

Example 1 Diagnosis of Malaria by IgM Capture Enzyme Immunoassay

In an embodiment of the present invention, in order to diagnose malaria by IgM capture immunological measurement method, goat-derived anti-human IgM antibody was coated on a fixed body, and the recombinant surface antigen purified from transformed yeast or Escherichia coli was wasabi. An enzyme-labeled antigen conjugate made by conjugation with peroxidase (HRP) was used.

The plate used in the present invention was prepared as follows. Chlorine-derived anti-human IgM antibody (Bethyl anti-human IgM mu chain-specific, affinity purified, USA) is diluted in phosphate buffer solution (pH 7.0) to 1 ~ 10 ㎍ / ㎖ and then 100 to 200 on polystyrene plate Each well was added to the wells and left at room temperature for 12 to 18 hours. The solution remaining in the wells was aspirated and removed, dissolved in 0.1% gelatin or 0.1% casein in a phosphate buffer solution and added to the wells for 250 hours each. After standing at room temperature, the solution was aspirated to remove and dried for 18 hours in a low temperature room.

Specifically, 100 μl of each solution is added to each well of a plate coated with a goat-derived anti-human IgM antibody in a phosphate buffer solution containing 0.1% bovine serum albumin as a sample dilution solution and antibody negative plasma. The sample and the antibody-positive plasma sample were added to each 20 ~ 20 ㎕ mixed well, then put into a reactor at 37 ℃ reacted for 60 minutes. After the reaction was completed, the well plate was washed five times using phosphate buffer (300 μl) containing 0.05% Tween 20. A conjugate of malaria surface antigen (prepared by the method of Preparation Example 1 or 2) and HRP purified from yeast or Escherichia coli was prepared as follows.

Specifically, the recombinant malaria fissile surface antigen isolated and purified by the method of Preparation Example 1 or 2 was prepared by using 1 L of 0.01 M sodium carbonate buffer (pH 9.6) at 4 ° C. Dialysis was carried out for 1 day with 3 exchanges. In addition, HRP to be conjugated to the surface antigen, first dissolved 5 mg of HRP in 0.5 ml of distilled water, and then 100 μl of 42 mg / ml sodium iodide (NaI04) was added to the HRP solution to form a tube with foil. In a wrapped state, HRP was oxidized by shaking for 30 minutes at room temperature. When HRP was sufficiently oxidized, 60 µl of 1M glycerol was added to the reaction solution, and the reaction was terminated by shaking the tube at room temperature for 30 minutes while wrapping the tube with foil.

For the conjugation reaction between HRP and surface antigen, the HRP reaction solution was removed using a PD10 column (Pharmacia) saturated with 0.01 M sodium carbonate buffer solution (pH 9.6). After the surface antigen solution was added to the HRP reaction solution thus oxidized, the tube was wrapped in foil to react with shaking overnight at room temperature to prepare an HRP-surface antigen conjugate. After the conjugation reaction between HRP and surface antigen was completed, 40.8 μl of 4 mg / ml NaBH4 was added to stabilize the HRP-surface antigen conjugate, and the reaction was carried out by shaking the tube at 4 ° C. for 2 hours while wrapping the tube with foil. In addition, in order to remove HRP in the reaction solution that remains unbound from the HRP-bound HRP-conjugated conjugate, a pros equilibrated with phosphate buffer using histidine residues present at the C-terminus of the surface antigen conjugated to HRP. Unbound HRP was removed with phosphate buffer after adsorption on the bond column. The HRP-surface antigen conjugate adsorbed on the probond column is desorbed by treatment with 1 M imidazole phosphate buffer to collect proteins, and the protein concentration of the HRP-surface antigen conjugate is measured using a bovine serum albumin (BSA) protein quantitative kit. After that, BSA at a final concentration of 1% was added and stored in the refrigerator.

Using a conjugate of malaria surface antigen and HRP prepared as described above, using a phosphate buffer solution (pH 7.0) containing 1% BSA and 0.05% Tween 20 (pH 7.0) in a ratio of 1: 100 to 1: 1000. After dilution, 100 μl of the enzyme-labeled antigen conjugate was added to the wells, and the reaction was carried out at 37 ° C. for 30 minutes. After the reaction, the well plate was washed five times with phosphate buffer solution containing 0.05% Tween 20, 100 μg / ml tetramethylbenzidine, 0.006% hydrogen peroxide and citric acid phosphate buffer (pH 4.5). Substrate solution containing 100 μl was added and developed in the dark for 30 minutes. After proper color development, 100 µl of the reaction stopper solution (1N sulfuric acid solution) was added to each well to terminate the color reaction, and then, at 96 nm at 650 nm using a 96 well plate reader (Molecular Devices) as a reference wavelength. Absorbance was measured. Tetramethylbenzidine, a color developing agent in the substrate solution, was decomposed by HRP of an enzyme-labeled antigen conjugate bound to human IgM to cause a color reaction, and the degree of color development was measured by absorbance to detect the presence of malaria-specific IgM and the amount of antibody.

In the above method, malaria was diagnosed in 216 malaria positive samples (plasma of malaria patient) and 353 negative samples (plasma of normal person). As a result, the IgM capture enzyme immunoassay using the recombinant antimalarial surface antigen of the present invention, when the positive cut-off is determined by determining the positive cut-off value to the negative sample average value of 0.05, the malaria patient 212 out of 216 plasma samples were positive and 351 out of 353 plasma samples of normal were negative, showing 98.1% sensitivity and 99.4% specificity (see FIG. 1).

Comparative Example 1 Comparison of IgM capture enzyme immunoassay and indirect enzyme immunoassay

In an embodiment of the present invention, goat-derived anti-human IgM antibody was coated on the fixture to diagnose malaria through IgM capture immunological assay. In addition, an enzyme-labeled antigen conjugate made by conjugating recombinant surface antigen purified from transformed yeast or Escherichia coli with horseradish peroxidase (HRP) was used (see the method of Example 1 above).

For comparison, preferred embodiments of our indirect enzyme immunological assay are as follows. First, 100 μl of the recombinant surface antigen solution of malaria isolated and purified in Preparation Example 1 or 2 in a 96-well plate (Nunc) was diluted in 0.1 M carbonate buffer (pH 9.5) at a concentration of 0.5 μg / ml. After aliquoting, the plate was sealed and left at 4 ° C. for 18 hours so that the antigens dispensed in the wells were attached to the well plates. Surface antigen remaining unattached to the well plate was removed by dispensing 300 μl of phosphate buffer solution containing 5% Goat serum per well and standing at 4 ° C. for 18 hours. The solution remaining in the well was removed and left at room temperature for 1 hour to dry the moisture, and then placed in a sealed container with a dehumidifying agent and stored in a low temperature 4 ℃ refrigerator. NaOH (1.15 mg / ml), KH 2 PO 4 (0.2 mg / ml), KCl (0.2 mg / ml), NaCl (8 mg / ml) in each well of a 96-well plate coated with a recombinant surface antigen of malaria prepared as described above ㎖), bovine serum (300 µl / ml), chimeric sal (0.2 mg / ml), and 100 µl of the sample dilution were added, and 10 µl of the antibody negative plasma sample and the antibody-positive plasma sample were added and mixed well. Into the reactor at 37 ℃, reacted for 60 minutes. After the reaction was completed, the well plate was washed five times using phosphate buffer (300 μl) containing 0.05% Tween 20. HRP-conjugated anti-human IgG antibody (Cappel) and anti-human IgM antibody (Bethy1) were derived from Tris (10 mM), NaCl (0.5M), CaC1 ₂ (1 mM), glycerol (0.5%), bovine 1: 40,000 using dilutions containing serum (0.2 mL / mL), goat serum (100 μL / mL), Tween 20 (0.05%), chimerosal (0.2 mg / mL) and phenol red (50 μg / mL) After diluting at a ratio of 100 μl each diluted conjugate was added to the wells and allowed to react at 37 ° C. for 60 minutes. After completion of the reaction, the well plate was washed five times with 0.05% Tween 20 / phosphate buffer, and contained 100 µg / ml of tetrametylbenzidine, 0.006% hydrogen peroxide and citric acid phosphate buffer (pH 4.5). 100 μl of the substrate solution was added and developed in the dark for 30 minutes. After proper color development, 100 µl of the reaction stopper solution (2N sulfuric acid solution) was added to each well to terminate the color reaction, and then, at 96 nm at 650 nm using a 96 well plate reader (Molecular Devices) as a reference wavelength. Absorbance was measured. Tetramethyl benzidine, a color developing agent in the substrate solution, is decomposed by HRP bound to anti-human IgG antibody and anti-human IgM antibody to cause color reaction, and the degree of color development is measured by absorbance. Was detected.

Malaria was diagnosed in 75 malaria positive samples (plasma of malaria patients) and 92 negative samples (plasma of normal persons) to compare the difference between the method used in the present invention and the indirect enzyme immunological measurement method described above. As a result, the IgM capture enzyme immunoassay using the recombinant antimalarial surface antigen of the present invention, when the positive cut-off is determined by determining the positive cut-off value to the negative sample average value of 0.05, the malaria patient 73 out of 75 plasma samples were positive and 92 out of 92 plasma samples were negative, showing 97.3% sensitivity and 100% specificity.

In contrast, the indirect enzyme immunological measurement method described above determined 59 out of 75 plasma samples of malaria patients when the same plasma was determined based on a positive cut-off value of 0.2 plus a negative sample mean. Positive and 85 of 92 normal plasma samples were negative, showing 78.6% sensitivity and 92.4% specificity (see FIG. 2).

<Comparative Example 2> Comparison of IgM capture enzyme immunoassay and antigen sandwich enzyme immunoassay for normal age groups

In an embodiment of the present invention, in order to diagnose malaria by IgM capture immunological measurement method, goat-derived anti-human IgM antibody was coated on a fixed body, and the recombinant surface antigen purified from transformed yeast or Escherichia coli was wasabi. An enzyme-labeled antigen conjugate made by conjugation with peroxidase (HRP) was used (see the method of Example 1 above).

For comparison, preferred embodiments of our antigen sandwich enzyme immunoassay method are those of a plate coated with malaria recombinant surface antigen prepared by the same method as the preferred embodiment of indirect enzyme immunological measurement method of Comparative Example 1. 30 μl of HRP-surface antigen conjugate solution was added to each well, and 100 μl of malaria positive sample (plasma of malaria patient) and negative sample (plasma of normal person) were mixed well. I was. The wells were washed by treating 300 μl of phosphate buffer solution containing Tween 20 five times, and then 100 μl of substrate solution containing 100 μg / ml of tetramethylbenzidine, 0.006% hydrogen peroxide and citric acid phosphate buffer (pH 4.5) was added. It was. Subsequently, the plate to which the substrate solution was added was developed in the dark for 30 minutes, and then 100 µl of 2 N sulfuric acid solution was added to each well to stop the reaction. The 96-well plate reader (Molecular Devices) used as a reference wavelength of 650 nm. Absorbance was measured at 450 nm. Tetramethylbenzidine, a color developing agent in the substrate solution, is decomposed by HRP of the HRP-surface antigen conjugate to generate a color reaction. The degree of color development was measured by absorbance to detect the presence or absence of the antibody.

In order to compare the difference between the method used in the present invention and the antigenic sandwich enzyme immunological measurement method described above, the plasma of 129 healthy subjects was diagnosed. The 129 normal people consisted of 26 teenagers, 43 in 20s, 33 in 30s, and 27 in 40s. As a result of diagnosis, the IgM capture enzyme immunoassay using the recombinant malaria surface antigen of the present invention is determined by determining the positive cut-off value by adding 0.05 to the negative sample mean. Of the 129 plasma samples, 129 were negative and showed 100% specificity. On the other hand, the antigen sandwich enzyme immunological measurement method described above is based on 127 out of 129 normal plasma samples when judged by determining the positive cut-off for the same plasma by adding 0.05 to the negative sample mean. Was negative and showed a specificity of 98.4%. In particular, 100% specificity was observed in those under 30 years of age born after the malaria epidemic, but 2 out of 27 samples were positive in the 40s born during the malaria epidemic, indicating a specificity of 92.6%. Showed a drop in specificity (see FIG. 3).

According to the immunological measurement method, diagnostic reagent, and diagnostic method for detecting malaria-specific IgM present in the blood using the antigen of the malaria protozoa of the present invention, since only malaria-specific IgM is detected, current malaria patients and past patients Malaria can be diagnosed separately.

Therefore, the immunological measurement method, diagnostic reagent and diagnostic method of malaria of the present invention is difficult to diagnose by antigen test due to the small number of parasites present in the blood, such as Plasmodium vivax , a malaria that occurs in Korea. Unlike the antigen test, it can be diagnosed in the latent state, so it can be very effectively used to diagnose malaria with high probability of recurrence in the latent state. In addition, the immunological measurement method, diagnostic reagent and diagnostic method of malaria of the present invention is useful for early diagnosis of malaria. In addition, malaria can be effectively diagnosed even in older and older malaria epidemics.

In addition, the immunological measurement method and diagnostic reagent of malaria of the present invention have a significantly improved specificity and sensitivity than the indirect enzyme immunological measurement method and diagnostic reagent described in Comparative Example 1, and also in Comparative Example 2 It has improved specificity over the described antigen sandwich enzyme immunological measurement methods and diagnostic reagents.

While the invention has been described in detail above with reference to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope and spirit of the invention, and such modifications and variations fall within the scope of the appended claims. It is also natural.

Claims (30)

In the immunological measurement method of malaria, An immunological measurement method for malaria, characterized by detecting malaria specific IgM present in blood isolated from humans using an antigen of malaria protozoa. The method of claim 1, wherein the immunological measurement method for detecting IgM is an IgM capture immunoassay. The method of claim 2, wherein the immunological measurement method for detecting IgM is an IgM capture enzyme immunoassay. The method of claim 1, wherein the anti-human IgM antibody is used to detect malaria specific IgM present in the blood. The method of claim 1, wherein the antigen of the malaria protozoa is a surface antigen of the malaria protozoa. The method of claim 1 wherein the immunologically measuring method which, in whole or in part characterized by Mero Zh surface protein (Merozoit surface protein) of an antigen of the malaria parasite is SUMO Plastic boxes rhodium ratio (Plasmodium vivax). The method of claim 6, wherein the immunological measuring method, characterized in that the C- terminal region of booties Zh surface protein (Merozoit surface protein) of an antigen of the malaria parasite is SUMO Plastic boxes rhodium ratio (Plasmodium vivax). 8. The method of claim 7, wherein the antigen is all or part of a PV200C polypeptide present at the C-terminal portion of the mesozoite surface protein. 9. The method of claim 8, wherein the amino acid sequence of the PV200C polypeptide is an amino acid sequence set forth in SEQ ID NO: 1. The method of claim 1, wherein the antigen of the malaria protozoa is a recombinant malaria surface antigen purified from transformed yeast or Escherichia coli. (i) attaching the anti-human IgM antibody to the fixture; (ii) binding the IgM by treating serum or plasma samples to the antibody-coated fixture; (iii) adding a labeled antigen conjugate to the above to react with IgM bound to the antibody attached to the fixture; And (iv) analyzing by using a label of a label antigen conjugate conjugated to IgM; IgM capture immunological measurement method comprising a. The antigen of claim 11, wherein the antigen of the labeled antigen conjugate of step (iii) is Plastic Sumo rhodium ratio box booties Zh surface protein immunologically measuring method which, in whole or in part, wherein the C- terminal region of the (Merozoit surface protein) of (Plasmodium vivax). A malaria diagnostic reagent comprising an antigen of malaria protozoa and detecting malaria specific IgM present in blood. 14. The malaria diagnostic reagent according to claim 13, wherein the antigen of malaria protozoa is a surface antigen of malaria protozoa. The method of claim 13 wherein the malaria antigen is a parasite Plastic Sumo rhodium ratio box (Plasmodium vivax) Mero Zh surface protein (Merozoit surface protein) in whole or in part to malaria diagnostic reagent as claimed in. The antimalarial diagnostic reagent according to claim 15, wherein the antigen of the malaria protozoa is the C-terminal portion of the merzoit surface protein of Plasmodium vivax . The diagnostic agent for malaria according to claim 16, wherein the antigen is all or part of the PV200C polypeptide present at the C-terminal portion of the mesozoite surface protein. 18. The diagnostic reagent for malaria according to claim 17, wherein the amino acid sequence of the PV200C polypeptide is an amino acid sequence set forth in SEQ ID NO: 1. Fixtures coated with an anti-human IgM antibody; Marker antigen conjugates; And a malaria diagnostic reagent comprising a substrate solution containing a coloring agent. 20. The diagnostic reagent for malaria according to claim 19, wherein the antigen of the labeled antigen conjugate is all or part of the merozite surface protein of malaria protoplasma plasmid bibox . 21. The method of claim 20, wherein the labeled antigen conjugate of the antigen is Plasmodium Plastic Sumo rhodium ratio box Mero Zh surface protein of the C- terminal region in whole or in part characterized in that, malaria diagnostic reagent according to the. 20. The diagnostic agent for malaria according to claim 19, wherein the label of the label antigen conjugate comprises horseradish peroxidase, alkaline phosphatase, colloidal gold, fluorescent substance, or pigment. 20. The diagnostic reagent for malaria according to claim 19, wherein the substrate solution is composed of a colorant and a buffer solution that react with the label of the label antigen conjugate to cause color development. (delete) (delete) (delete) (delete) (delete) (delete) (delete)