Colloidal gold immunochromatography test paper for rapidly detecting pigeon type I paramyxovirus and preparation method of colloidal gold immunochromatography test paper

Abstract

The invention discloses colloidal gold immunochromatography test paper for rapidly detecting pigeon I-type paramyxovirus and a preparation method of the colloidal gold immunochromatography test paper. Viral diseases of pigeons, especially pigeon plague virus and pigeon adenovirus, are rapidly detected on the basis of a newly developed test strip, and the operation is simple and convenient; according to the design of antigens and antibodies of the pigeon plague virus and the pigeon adenovirus, rapid detection of the pigeon plague virus and the pigeon adenovirus can be conveniently carried out, and the obtained test strip is high in specificity and high in sensitivity. The pigeon I-type paramyxovirus detection kit has the advantages that the pigeon I-type paramyxovirus detection kit does not have cross reaction with chicken infectious bronchitis, chicken egg drop syndrome, chicken Marek's disease, avian influenza, fowl adenovirus, infectious bursal disease, escherichia coli, salmonella and other samples, but can have cross reaction with Newcastle disease, so that the pigeon I-type paramyxovirus detection kit can be used for diagnosing pigeon I-type paramyxovirus and Newcastle disease.

Description

Technical Field

[0001] The invention relates to the technical field of pigeon breeding, and in particular to a colloidal gold immunochromatographic test paper for rapidly detecting pigeon type I paramyxovirus and a preparation method thereof. Background Art

[0002] Some pigeon diseases, such as paramyxovirus type 1 and adenovirus, can generally be initially diagnosed based on clinical symptoms and postmortem lesions. However, definitive diagnosis requires further methods. Currently, diagnostic methods for viral diseases utilize serological and molecular biological methods. In addition to classic serological methods such as the hemagglutination (HA) test, hemagglutination inhibition (HI) test, fluorescent antibody assay (IFA), agar diffusion test (AGP), latex agglutination test (LAT), embryo neutralization test, and plaque neutralization test, serological methods also include ELISAs using monoclonal antibodies. While HA and HI are commonly used by pigeon farms for antibody monitoring and diagnosis of Newcastle disease due to their simplicity, these tests are often subject to errors due to various reasons. Furthermore, HI is not sensitive enough for detecting low levels of antibodies, making it somewhat unreliable. Fluorescent antibody assays are simple, rapid, sensitive, accurate, and specific, making them suitable for rapid diagnosis. The antigen can be stored for long periods at -70°C, and the entire assay only takes one hour, making them suitable for large-scale qualitative antibody monitoring in SPF pigeons. The ELIAS diagnostic method is currently the most researched and fastest-developing technology. Various forms of ELIAS are widely used for antigen and antibody detection. Compared with other serological methods, ELIAS offers excellent sensitivity, high specificity, ease of use, reusability, and reduced antigen usage. Furthermore, the results can be easily stored over time, making it particularly suitable for serological diagnosis and epidemiological surveys of NDV in primary veterinary settings and pigeon farms. With the rapid development of molecular biology techniques, nucleic acid-based techniques are now also being used for the detection of PPMV-I, with PCR, nucleic acid probes, restriction endonuclease fragment length polymorphism analysis (RFLP), and sequence analysis being particularly widely used. While these methods have their advantages, they are also time-consuming and labor-intensive, requiring specialized equipment and personnel, making them difficult to implement in primary veterinary settings.

[0003] Colloidal gold immunochromatography (GICA) is a simple and rapid immunological detection technique developed in the early 1990s based on immunofiltration technology. It is a solid-phase labeled immunoassay that organically combines colloidal gold labeling, immunoassay, and chromatographic analysis. In recent years, continuous improvements have led to a large number of commercialized test strips being widely used in human medicine, sparking a surge in research and application of colloidal gold immunoassay technology. However, the number of products available for detecting viral diseases in pigeons is limited, and the development of more colloidal gold immunochromatographic detection products is needed. Summary of the Invention

[0004] Therefore, based on the above background, the present invention provides a colloidal gold immunochromatographic test paper for rapid detection of pigeon type I paramyxovirus and a preparation method thereof, which has fast and simple operation, sensitive detection and high accuracy.

[0005] The technical solution provided by the present invention is:

[0006] A colloidal gold immunochromatographic test paper for rapid detection of pigeon paramyxovirus type I and a preparation method thereof, wherein the preparation comprises the following steps:

[0007] S1: Pre-treating a pigeon body fluid or tissue sample or a swab sample to obtain a sample liquid;

[0008] S2: Use a dropper to vertically add 3-4 drops of the sample solution obtained in step 1) to the sample well on the test strip. Wait for more than 3 minutes and less than 20 minutes, and observe the results in the inspection window in the middle of the test card. If the two test lines of the test strip appear at the same time, it is judged as positive; if the quality control line reacts but the test line does not react, it is judged as negative;

[0009] The preparation method of the test strip comprises the following steps:

[0010] (1) Preparation of immune antigens:

[0011] The F gene fragment of pigeon paramyxovirus type 1 was connected to the PGEX-4T-1 prokaryotic expression vector. After IPTG induction and analysis, the F protein was efficiently expressed in Escherichia coli BL21. The inclusion body protein was extracted as an immune antigen.

[0012] (2) Preparation of antiviral monoclonal antibodies

[0013] (3) Adhere the nitrocellulose membrane to the middle of the PVC sheet;

[0014] (4) applying the purified antiviral monoclonal antibody to a nitrocellulose membrane to coat an area of ​​the nitrocellulose membrane to obtain a detection line;

[0015] (5) coating another area of ​​the nitrocellulose membrane with purified goat anti-mouse antibody to obtain a quality control line;

[0016] (6) Preparation of colloidal gold labeled protein solution of antiviral monoclonal antibodies:

[0017] (7) immersing the glass fiber membrane in a colloidal gold-labeled protein solution of an antiviral monoclonal antibody, soaking it in water, and drying it to obtain a colloidal gold binding pad; after drying the colloidal gold binding pad at 37° C. to a constant weight, adhering it to the end of the nitrocellulose membrane away from the quality control line;

[0018] (8) immersing the sample pad in a 0.05 M Tris solution with a pH of 8.5 to obtain a treated sample pad;

[0019] (9) The treated sample pad is adhered to one end of the colloidal gold binding pad, and the other end is adhered to the nitrocellulose membrane; the treated sample pad is adhered to one end of the PVC rubber plate; the absorbent pad is adhered to the nitrocellulose membrane and adhered to the other end of the PVC rubber plate.

[0020] Furthermore, the preparation of the colloidal gold labeled protein solution in step (6) comprises the following steps:

[0021] ①Prepare colloidal gold solution by citric acid reduction method;

[0022] ② After adjusting the pH value of the colloidal gold solution prepared in step ① to 9.0-9.5, the colloidal gold solution and the antiviral monoclonal antibody were mixed, stirred, and then a buffer solution was added to adjust the concentration to 1%. After low-speed centrifugation, the supernatant was collected;

[0023] ③ After centrifuging the supernatant obtained in step ②, the precipitate was taken and dissolved with 0.015M colloidal gold solution with a pH of 7.4 to obtain a colloidal gold-labeled protein solution of antiviral monoclonal antibodies, which was stored at low temperature for future use.

[0024] Furthermore, the colloidal gold complex solution of 0.015 M and pH=7.4 is a PBS solution containing 20 g sucrose, 1.1 g BSA, 1 g PVP, and 0.02 g sodium azide in 1 L PBS solution.

[0025] Furthermore, the 0.05 M Tris solution with a pH of 8.5 in step (8) refers to a Tris solution containing 1 g BSA, 1 g PVP, 500 mL Tween-20, and 0.02 g sodium azide in 1 L Tris solution.

[0026] Furthermore, the preparation of the antibody in step (2) includes anti-pigeon type I paramyxovirus F protein monoclonal antibody and anti-pigeon adenovirus pigeon adenovirus FADV4 type Fiber2 protein monoclonal antibody;

[0027] Preparation of anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies: BALB / C mice were immunized with anti-pigeon type 1 paramyxovirus F protein to prepare anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies according to conventional monoclonal antibody preparation techniques, and then purified to obtain purified anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies;

[0028] Preparation of monoclonal antibodies against pigeon adenovirus FADV4 type Fiber2 protein:

[0029] ① The gold-labeled antibody is a monoclonal antibody 5A3 against the Fiber2 protein of FADV4 avian adenovirus, obtained by preparing mouse ascites using hybridoma cell line 5A3 and purifying IgG via a Protein G column;

[0030] ②The detection antibody is the monoclonal antibody 1D7 against the Fiber2 protein of FADV4 avian adenovirus, which is obtained by preparing mouse ascites using the hybridoma cell line 1D7 and then purifying IgG using a Protein G column.

[0031] The beneficial effects achieved by the present invention are:

[0032] The invention is based on a newly developed test strip to quickly detect viral diseases of pigeons, especially pigeon plague virus and pigeon adenovirus, with simple and convenient operation;

[0033] Among them, the design of antigens and antibodies for pigeon plague virus and pigeon adenovirus can facilitate the rapid detection of pigeon plague virus and pigeon adenovirus. The resulting test strips have strong specificity and do not cross-react with other samples such as infectious bronchitis, egg drop syndrome, chicken Marek's, avian influenza, avian adenovirus, infectious bursal disease, Escherichia coli, Salmonella, etc., but can cross-react with Newcastle disease. Therefore, it can be used for the diagnosis of pigeon type 1 paramyxovirus disease and chicken Newcastle disease.

[0034] Figures in the specification

[0035] Figure 1 This is a schematic diagram of the structure of the test strip of the present invention. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0037] Example 1: A method for preparing a colloidal gold immunochromatographic test paper for rapid detection of pigeon paramyxovirus type I, comprising the following steps:

[0038] S1: Pre-treating a pigeon body fluid or tissue sample or a swab sample to obtain a sample liquid;

[0039] The preprocessing operations for samples from different sources are as follows:

[0040] Body fluid samples (serum / plasma): directly draw 80–100 μL for later use;

[0041] Tissue samples: 0.1 g of tissue was added to 1 mL of PBS buffer, ground, and centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected.

[0042] Swab sample: Dip the swab into 0.5 mL of sample buffer (PBS buffer), squeeze it, discard the swab, and collect the liquid.

[0043] S2: Use a dropper to vertically add 3-4 drops of the sample solution obtained in step 1) to the sample well on the test strip. Wait for more than 3 minutes and less than 20 minutes, and observe the results in the inspection window in the middle of the test card. If the two test lines of the test strip appear at the same time, it is judged as positive; if the quality control line reacts but the test line does not react, it is judged as negative;

[0044] The preparation method of the test strip comprises the following steps:

[0045] (1) Preparation of immune antigens:

[0046] The F gene fragment of pigeon paramyxovirus type 1 was connected to the PGEX-4T-1 prokaryotic expression vector. After IPTG induction and analysis, the F protein was efficiently expressed in Escherichia coli BL21. The inclusion body protein was extracted as an immune antigen.

[0047] (2) Preparation of antiviral monoclonal antibodies

[0048] The preparation of antibodies includes monoclonal antibodies against the F protein of pigeon paramyxovirus type 1 and monoclonal antibodies against the Fiber2 protein of pigeon adenovirus FADV type 4;

[0049] Preparation of anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies: BALB / C mice were immunized with anti-pigeon type 1 paramyxovirus F protein to prepare anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies according to conventional monoclonal antibody preparation techniques, and then purified to obtain purified anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies;

[0050] Preparation of monoclonal antibodies against pigeon adenovirus FADV4 type Fiber2 protein:

[0051] ① The gold-labeled antibody is a monoclonal antibody 5A3 against the Fiber2 protein of FADV4 avian adenovirus, obtained by preparing mouse ascites using hybridoma cell line 5A3 and purifying IgG via a Protein G column;

[0052] ②The detection antibody is the monoclonal antibody 1D7 against the Fiber2 protein of FADV4 avian adenovirus, which is obtained by preparing mouse ascites using the hybridoma cell line 1D7 and purifying IgG using a Protein G column.

[0053] (3) Adhere the nitrocellulose membrane to the middle of the PVC sheet;

[0054] (4) applying the purified antiviral monoclonal antibody to a nitrocellulose membrane to coat an area of ​​the nitrocellulose membrane to obtain a detection line;

[0055] (5) coating another area of ​​the nitrocellulose membrane with purified goat anti-mouse antibody to obtain a quality control line;

[0056] (6) Preparation of colloidal gold labeled protein solution of antiviral monoclonal antibodies:

[0057] Gold solution was prepared by citric acid reduction method: 100 mL of 0.01% chloroauric acid aqueous solution was taken, heated to reflux under magnetic stirring, and after boiling, 2.5 mL of freshly prepared 1% trisodium citrate aqueous solution was quickly added. Heating was continued until the solution turned purple-red. After cooling, the volume was restored to the original volume with distilled water to obtain a colloidal gold solution with colloidal gold particles with a diameter of 20 to 30 nm. The pH value of the colloidal gold solution was adjusted to 9.0-9.5 with 0.1 mol / L K2CO3, and 1 mL of colloidal gold and 50 μg of purified antiviral monoclonal antibody were taken and stirred. After 40 minutes, 5% SA was added to make the final concentration 1%; the solution was centrifuged at 3000 rpm for 20 minutes, and the precipitate formed by the agglomerated gold colloid particles was discarded to obtain a supernatant; finally, the supernatant was centrifuged at 12000 rpm for 30 minutes at a temperature of 4°C to obtain a precipitate, which was dissolved with 100 μL of 0.015 M colloidal gold solution with a pH of 7.4 and stored at 4°C to obtain a colloidal gold-labeled protein solution of anti-pigeon type I paramyxovirus F protein monoclonal antibody. The colloidal gold complex solution of 0.015 M and pH = 7.4 refers to a PBS solution containing 20 g sucrose, 1.1 g BSA, 1 g PVP, and 0.02 g sodium azide in 1 L PBS solution.

[0058] (7) immersing the glass fiber membrane in a colloidal gold-labeled protein solution of an antiviral monoclonal antibody, soaking it in water, and drying it to obtain a colloidal gold binding pad; after drying the colloidal gold binding pad at 37° C. to a constant weight, adhering it to the end of the nitrocellulose membrane away from the quality control line;

[0059] (8) immersing the sample pad in a 0.05 M Tris solution with a pH of 8.5 to obtain a treated sample pad;

[0060] The 0.05 M, pH 8.5 Tris solution refers to a Tris solution containing 1 g of BSA, 1 g of PVP, 500 mL of Tween-20, and 0.02 g of sodium azide in 1 L of Tris solution.

[0061] (9) The treated sample pad is adhered to one end of the colloidal gold binding pad, and the other end is adhered to the nitrocellulose membrane; the treated sample pad is adhered to one end of the PVC rubber plate; the absorbent pad is adhered to the nitrocellulose membrane and adhered to the other end of the PVC rubber plate.

[0062] The structure of the test strip prepared according to the above preparation method is as follows Figure 1 As shown, it comprises a PVC sheet 1, a nitrocellulose membrane 2, a colloidal gold conjugate pad 3, a sample pad 4, and an absorbent pad 5. The PVC sheet 1 has the sample pad 4 attached to one end and the absorbent pad 5 attached to the other end. The colloidal gold conjugate pad 3 and the nitrocellulose membrane 2 are sequentially attached to the middle portion. The colloidal gold conjugate pad 3 is adhered to the sample pad 4 at one end and to the nitrocellulose membrane 2 at the other end, which is in turn adhered to the absorbent pad 5. The colloidal gold conjugate pad 3 is coated with a colloidal gold-labeled protein containing a monoclonal antibody against the F protein of pigeon paramyxovirus type I. A test line 6 and a quality control line 7 are sequentially provided on the nitrocellulose membrane 2 along the sample flow direction. The test line 6 is a linearly coated purified antiviral monoclonal antibody.

[0063] Conduct test on the test strips prepared above:

[0064] ① Randomly select 10 test strips from the same batch of the test kit. Add 100 μL of saline to the sample wells, place them flat, and wait 3–5 minutes to observe the results in the central inspection window of the test card. Reading the results should take no longer than 20 minutes. If the control line reacts but the test line does not, the test strip is considered valid. If both lines react simultaneously, or if only the test line reacts while the control line does not, the test strip is considered invalid. Results: All 10 test strips showed red bands on the control line, while none on the test line, indicating that all the prepared test strips were valid.

[0065] ② Positive coincidence test: 10 test strips were randomly selected from the same batch of the test kit and tested for 10 samples positive for pigeon paramyxovirus type 1. 100 μL of the sample was added to the sample well of the test strip, which was placed flat. The test strip was observed for 3–5 minutes after reading the results in the central inspection window, but no more than 20 minutes were allowed. A positive result was determined when both test lines appeared simultaneously. Results: Red bands appeared on both test lines on all 10 test strips, indicating a 100% positive coincidence rate.

[0066] Negative coincidence test: 10 test strips were randomly selected from the same batch of the test kit and tested against 10 negative samples of pigeon paramyxovirus type 1. 100 μL of the negative sample was added to the sample well of the test strip, which was then placed flat. The test strip was then observed for 3–5 minutes in the central inspection window, but the reading should not exceed 20 minutes. A negative result was determined when the control line showed a reaction but the test line did not. Results: All 10 test strips showed a red band on the control line and none on the test line, indicating a 100% negative coincidence rate.

[0067] ③ Three samples of pigeon Paramyxovirus type 1-positive and three samples of pigeon Paramyxovirus-negative allantoic fluid were tested using test strips from different batches. Each sample was tested five times to determine if the test results were reproducible. Results: The test results were consistent, indicating good reproducibility.

[0068] ④ Three samples of pigeon Paramyxovirus type I allantoic fluid with different hemagglutination titers were diluted in multiples and tested using test strips to compare the sensitivity of the hemagglutination test and the test strips. The test strips prepared by the present invention tested positive when the sample with a hemagglutination titer of 28 was diluted 32-fold, and when the sample with a hemagglutination titer of 27 was diluted 16-fold. In other words, the test strips prepared by the present invention can detect virus fluids with a hemagglutination titer of 23 or higher.

[0069] The present invention and its embodiments are described above. Such description is not restrictive. The embodiment shown in the embodiment is only one embodiment of the present invention, and the actual structure is not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the invention, without creatively designing a structure and embodiment similar to the technical solution, they shall fall within the scope of protection of the present invention.

Claims

1. A colloidal gold immunochromatographic test paper for rapid detection of pigeon type I paramyxovirus, characterized in that: It includes a PVC rubber plate, a nitrocellulose membrane, a colloidal gold binding pad, a sample pad and a water absorbent pad; A sample pad is adhered to one end of the PVC rubber plate, a water-absorbing pad is adhered to the other end, and a colloidal gold binding pad and a nitrocellulose membrane are adhered to the middle of the plate in sequence; One end of the colloidal gold conjugate pad is adhered to the sample pad, and the other end thereof is adhered to the nitrocellulose membrane, which is adhered to the absorbent pad; the colloidal gold conjugate pad is coated with colloidal gold-labeled protein with antiviral monoclonal antibodies; Detection lines and quality control lines are arranged on the nitrocellulose membrane in sequence along the sample flow direction; The test line is a purified antiviral monoclonal antibody coated in a linear form.

2. The method for preparing a colloidal gold immunochromatographic test paper for rapid detection of pigeon type I paramyxovirus according to claim 1, wherein The preparation includes the following steps: S1: Pre-treating a pigeon body fluid or tissue sample or a swab sample to obtain a sample liquid; S2: Use a dropper to vertically add 3-4 drops of the sample solution obtained in step 1) to the sample well on the test strip. Wait for more than 3 minutes and less than 20 minutes, and observe the results in the inspection window in the middle of the test card. If the two test lines of the test strip appear at the same time, it is judged as positive; if the quality control line reacts but the test line does not react, it is judged as negative; The preparation method of the test strip comprises the following steps: (1) Preparation of immune antigens: The F gene fragment of pigeon paramyxovirus type 1 was connected to the PGEX-4T-1 prokaryotic expression vector. After IPTG induction and analysis, the F protein was efficiently expressed in Escherichia coli BL21. The inclusion body protein was extracted as an immune antigen. (2) Preparation of antiviral monoclonal antibodies (3) Adhere the nitrocellulose membrane to the middle of the PVC sheet; (4) applying the purified antiviral monoclonal antibody to a nitrocellulose membrane to coat an area of ​​the nitrocellulose membrane to obtain a detection line; (5) coating another area of ​​the nitrocellulose membrane with purified goat anti-mouse antibody to obtain a quality control line; (6) Preparation of colloidal gold labeled protein solution of antiviral monoclonal antibodies: (7) immersing the glass fiber membrane in a colloidal gold-labeled protein solution of an antiviral monoclonal antibody, soaking it in water, and drying it to obtain a colloidal gold binding pad; after drying the colloidal gold binding pad at 37° C. to a constant weight, adhering it to the end of the nitrocellulose membrane away from the quality control line; (8) immersing the sample pad in a 0.05 M Tris solution with a pH of 8.5 to obtain a treated sample pad; (9) The treated sample pad is adhered to one end of the colloidal gold binding pad, and the other end is adhered to the nitrocellulose membrane; the treated sample pad is adhered to one end of the PVC rubber plate; the absorbent pad is adhered to the nitrocellulose membrane and adhered to the other end of the PVC rubber plate.

3. the method for detecting pigeon disease by colloidal gold immunochromatography according to claim 2, is characterized in that, The preparation of the colloidal gold labeled protein solution in step (6) comprises the following steps: ①Prepare colloidal gold solution by citric acid reduction method; ② After adjusting the pH value of the colloidal gold solution prepared in step ① to 9.0-9.5, the colloidal gold solution and the antiviral monoclonal antibody were mixed, stirred, and then a buffer solution was added to adjust the concentration to 1%. After low-speed centrifugation, the supernatant was collected; ③ After centrifuging the supernatant obtained in step ②, the precipitate was taken and dissolved with 0.015M colloidal gold solution with a pH of 7.4 to obtain a colloidal gold-labeled protein solution of antiviral monoclonal antibodies, which was stored at low temperature for future use.

4. the method for detecting pigeon disease by colloidal gold immunochromatography according to claim 3, is characterized in that, The 0.015 M colloidal gold complex solution with a pH of 7.4 is a PBS solution containing 20 g sucrose, 1.1 g BSA, 1 g PVP, and 0.02 g sodium azide in 1 L of PBS solution.

5. the method for detecting pigeon disease by colloidal gold immunochromatography according to claim 2, is characterized in that, The 0.05 M, pH 8.5 Tris solution in step (8) refers to a Tris solution containing 1 g BSA, 1 g PVP, 500 mL Tween-20, and 0.02 g sodium azide in 1 L of Tris solution.

6. the method for detecting pigeon disease by colloidal gold immunochromatography according to claim 2, is characterized in that, The preparation of the antibodies in step (2) includes anti-pigeon type I paramyxovirus F protein monoclonal antibody and anti-pigeon adenovirus pigeon adenovirus FADV4 type Fiber2 protein monoclonal antibody; Preparation of anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies: BALB / C mice were immunized with anti-pigeon type 1 paramyxovirus F protein to prepare anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies according to conventional monoclonal antibody preparation techniques, and then purified to obtain purified anti-pigeon type 1 paramyxovirus F protein monoclonal antibodies; Preparation of monoclonal antibodies against pigeon adenovirus FADV4 type Fiber2 protein: ① The gold-labeled antibody is a monoclonal antibody 5A3 against the Fiber2 protein of FADV4 avian adenovirus, obtained by preparing mouse ascites using hybridoma cell line 5A3 and purifying IgG via a Protein G column; ②The detection antibody is the monoclonal antibody 1D7 against the Fiber2 protein of FADV4 avian adenovirus, which is obtained by preparing mouse ascites using the hybridoma cell line 1D7 and purifying IgG using a Protein G column.

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