CN117783520A - Dengue virus and plasmodium falciparum plasmodium vivax antibody preparation method and detection kit thereof - Google Patents

Abstract

The invention relates to the field of antibody antigen preparation, in particular to an antigen detection kit for simultaneously detecting dengue virus and metazate and malignant malaria, which simultaneously contains dengue virus antibodies and metazate and malignant malaria antibodies. The performance of the product meets the requirements of quality standards (the detection result of a negative reference product is negative, the detection result of a positive reference product is positive), the product specificity and the interference test meet the requirements of yield and quality (the kit is not affected by serum matrixes such as dry triglyceride, bilirubin and the like due to no cross reaction with respiratory tract related diseases), and the acceleration stability test of the product meets the requirements of 1 month.

Description

Dengue virus and plasmodium falciparum plasmodium vivax antibody preparation method and detection kit thereof

Technical Field

The invention relates to the field of pathogen detection, in particular to a preparation method of dengue virus and plasmodium falciparum plasmodium vivax antibodies and a detection kit thereof.

Background

Dengue is an acute insect-borne infectious disease caused by dengue virus transmitted by mosquito. After dengue virus infection, recessive infection, dengue hemorrhagic fever and dengue hemorrhagic fever can be caused, and the dengue hemorrhagic fever is rare in China. Typical dengue clinical manifestations are onset of symptoms such as flash, high fever, headache, severe soreness of muscles and bone joints, rash, bleeding tendency, lymphadenectasis, decreased white blood cell count, thrombocytopenia, etc. in some patients. The disease is mainly prevalent in tropical and subtropical areas, and Guangdong, hong Kong, australian and the like of China are dengue endemic areas. Because the disease is transmitted by aedes, the epidemic has certain seasonality, generally 5-11 months per year, and the peak is 7-9 months. In the new epidemic area, the population is generally susceptible, but the onset is mainly adult, in the local epidemic area, the onset is mainly children.

Plasmodium falciparum hosts one of four plasmodium species in humans, the causative agent of malaria. Plasmodium falciparum hosts human and female anopheles mosquitoes. The split proliferation and gamete reproduction are started in human body. Gamete reproduction and spore proliferation are completed in the mosquito body. The development sites in human body are liver cells and red blood cells

Disclosure of Invention

Aiming at the actual detection requirements of dengue virus and plasmodium falciparum, the invention provides a preparation method of antibodies of dengue virus and plasmodium falciparum and a detection kit thereof.

The invention is realized by the following technical scheme:

the invention provides an antigen detection kit for simultaneously detecting dengue virus, metazate and malignant malaria, which simultaneously contains dengue virus antibodies and metazate and malignant malaria antibodies.

The preparation method of the dengue virus antibody comprises the following steps:

1 selection of the Gene of interest

2 construction of recombinant plasmid

2.1 acquisition of the Gene of interest

(1) Designing a primer, designing a primer required for amplifying a target gene by taking a DV-NS1 gene fragment synthesized by the gene as a template, and adding enzyme cutting sites BamH I and Xho I at the 5' end of the primer according to the selected insertion site on the vector;

primer 1: NS1-F:5' -CGggatccGACTCTGGTTGCATTGTTT-3’(BamH I)

Primer 2: NS1-R:5' -CCctcgagCGCGGTCACCAGGCTGTTAA-3’(Xho I)

After the PCR amplified product is recovered, the digestion product is recovered through BamH I and Xho I double digestion, and then the digestion product is connected to a vector pET32a after BamH I and Xho I digestion, DH5 alpha is converted, and the T7/T7TER primer is used for PCR identification of the recombinant plasmid, wherein the theoretical value is 1753bp. The obtained positive transformant extracts recombinant plasmid, converts escherichia coli expression strain Rosetta, and obtains DV-NS1 recombinant antigen expression strain: R/pET32a-DV-NS1.

3 recombinant protein expression purification

3.1 Induction of expression

3.2 high pressure crushing

3.3 protein purification

Performing affinity chromatography by using NI ion chromatographic column, and balancing the chromatographic column with 10 times of column volume of ultrapure water; then, the column is equilibrated with 10 times of column volume equilibration buffer (20 mM PBS pH 7.4), and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM PBS pH 7.4); finally, the target protein was eluted with elution buffer (20 mM PBS-150mM IM pH 7.4).

The collected protein samples were placed in dialysis bags, placed in beakers containing dialysate (20 mM PBS, pH 7.4), and dialyzed overnight at 4 ℃. SDS-PAGE detection was performed on the purified proteins. The preparation method of the metaday and malignant malaria antibody comprises the following steps:

preparation of recombinant protein of histidine-rich protein 2 of plasmodium falciparum

1. Selection of the Gene of interest

Referring to Genbank, AAC47453.1 HRP2 amino acid sequence, carrying out codon optimization and gene synthesis on the amino acid sequence, constructing a prokaryotic expression vector pET24a, transforming escherichia coli, carrying out recombinant expression and purification to obtain a large amount of high-purity and strong-antigenicity HRP2;

2. recombinant plasmid construction

3. Recombinant protein expression purification (II) preparation of merozoite surface protein-1 recombinant protein of plasmodium vivax

1. Selection of the Gene of interest

AAN86236.1 sequence, through analysis, select its antigen dominant epitope, and the region with strong conservation, carry on codon optimization, gene synthesis, construct to prokaryotic expression vector pET24a, transform E.coli, carry on recombinant expression, purify and obtain a large amount of high purity, strong antigenic MSP-1 recombinant proteins;

2. recombinant plasmid construction

3. Preparation of recombinant protein expression purification (tri) histidine-rich protein 2 and merozoite surface protein-1 antibodies

1. Immunization of mice

2. Antibody preparation

3. Antibody purification

4. Antibody validation

A double antibody sandwich method is adopted to verify the pairing effect of antibodies; respectively labeling 10 antibodies by HRP, and carrying out double-antibody sandwich detection by adopting a chessboard method; coating corresponding antibody, adding pfHRP2 (or pvMSP-1) protein, adding HRP marked antibody to form antibody-antigen-enzyme mark antibody complex, and detecting OD450 by enzyme mark instrument under the action of substrate color developing liquid.

The beneficial effects are that:

the performance of the product meets the requirements of quality standards (the detection result of a negative reference product is negative, the detection result of a positive reference product is positive), the product specificity and the interference test meet the requirements of yield and quality (the kit is not affected by serum matrixes such as dry triglyceride, bilirubin and the like due to no cross reaction with respiratory tract related diseases), and the acceleration stability test of the product meets the requirements of 1 month.

Drawings

Fig. 1: and (5) PCR identification results.

Fig. 2: SDS-PAGE induced expression identification.

Fig. 3: SDS-PAGE purification results.

Fig. 4: and (5) PCR identification results.

Fig. 5: SDS-PAGE induced expression identification.

Fig. 6: SDS-PAGE purification results.

Fig. 7: and (5) PCR identification results.

Fig. 8: SDS-PAGE induced expression identification.

Fig. 9: SDS-PAGE purification results.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

The antigen detection kit for simultaneously detecting dengue virus, metaday and malignant malaria provided by the embodiment simultaneously contains dengue virus antibodies, metaday and malignant malaria antibodies.

The embodiment provides dengue virus antigen preparation and colloidal gold detection kit

Preparation of dengue recombinant protein

1 selection of the Gene of interest

The non-structural protein NS1 of the dengue virus plays an important role in the virus infection, replication and transmission process, has higher stability in 4 dengue virus typing, and is an important marker for early diagnosis of dengue virus infection. Referring to an NS1 amino acid sequence in Genbank ADI80655.1, codon optimization and gene synthesis are carried out on the sequence, a prokaryotic expression vector pET32a is constructed, escherichia coli is transformed, recombinant expression and purification are carried out, and a large amount of NS1 with high purity and strong antigenicity is obtained, so that the development requirement of an antibody detection kit is met.

2 construction of recombinant plasmid

2.1 acquisition of the Gene of interest

(2) Designing a primer, designing a primer required for amplifying a target gene by taking a DV-NS1 gene fragment synthesized by the gene as a template, and adding enzyme cutting sites BamH I and Xho I at the 5' end of the primer according to the selected insertion site on the vector;

primer 1: NS1-F:5' -CGggatccGACTCTGGTTGCATTGTTT-3’(BamH I)

Primer 2: NS1-R:5' -CCctcgagCGCGGTCACCAGGCTGTTAA-3’(Xho I)

The reaction system was added as in Table 1 and amplification was performed by the amplification procedure of Table 2

TABLE 1 PCR reaction System

TABLE 2 amplification procedure

After the PCR amplified product is recovered, the BamH I and Xho I double digestion is carried out, then the digestion product is recovered, the digestion product is connected to a vector pET32a after the BamH I and Xho I digestion, DH5 alpha is converted, the T7/T7TER primer is used for PCR identification of the recombinant plasmid, the theoretical value is 1753bp, and the identification result is as shown in figure 1 and is consistent with the theoretical value. The obtained positive transformant extracts recombinant plasmid, converts escherichia coli expression strain Rosetta, and obtains DV-NS1 recombinant antigen expression strain: R/pET32a-DV-NS1.

3 recombinant protein expression purification

3.1 Induction of expression

10. Mu.l of recombinant plasmid expressing strain was inoculated into 5ml containing 200ug/ml ampicillin sodium (AMP) (from Soy pal)In LB medium, shaking culture is carried out at 37 ℃ at 200rpm for overnight (about 16 hours), 200 mu L of bacterial liquid is added into 60ml of LB medium containing AMP with the same concentration, shaking culture is carried out at 37 ℃ at 200rpm for overnight, all bacterial liquid is inoculated into 1L of LB medium containing AMP with the same concentration, shaking culture is carried out at 28 ℃ at 150rpm for 2-2.5 hours, and OD is measured ₆₀₀ Between 0.5 and 0.8, inducer IPTG was added at a final concentration of 0.5mM, and the shaking culture was continued at 28℃and 150rpm for 4.5 hours. After fermentation is completed, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected. The expression and identification results are shown in figure 2, and the theoretical size of the target protein is 58.7kD, and the results show that the target protein accords with the theory.

3.2 high pressure crushing

Bacterial pellet was resuspended in 30ml 20mM PBS (pH 7.4) and broken at low temperature and high pressure, parameters set: the pressure value is 910-980bar, the cycle is 3 times, the temperature is set to 4 ℃, and the actual operation temperature is not higher than 13 ℃. After the completion of the crushing, the crushed product was centrifuged at 9000rpm at low temperature for 30min, and the supernatant was collected.

3.3 protein purification

Performing affinity chromatography by using NI ion chromatographic column, and balancing the chromatographic column with 10 times of column volume of ultrapure water; then, the column is equilibrated with 10 times of column volume equilibration buffer (20 mM PBS pH 7.4), and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM PBS pH 7.4); finally, the target protein was eluted with elution buffer (20 mM PBS-150mM IM pH 7.4).

The collected protein samples were placed in dialysis bags, placed in beakers containing dialysate (20 mM PBS, pH 7.4), and dialyzed overnight at 4 ℃. SDS-PAGE detection is carried out on the purified protein, and the result is shown in figure 3, and the DV-NS1 purity is more than 95%.

Preparation of colloidal gold detection kit

According to the principle of gold-labeled immunochromatography test, an anti-human IgG antibody and an anti-human IgM antibody are respectively coated on a nitrocellulose membrane, and dengue antigens are fixed on a gold-labeled pad. When dengue specific IgG and IgM antibodies are contained in the sample, a complex is formed with the gold-labeled antigen, the complex moves forwards under the action of chromatography and is combined with the antibodies coated on the detection line, and the complex is coagulated and developed to be a positive result; when the sample does not contain dengue specific IgG and IgM antibodies, a complex cannot be formed at the detection line, no red band appears, and the result is a negative result.

Regardless of whether the sample contains the dengue IgM/IgG antibody to be detected, the gold-labeled quality control protein (biotinylated BSA) is combined with the antibody (streptavidin) coated at the quality control line to form a complex for coacervation and color development (quality control line, C).

1. Colloidal gold preparation and protein labelling

And (3) preparing colloidal gold by adopting a sodium citrate reduction method, respectively labeling the colloidal gold by using dengue recombinant antigen and biotinylated BSA, and purifying the protein labeled by the colloidal gold by adopting a speed centrifugation method. Concentrating according to daily experience value of 10:1, and fixing 10% of solid phase on glass fiber.

2. Preparation of nitrocellulose membrane and assembly of test strip

And respectively selecting mouse anti-human IgM monoclonal antibody, mouse anti-human IgG monoclonal antibody and streptavidin to respectively score as detection lines (T2 and T1) and a quality control line (C) of the test paper, finishing coating of the nitrocellulose membrane, finishing gradient coating according to coating concentration of 0.2mg/mL, 0.4mg/mL, 0.6mg/mL and 1.0mg/mL, and assembling a colloidal gold test strip for testing. The result shows that when the coating concentration of the protein is 0.4mg/mL, the detection results of the positive sample and the negative sample meet the requirements, the coating concentration is further improved, the detection result of the negative sample shows false positive, and the reagent with the coating concentration of 0.4mg/mL has good sensitivity and specificity and is suitable coating concentration.

3. Preparation of sample dilutions

According to the experimental requirements, three sample dilutions were prepared, physiological saline, 15mM PBS buffer, and 20mM PBS buffer, respectively, were added dropwise to the test strips, and were tested 10 times in parallel, and the dilutions were investigated in terms of membrane surface analysis speed, band color development, background color, presence or absence of false positives, and the like. The results showed that the 20mM PBS buffer results were satisfactory and optimal and could be used as a sample diluent.

4. Analysis of product Performance

Enterprise reference preparation, positive reference: 13 samples determined by the dengue IgM/IgG antibody detection kit are selected, less samples or samples with abnormal appearance are removed, 10 samples are selected to be made into positive reference products, and the positive reference products are respectively numbered as P1-P10. Negative reference: the samples with fewer numbers or abnormal appearance are removed from 20 dengue virus IgM/IgG antibody negative samples (which do not contain St.Louis encephalitis virus positive antibody positive samples, west Nile virus positive antibody positive samples, japanese encephalitis virus antibody positive samples and yellow fever virus antibody positive samples), 15 samples are selected to be made into negative reference products, and the numbers are respectively N1-N15; the test paper strip is used for detecting, positive reference products can be detected completely, and the detection results of negative reference products are all negative.

5. Specificity verification

The cross-reactive virus (St.Louis encephalitis virus, west Nile virus, japanese encephalitis virus, yellow fever virus) -IgM/IgG antibody specific samples were 5 samples each positive confirmed with four IgM/IgG antibody detection kits. Another 20 samples of negative blood from common people were collected and analyzed for specificity verification. The results show that: the reagent has cross reaction with antibody positive samples of the St.Louis encephalitis virus, the West Nile virus, the Japanese encephalitis virus and the yellow fever virus, and does not have cross reaction with normal human serum.

6. Serum matrix interference validation

In clinical examination, common interfering substances in a sample, such as triglyceride and bilirubin, are easy to interfere with detection results. The potential maximum concentration of each substance in human body is obtained by reference to the standard WS/T416-2013 interference experimental guideline of the sanitation industry through document retrieval and clinical institution collection, and the concentration is used as a reference for research. The maximum concentration of triglyceride in human body is about 63.10mmol/L, 64.0mmol/L,36.0mmol/L, 24.0mmol/L, 12.0mmol/L, 6.0mmol/L, 3.0mmol/L, 1.0mmol/L and 1000 mu mol/L of bilirubin are added into collected IgM/IgG antibody negative and weak positive samples, and whether the detection result is interfered is verified. The results show that: when the concentration of triglyceride is 6.0mmol/L or lower than the concentration, bilirubin is 1000 mu mol/L or lower than the concentration, the method has no interference to the test result and does not influence the interpretation of the final result, and can be used for sample detection.

7. Mercaptoethanol destruction assay

2-mercaptoethanol reduces the-S-S-bond to-SH, destroying the IgM/IgG structure, and rendering it inactive. Dengue virus IgM/IgG antibody positive serum samples were selected for 5 cases. The blood positive samples are divided into a test group and a control group, wherein the test group is added with equivalent 0.2 mol/L2-mercaptoethanol, the control group is added with equivalent PBS, and the mixture is simultaneously placed at 37 ℃ for 1h. The results show that after the sample containing IgM/IgG antibodies is treated by 2-mercaptoethanol, the detection result of the antibodies is changed from positive to negative. Thus affecting the interpretation of the results and thus not allowing the presence of mercaptoethanol throughout the strip preparation and detection process.

8. Accelerated stability test

Sealing the test strip with plastic bag and aluminum foil, adding desiccant, storing at 37deg.C, detecting 1 time every 1 week, detecting stability for 6 times, and setting up normal temperature control group. The result shows that the colloidal gold finished test strip sealed by the aluminum foil bag can still reach the above performance indexes of the product after being stored for 6 weeks at 37 ℃, and the result has no obvious difference from the control group, thus indicating that the test strip has better accelerated stability experiment.

The dengue IgM and IgG antibody detection kit prepared from the recombinant protein meets the requirements of quality standards on product performance (the detection result of a negative reference is negative, the detection result of a positive reference is positive), the product specificity and the interference test meet the requirements of yield and quality (cross reaction with St.Louis encephalitis virus, west Nile virus, japanese encephalitis virus and yellow fever virus exists, the kit is not influenced by serum matrixes such as dry triglyceride and bilirubin), and the acceleration stability test of the product meets the requirements of 1 month.

Example 2

The method for preparing the metaday and malignant malaria antibody in the embodiment comprises the following steps:

1. preparation of histidine-rich protein 2 recombinant protein of plasmodium falciparum

1. Selection of the Gene of interest

Referring to Genbank, AAC47453.1 HRP2 amino acid sequence, carrying out codon optimization and gene synthesis on the amino acid sequence, constructing a prokaryotic expression vector pET24a, transforming escherichia coli, carrying out recombinant expression and purification to obtain a large amount of high-purity and strong-antigenicity HRP2;

2. recombinant plasmid construction

2.1 acquisition of the Gene of interest

(3) Designing a primer, designing a primer required for amplifying a target gene by taking a gene synthesis pfHRP2 gene fragment as a template, and adding enzyme cutting sites NdeI and Xho I at the 5' end of the primer according to a selected insertion site on a vector;

primer 1: pf-F:5' -GGAATTCCATATGGTTTCTTTCTCTAAAAACAA-3’(NdeI)

Primer 2: pf-R:5' -CCCTCGAGGTGACGCAGGCAGTGGGTAGCA-3’(Xho I)

(4) PCR reaction

Adding a reaction system according to Table 1, and performing amplification by the amplification procedure of Table 2;

TABLE 1 PCR reaction System

PCR reaction component	50 μl system
		2*Taq mix	25
Upstream primer	1
		Downstream primer	1
DNA template	1
		Adding ddH 2 O to	Up to50μl

TABLE 2 amplification procedure

(5) Recombinant vector construction

Recovering PCR amplified products, performing double digestion by Nde I and XhoI, recovering digested products, connecting the digested products to a vector pET24a after digestion by Nde I and XhoI, transforming DH5 alpha, and identifying recombinant plasmids by using a T7/T7TER universal primer PCR, wherein the theoretical value is 1138bp, and the identification result is as shown in figure 4 and is consistent with the theoretical value; the obtained positive transformant extracts recombinant plasmid, converts escherichia coli expression strain Rosetta, and obtains pfHRP2 recombinant antigen expression strain: R/pET24a-pfHRP2;

3. recombinant protein expression purification

3.1 Induction of expression

10. Mu.l of recombinant plasmid-expressing strain was inoculated into 5ml of LB medium containing 100ug/ml kanamycin (Kan) (purchased from Soy pal), shaking culture was carried out at 37℃and 200rpm for overnight (about 16 hours), 200. Mu.l of the strain was added to 60ml of LB medium containing the same concentration of Kan, shaking culture was carried out at 37℃and 200rpm for overnight, the whole strain was inoculated into 1L of LB medium containing the same concentration of Kan, shaking culture was carried out at 28℃and 150rpm for 2 to 2.5 hours, and OD was measured ₆₀₀ Between 0.5 and 0.8, inducer IPTG was added at a final concentration of 0.5mM, and the shaking culture was continued at 28℃and 150rpm for 4.5 hours. After fermentation is completed, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected. The expression and identification results are shown in figure 5, the theoretical size of the target protein is 33.8kD, and the results show that the target protein accords with the theory.

3.2 high pressure crushing

Bacterial pellet was resuspended in 30ml 20mM PBS (pH 7.4) and broken at low temperature and high pressure, parameters set: the pressure value is 910-980bar, the cycle is carried out for 3 times, the temperature is set to be 4 ℃, and the actual running temperature is not higher than 13 ℃; after crushing, centrifuging the crushed product at 9000rpm for 30min at low temperature, and collecting a supernatant;

3.3 protein purification

Performing affinity chromatography by using NI ion chromatographic column, and balancing the chromatographic column with 10 times of column volume of ultrapure water; then, the column is equilibrated with 10 times of column volume equilibration buffer (20 mM PBS pH 7.4), and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM PBS pH 7.4); finally eluting the target protein by using an elution buffer (20 mM PBS-250mM IM pH 7.4);

the collected protein samples were placed in dialysis bags, placed in beakers containing dialysate (20 mM PBS, pH 7.4), and dialyzed overnight at 4 ℃; SDS-PAGE detection is carried out on the purified protein, and the result is shown in FIG. 6, and the purity of pfHRP2 is more than 95%.

2. Preparation of merozoite surface-1 recombinant protein of plasmodium vivax

1. Selection of the Gene of interest

AAN86236.1 sequence, through analysis, select its antigen dominant epitope, and the region with strong conservation, carry on codon optimization, gene synthesis, construct to prokaryotic expression vector pET24a, transform E.coli, carry on recombinant expression, purify and obtain a large amount of high purity, strong antigenic MSP-1 recombinant proteins;

2. recombinant plasmid construction

1.1 acquisition of the Gene of interest

(1) Designing a primer, designing a primer required for amplifying a target gene by taking a gene synthesis pvMSP-1 gene fragment as a template, and adding enzyme cutting sites NdeI and Xho I at the 5' end of the primer according to a selected insertion site on a vector;

primer 1: pv-F:5' -GGAATTCcatatgAAACTGGAAGAATACAAAAA-3’(NdeI)

Primer 2: pv-R:5' -CCctcgagAGAGCAGAAAACACCTTCGAA-3’(Xho I)

(2) PCR reaction

The reaction system was added as in Table 1 and amplification was performed by the amplification procedure of Table 2

TABLE 1 PCR reaction System

PCR reaction component	50 μl system
		2*Taq mix	25
Upstream primer	1
		Downstream primer	1
DNA template	1
		Adding ddH 2 O to	Up to50μl

TABLE 2 amplification procedure

(3) Recombinant vector construction

Recovering PCR amplified products, performing double digestion by Nde I and XhoI, recovering digested products, connecting the digested products to a vector pET24a after digestion by Nde I and XhoI, transforming DH5 alpha, and identifying recombinant plasmids by using a T7/T7TER universal primer PCR, wherein the theoretical value is 650bp, and the identification result is as shown in figure 7 and is consistent with the theoretical value; the obtained positive transformant extracts recombinant plasmid, converts escherichia coli expression strain Rosetta, and obtains pfHRP2 recombinant antigen expression strain: R/pET24a-pvMSP-1;

3. recombinant protein expression purification

3.1 Induction of expression

10. Mu.l of recombinant plasmid-expressing strain was inoculated into 5ml of LB medium containing 100ug/ml kanamycin (Kan) (purchased from Soy pal), shaking culture was carried out at 37℃and 200rpm for overnight (about 16 hours), 200. Mu.l of the strain was added to 60ml of LB medium containing the same concentration of Kan, shaking culture was carried out at 37℃and 200rpm for overnight, the whole strain was inoculated into 1L of LB medium containing the same concentration of Kan, shaking culture was carried out at 28℃and 150rpm for 2 to 2.5 hours, and OD was measured ₆₀₀ Between 0.5 and 0.8, inducer IPTG was added at a final concentration of 0.5mM, and the shaking culture was continued at 28℃and 150rpm for 4.5 hours. After fermentation is completed, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected. The expression and identification results are shown in figure 8, and the theoretical size of the target protein is 16.7kD, and the results show that the target protein accords with the theory.

3.2 high pressure crushing

Bacterial pellet was resuspended in 30ml 20mM PBS (pH 7.4) and broken at low temperature and high pressure, parameters set: the pressure value is 910-980bar, the cycle is carried out for 3 times, the temperature is set to be 4 ℃, and the actual running temperature is not higher than 13 ℃; after crushing, centrifuging the crushed product at 9000rpm for 30min at low temperature, and collecting a supernatant;

3.3 protein purification

Performing affinity chromatography by using NI ion chromatographic column, and balancing the chromatographic column with 10 times of column volume of ultrapure water; then, the column is equilibrated with 10 times of column volume equilibration buffer (20 mM PBS pH 7.4), and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM PBS pH 7.4); finally, the target protein was eluted with elution buffer (20 mM PBS-250mM IM pH 7.4).

The collected protein samples were placed in dialysis bags, placed in beakers containing dialysate (20 mM PBS, pH 7.4), and dialyzed overnight at 4 ℃.

SDS-PAGE detection is carried out on the purified protein, and the result is shown in figure 9, and the purity of the pvMSP-1 is more than 95%.

3. Preparation of histidine-rich protein 2 and merozoite surface protein-1 antibodies

1. Immunization of mice

Subcutaneous multipoint injection immunization was performed with histidine-rich protein 2 (HRP-2) and merozoite surface protein-1 (MSP-1) as immunogens, and 6-8 week old female balb/c mice were selected, respectively. After 3 times of immunization, tail cutting and blood sampling are carried out, serum is collected, an indirect ELISA method is used for detecting the titer of immune serum, and after the titer is qualified, the spleen of the mouse is taken for experiment.

2. Antibody preparation

Spleen cells of immunized mice and mouse myeloma cells (SP 2/0) were mixed in a ratio of 10:1 using a hybridoma preparation platform and fused with PEG. After the fusion cells are screened by HAT culture medium, positive screening is carried out by ELISA method, and positive cells are subcloned by limiting dilution method, so that the singleness of each cell line is ensured. Two proteins were screened to obtain 5 cell lines each. The 10 antibodies were subjected to amplification culture according to 5X 10 ⁵ -10 ⁶ Injecting the obtained product into abdominal cavity of mice, preparing ascites, collecting ascites for 7d, centrifuging, and freezing at-20deg.C.

3. Antibody purification

The purification method of Protein-A column is adopted to purify the antibody according to the processes of crude treatment, balancing, loading, washing impurities, eluting, neutralizing and dialyzing. Collecting eluent, dialyzing, replacing buffer solution, centrifuging, collecting supernatant, and storing at-20deg.C.

4. Antibody validation

The paired effect of the antibody is verified by adopting a double antibody sandwich method. And respectively labeling 10 antibodies by HRP, and performing double-antibody sandwich detection by adopting a chessboard method. Coating corresponding antibody, adding pfHRP2 (or pvMSP-1) protein, adding HRP marked antibody to form antibody-antigen-enzyme mark antibody complex, and detecting OD450 by enzyme mark instrument under the action of substrate color developing liquid.

HRP-2 antibody pairing results

MSP-1 pairing results

Analysis of results:

the enzyme-immune result shows that the antibody 1 is used as a coating antibody, the antibody 2 is used as a labeling antibody, the linearity and the sensitivity for detecting the plasmodium falciparum HRP2 protein are best, and the detection background value is low.

The antibody 10 is used as a coating antibody, the antibody 6 is used as a labeling antibody, the linearity and the sensitivity for detecting the plasmodium vivax MSP-1 protein are best, and the detection background value is low.

1. Development of colloidal gold detection kit

The reagent utilizes the principle of colloidal gold immunochromatography, and a nitrocellulose membrane is coated with an anti-plasmodium vivax monoclonal antibody (merozoite surface protein-1 (MSP-1)), a plasmodium falciparum monoclonal antibody (anti-histidine-rich protein II) and a goat anti-mouse IgG antibody, and an anti-plasmodium vivax pairing antibody and an anti-plasmodium falciparum pairing monoclonal antibody are fixed on a gold-labeled pad. If the P.v. merozoite surface protein antigen exists in the sample, the p.v. merozoite surface protein antigen reacts with the pre-marked anti-plasmodium vivax antibody to form a compound, the compound is captured by the anti-plasmodium vivax antibody pre-fixed on a membrane under the action of chromatography, and a mauve P.v. (T1) band is formed in a detection area, and is a positive result of plasmodium vivax; if p.f. plasmodium falciparum histidine-rich protein (HRP-2) antigen exists in the sample, the p.f. plasmodium falciparum histidine-rich protein reacts with the pre-labeled anti-plasmodium falciparum antibody to form a complex, the complex is captured by the anti-plasmodium falciparum antibody pre-immobilized on a membrane under the action of chromatography, and a mauve p.f. (T2) band is formed in a detection zone, so that the p.f. plasmodium falciparum positive result is obtained; if p.f. plasmodium falciparum antigen and p.v. plasmodium vivax antigen are present in the blood sample, the p.f. plasmodium falciparum antigen and the p.v. plasmodium vivax antigen react with the pre-labeled anti-plasmodium falciparum antibody and plasmodium vivax antibody respectively to form a complex, and the complex is captured with the anti-plasmodium falciparum antibody and the plasmodium vivax antibody which are pre-immobilized on the membrane respectively under the action of chromatography to form a mauve p.f. (T2) band and a mauve p.v. (T1) band in a detection zone, which is a mixed positive result of plasmodium falciparum and plasmodium vivax;

if the p.v. plasmodium vivax antigen or the p.f. plasmodium falciparum antigen is not present in the sample, the quality control region (C) forms a mauve C band, and no p.v. (T1) or p.f. (T2) mauve band is formed in the detection region, which is a negative result; 2. colloidal gold preparation and protein labelling

Colloidal gold is prepared by adopting a sodium citrate reduction method, plasmodium vivax and plasmodium falciparum marked antibodies are respectively used for marking the colloidal gold, and the protein marked by the colloidal gold is purified by adopting a speed centrifugation method. Concentrating according to daily experience value of 10:1, and fixing 10% of solid phase on glass fiber.

2. Preparation of nitrocellulose membrane and assembly of test strip

And respectively selecting plasmodium vivax and plasmodium falciparum coated antibodies and goat anti-mouse IgG antibody streaks as detection lines (T1 and T2) and a quality control line (C) of the test paper to finish coating of the nitrocellulose membrane, respectively finishing gradient coating according to coating concentration of 0.2mg/mL, 0.4mg/mL, 0.6mg/mL and 1.0mg/mL, and assembling a colloidal gold test strip for testing. The result shows that when the coating concentration of the protein is 0.6mg/mL, the detection results of the positive sample and the negative sample meet the requirements, the coating concentration is further improved, the detection result of the negative sample shows false positive, and the reagent with the coating concentration of 0.6mg/mL has good sensitivity and specificity and is suitable coating concentration.

3. Preparation of sample dilutions

According to the experimental requirements, three sample dilutions were prepared, physiological saline, 15mM PBS buffer, and 20mM PBS buffer, respectively, were added dropwise to the test strips, and were tested 10 times in parallel, and the dilutions were investigated in terms of membrane surface analysis speed, band color development, background color, presence or absence of false positives, and the like. The results showed that the 20mM PBS buffer results were satisfactory and optimal and could be used as a sample diluent.

4. Analysis of product Performance

Enterprise reference preparation, positive reference: 13 positive samples determined by plasmodium vivax and plasmodium falciparum detection kits are selected, less samples or samples with abnormal appearance are removed, 10 positive reference samples are selected and manufactured, and the positive reference samples are respectively numbered as P1-P10. Negative reference: removing less samples or samples with abnormal appearance from 20 negative samples (including positive samples of hepatitis B virus, treponema pallidum and human immunodeficiency virus), and selecting 15 samples as negative reference products with the numbers of N1-N15; the test paper strip is used for detecting, positive reference products can be detected completely, and the detection results of negative reference products are all negative.

5. Specificity verification

The cross-reactive virus (hepatitis b virus, treponema pallidum and human immunodeficiency virus positive samples) specific samples are positive samples confirmed by four detection kits. Another 20 samples of negative blood from common people were collected and analyzed for specificity verification. The results show that: the reagent has no cross reaction with positive samples of hepatitis B virus, treponema pallidum and human immunodeficiency virus, and has no cross reaction with normal human serum.

6. Serum matrix interference validation

In clinical examination, common interfering substances in a sample, such as triglyceride and bilirubin, are easy to interfere with detection results. The potential maximum concentration of each substance in human body is obtained by reference to the standard WS/T416-2013 interference experimental guideline of the sanitation industry through document retrieval and clinical institution collection, and the concentration is used as a reference for research. The maximum concentration of triglyceride in human body is about 63.10mmol/L, 64.0mmol/L,36.0mmol/L, 24.0mmol/L, 12.0mmol/L, 6.0mmol/L, 3.0mmol/L, 1.0mmol/L and 1000. Mu. Mol/L of bilirubin are added into the collected negative and weak positive samples respectively, and whether the detection result is interfered is verified. The results show that: when the concentration of triglyceride is 6.0mmol/L or lower than the concentration, bilirubin is 1000 mu mol/L or lower than the concentration, the method has no interference to the test result and does not influence the interpretation of the final result, and can be used for sample detection.

7. Accelerated stability test

Sealing the test strip with plastic bag and aluminum foil, adding desiccant, storing at 37deg.C, detecting 1 time every 1 week, detecting stability for 6 times, and setting up normal temperature control group. The result shows that the colloidal gold finished test strip sealed by the aluminum foil bag can still reach the above performance indexes of the product after being stored for 6 weeks at 37 ℃, and the result has no obvious difference from the control group, thus indicating that the test strip has better accelerated stability experiment.

In conclusion, the plasmodium vivax and plasmodium falciparum test kit prepared by preparing recombinant protein histidine-rich protein 2 and merozoite surface protein-1 antibody has the product performance meeting the requirements of quality standard (the negative reference detection result is all negative, the positive reference detection result is all positive), the product specificity and the interference test meet the yield and quality requirements (the positive samples of hepatitis B virus, treponema pallidum and human immunodeficiency virus are not in cross reaction, the kit is not influenced by serum matrixes such as dry triglyceride, bilirubin and the like), and the accelerated stability test of the product meets the requirements of 1 month. The product has detailed research and development parameters, clear preparation process and clear preparation key points, and achieves the aim of project research and development.

It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that various changes, modifications, additions and substitutions can be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims (3)

1. An antigen detection kit for simultaneously detecting dengue virus and metazate and malignant malaria is characterized by simultaneously containing dengue virus antibodies and metazate and malignant malaria antibodies.

2. The antigen detection kit as claimed in claim 1, wherein the dengue virus antibody preparation method comprises the steps of:

1. selection of the Gene of interest

Referring to Genbank, carrying out codon optimization and gene synthesis on an NS1 amino acid sequence in ADI80655.1, constructing a prokaryotic expression vector pET32a, and transforming escherichia coli for recombinant expression;

2. recombinant plasmid construction

2.1 acquisition of the Gene of interest

(1) Designing a primer, designing a primer required for amplifying a target gene by taking a DV-NS1 gene fragment synthesized by the gene as a template, and adding enzyme cutting sites BamH I and Xho I at the 5' end of the primer according to the selected insertion site on the vector;

primer 1: NS1-F:5' -CGggatccGACTCTGGTTGCATTGTTT-3’(BamH I)

Primer 2: NS1-R:5' -CCctcgagCGCGGTCACCAGGCTGTTAA-3’(Xho I)

The reaction system was added as in Table 1 and amplification was performed by the amplification procedure of Table 2

TABLE 1 PCR reaction System

TABLE 2 amplification procedure

After the PCR amplified product is recovered, the digestion product is recovered through BamH I and Xho I double digestion, and then the digestion product is connected to a vector pET32a after BamH I and Xho I digestion, DH5 alpha is converted, and the T7/T7TER primer is used for PCR identification of the recombinant plasmid, wherein the theoretical value is 1753bp. The obtained positive transformant extracts recombinant plasmid, converts escherichia coli expression strain Rosetta, and obtains DV-NS1 recombinant antigen expression strain: R/pET32a-DV-NS1.

3. Recombinant protein expression purification

3.1 Induction of expression

10 mu l recombinant plasmid expression strain is inoculated into 200ug/ml ampicillin sodium(AMP) (from Soy pal) in 5ml LB medium, shaking culture at 37 ℃ C., 200rpm overnight (about 16 hours), adding 200. Mu.l of the bacterial liquid to 60ml LB medium containing the same concentration of AMP, shaking culture at 37 ℃ C., 200rpm overnight, inoculating the whole bacterial liquid to 1LLB medium containing the same concentration of AMP, shaking culture at 28 ℃ C., 150rpm for 2-2.5 hours, and measuring OD ₆₀₀ Between 0.5 and 0.8, inducer IPTG was added at a final concentration of 0.5mM, and the shaking culture was continued at 28℃and 150rpm for 4.5 hours. After fermentation is completed, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected.

3.2 high pressure crushing

Bacterial pellet was resuspended in 30ml 20mM PBS (pH 7.4) and broken at low temperature and high pressure, parameters set: the pressure value is 910-980bar, the cycle is 3 times, the temperature is set to 4 ℃, and the actual operation temperature is not higher than 13 ℃. After the completion of the crushing, the crushed product was centrifuged at 9000rpm at low temperature for 30min, and the supernatant was collected.

3.3 protein purification

Performing affinity chromatography by using NI ion chromatographic column, and balancing the chromatographic column with 10 times of column volume of ultrapure water; then, the column is equilibrated with 10 times of column volume equilibration buffer (20 mM PBS pH 7.4), and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM PBS pH 7.4); finally, the target protein was eluted with elution buffer (20 mM PBS-150mM IM pH 7.4).

The collected protein samples were placed in dialysis bags, placed in beakers containing dialysate (20 mM PBS, pH 7.4), and dialyzed overnight at 4 ℃. SDS-PAGE detection was performed on the purified proteins.

3. The antigen detection kit as claimed in claim 1, wherein the method for preparing the metazate, malignant malaria antibody comprises the following steps:

1. preparation of histidine-rich protein 2 recombinant protein of plasmodium falciparum

4. Selection of the Gene of interest

Referring to Genbank, AAC47453.1 HRP2 amino acid sequence, carrying out codon optimization and gene synthesis on the amino acid sequence, constructing a prokaryotic expression vector pET24a, transforming escherichia coli, carrying out recombinant expression and purification to obtain a large amount of high-purity and strong-antigenicity HRP2;

5. recombinant plasmid construction

2.1 acquisition of the Gene of interest

(2) Designing a primer, designing a primer required for amplifying a target gene by taking a gene synthesis pfHRP2 gene fragment as a template, and adding enzyme cutting sites NdeI and Xho I at the 5' end of the primer according to a selected insertion site on a vector;

primer 1: pf-F:5'-GGAATTCCATATGGTTTCTTTCTCTAAAAACAA-3' (NdeI)

Primer 2: pf-R:5'-CCCTCGAGGTGACGCAGGCAGTGGGTAGCA-3' (Xho I)

(3) PCR reaction

Adding a reaction system according to Table 1, and performing amplification by the amplification procedure of Table 2;

TABLE 1 PCR reaction System

PCR reaction component 50 μl system 2*Taq mix 25 Upstream primer 1 Downstream primer 1 DNA template 1 Adding ddH ₂ O to Up to50μl

TABLE 2 amplification procedure

(4) Recombinant vector construction

Recovering PCR amplified products, performing double digestion by Nde I and XhoI, recovering digested products, connecting the digested products to a vector pET24a after digestion by Nde I and XhoI, transforming DH5 alpha, and identifying recombinant plasmids by using a T7/T7TER universal primer PCR, wherein the theoretical value is 1138bp, and the identification result is as shown in figure 1 and is consistent with the theoretical value; the obtained positive transformant extracts recombinant plasmid, converts escherichia coli expression strain Rosetta, and obtains pfHRP2 recombinant antigen expression strain: R/pET24a-pfHRP2;

6. recombinant protein expression purification

3.1 Induction of expression

10. Mu.l of recombinant plasmid expression strain is inoculated into 5ml of LB culture medium containing 100ug/ml kanamycin (Kan), shaking culture is carried out at 37 ℃ and 200rpm for overnight, 200. Mu.l of bacterial liquid is added into 60ml of LB culture medium containing the same concentration Kan, shaking culture is carried out at 37 ℃ and 200rpm for overnight, all bacterial liquid is inoculated into 1L of LB culture medium containing the same concentration Kan, shaking culture is carried out at 28 ℃ and 150rpm for 2-2.5 hours, and OD is measured ₆₀₀ Adding inducer IPTG with final concentration of 0.5mM between 0.5 and 0.8, and continuously culturing at 28deg.C and 150rpm for 4.5 hr; after fermentation is completed, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected;

3.2 high pressure crushing

Bacterial pellet was resuspended in 30ml 20mM PBS (pH 7.4) and broken at low temperature and high pressure, parameters set: the pressure value is 910-980bar, the cycle is carried out for 3 times, the temperature is set to be 4 ℃, and the actual running temperature is not higher than 13 ℃; after crushing, centrifuging the crushed product at 9000rpm for 30min at low temperature, and collecting a supernatant;

3.3 protein purification

Performing affinity chromatography by using NI ion chromatographic column, and balancing the chromatographic column with 10 times of column volume of ultrapure water; then, the column is equilibrated with 10 times of column volume equilibration buffer (20 mM PBS pH 7.4), and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM PBS pH 7.4); finally eluting the target protein by using an elution buffer (20 mM PBS-250mM IM pH 7.4);

the collected protein samples were placed in dialysis bags, placed in beakers containing dialysate (20 mM PBS, pH 7.4), and dialyzed overnight at 4 ℃; SDS-PAGE detection is carried out on the purified protein, and the result is shown in figure 3, wherein the purity of pfHRP2 is more than 95%;

2. preparation of merozoite surface-1 recombinant protein of plasmodium vivax

1. Selection of the Gene of interest

AAN86236.1 sequence, through analysis, select its antigen dominant epitope, and the region with strong conservation, carry on codon optimization, gene synthesis, construct to prokaryotic expression vector pET24a, transform E.coli, carry on recombinant expression, purify and obtain a large amount of high purity, strong antigenic MSP-1 recombinant proteins;

2. recombinant plasmid construction

1.1 acquisition of the Gene of interest

(1) Designing a primer, designing a primer required for amplifying a target gene by taking a gene synthesis pvMSP-1 gene fragment as a template, and adding enzyme cutting sites NdeI and Xho I at the 5' end of the primer according to a selected insertion site on a vector;

primer 1: pv-F:5' -GGAATTCcatatgAAACTGGAAGAATACAAAAA-3’(NdeI)

Primer 2: pv-R:5' -CCctcgagAGAGCAGAAAACACCTTCGAA-3’(Xho I)

(2) PCR reaction

The reaction system was added as in Table 1 and amplification was performed by the amplification procedure of Table 2

TABLE 1 PCR reaction System

TABLE 2 amplification procedure

(3) Recombinant vector construction

Recovering PCR amplified products, carrying out double digestion by Nde I and XhoI, recovering digested products, connecting the digested products to a vector pET24a after digestion by Nde I and XhoI, carrying out DH5 alpha conversion, and carrying out PCR identification on recombinant plasmids by using T7/T7TER universal primers, wherein the theoretical value is 650bp, and the identification result is as shown in figure 1 and is consistent with the theoretical value; the obtained positive transformant extracts recombinant plasmid, converts escherichia coli expression strain Rosetta, and obtains pfHRP2 recombinant antigen expression strain: R/pET24a-pvMSP-1;

3. recombinant protein expression purification

3.1 Induction of expression

10. Mu.l of recombinant plasmid expression strain is inoculated into 5ml of LB culture medium containing 100ug/ml kanamycin (Kan), shaking culture is carried out at 37 ℃ and 200rpm for overnight, 200. Mu.l of bacterial liquid is added into 60ml of LB culture medium containing the same concentration Kan, shaking culture is carried out at 37 ℃ and 200rpm for overnight, all bacterial liquid is inoculated into 1L of LB culture medium containing the same concentration Kan, shaking culture is carried out at 28 ℃ and 150rpm for 2-2.5 hours, and OD is measured ₆₀₀ Adding inducer IPTG with final concentration of 0.5mM between 0.5 and 0.8, and continuously culturing at 28deg.C and 150rpm for 4.5 hr; after fermentation is completed, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected;

3.2 high pressure crushing

Bacterial pellet was resuspended in 30ml 20mM PBS (pH 7.4) and broken at low temperature and high pressure, parameters set: the pressure value is 910-980bar, the cycle is carried out for 3 times, the temperature is set to be 4 ℃, and the actual running temperature is not higher than 13 ℃; after crushing, centrifuging the crushed product at 9000rpm for 30min at low temperature, and collecting a supernatant;

3.3 protein purification

Performing affinity chromatography by using NI ion chromatographic column, and balancing the chromatographic column with 10 times of column volume of ultrapure water; then, the column is equilibrated with 10 times of column volume equilibration buffer (20 mM PBS pH 7.4), and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM PBS pH 7.4); finally eluting the target protein by using an elution buffer (20 mM PBS-250mM IM pH 7.4);

the collected protein samples were placed in dialysis bags, placed in beakers containing dialysate (20 mM PBS, pH 7.4), and dialyzed overnight at 4 ℃; performing SDS-PAGE detection on the purified protein, wherein the purity of the pvMSP-1 is more than 95%;

3. preparation of histidine-rich protein 2 and merozoite surface protein-1 antibodies

1. Immunization of mice

Taking histidine-rich protein 2 (HRP-2) and merozoite surface protein-1 (MSP-1) as immunogens, selecting female balb/c mice of 6-8 weeks old, and performing subcutaneous multipoint injection immunization respectively; after 3 times of immunization, tail cutting and blood sampling are carried out, serum is collected, an indirect ELISA method is used for detecting the titer of immune serum, and after the titer is qualified, the spleen of the mouse is taken for experiment;

2. antibody preparation

Mixing spleen cells of immunized mice and myeloma cells (SP 2/0) of the mice according to a ratio of 10:1 by utilizing a hybridoma preparation platform, and fusing by using PEG; screening fusion cells by HAT culture medium, performing positive screening by ELISA method, and subcloning positive cells by limiting dilution method to ensure the singleness of each cell line; screening two proteins to obtain 5 cell strains; the 10 antibodies were subjected to amplification culture according to 5X 10 ⁵ -10 ⁶ Injecting the obtained product into abdominal cavity of mice, preparing ascites, collecting ascites for 7d, centrifuging, and preserving at-20deg.C;

3. antibody purification

Purifying the antibody by adopting a purification method of a Protein-A column according to the processes of crude treatment, balancing, loading, washing impurities, eluting, neutralizing and dialyzing; collecting eluent for dialysis, replacing buffer solution, centrifuging to obtain supernatant, and freezing at-20deg.C;

4. antibody validation

A double antibody sandwich method is adopted to verify the pairing effect of antibodies; respectively labeling 10 antibodies by HRP, and carrying out double-antibody sandwich detection by adopting a chessboard method; coating corresponding antibody, adding pfHRP2 (or pvMSP-1) protein, adding HRP marked antibody to form antibody-antigen-enzyme mark antibody complex, and detecting OD450 by enzyme mark instrument under the action of substrate color developing liquid.