Renal syndrome hemorrhagic fever (HFRS) ELISA kit instruction manual
Hemorrhagic fever with renal syndrome (HFRS) ELISA kit instruction manual Kit component IgM antibody: Leuconostoc Mesenteroides,Leuconostoc Mesenteroide,Leuconostoc Mesenteroides Powder,Leuconostoc Mesenteroides Treatment Jiangsu Biodep Biotechnology Co. ,Ltd. , https://www.mbioda.com
Hemorrhagic fever with renal syndrome (hereinafter referred to as hemorrhagic fever) is a natural epidemic disease caused by Hantavirus, and is a Class B infectious disease prescribed by the Law of the People's Republic of China on the Prevention and Control of Infectious Diseases.
The annual incidence of hemorrhagic fever with renal syndrome in China accounts for more than 90% of the reported cases of Hantavirus infection in the world, and is the country most seriously affected by Hantavirus. Cases have occurred in 31 provinces, municipalities and autonomous regions in mainland China. The highest annual incidence in China has exceeded 110,000. In the past decade, the number of reported cases in China has been around 20,000 to 50,000. New epidemic areas have emerged, and there have been outbreaks, and the types of old epidemic areas have also changed.
Therefore, the epidemiological monitoring of hemorrhagic fever with renal syndrome is of great significance. The original ELISA kit for hemorrhagic fever with renal syndrome imported from Germany IBL has passed the European CE certification, which can provide hemorrhagic fever investigation and serological testing. Reliable basis.
Intended Use <br>This enzyme-free kit can be used for the qualitative detection of anti-Hantavirus IgG and IgM in the serum of patients with epidemic hemorrhagic fever (EHF).
Introduction to Preface <br>Emergent Hemorrhagic Fever (EHF), also known as Hemorrhagic Fever with Nephrotic Syndrome (HFRS), is caused by the Hantaan type. Compared with epidemic nephropathy (contagious due to close contact with the Pumara virus), HFRS is more contagious and is usually accompanied by uremia, respiratory paralysis, and bleeding associated with typical shock syndrome, and even death. The initial symptoms of this disease are pandemic-like symptoms. Typical symptoms of HFRS at first are thrombocytopenia and leukocytosis. Hantavirus is mainly distributed in Asia and the Balkan states and is transmitted to humans through direct contact with rodents, rodent excretions or secretions. Soldiers, farmers and campers are particularly vulnerable.
Experimental principle <br> The Hantan virus nucleocapsid protein of Hantan-type virus was coated in the well. In order to avoid interference with non-specific factors caused by IgG antibodies or rheumatoid factors when detecting IgM antibodies, patient sera must be incubated with rheumatoid factor IgG sorbents.
During the incubation of the experimental process, specific antibodies against recombinant hantavirus antigens bind to the solid phase coated plates. After washing the plates, we detected specific IgG and IgM with peroxidase-labeled anti-human IgG and IgM antibodies, respectively. The color is developed after the addition of the substrate solution, and the intensity of the color exhibited by the solution is proportional to the amount of bound antibody. The OD value was then read using a microplate reader.
Storage and Stability <br>The kit should be transported and stored at 2~8°C to avoid high temperatures and direct sunlight. Unopened reagents can be stored until the expiration date. The coated strip can be stored in the closed bag at 2~8 °C until it is valid. The storage and stability of the prepared reagents are set forth in the corresponding sections.
The sealed microplate is sealed and stored at 2-8 °C until it is valid.
Sample collection and storage <br>human serum Pay attention to the routine precautions for collecting whole blood by venipuncture. The chemical integrity of the blood sample should be maintained from the moment the blood sample is collected to the time the test is performed. Do not use obvious hemolysis, jaundice, and lipemia samples. If the sample is turbid, it should be centrifuged to remove all particulate matter before the experiment. Human serum samples are used in this test. store 2-8 ° C ≤-20°C Avoid heat and direct sunlight to avoid repeated freezing. stability 5 days 12mon specification symbol ingredient 1 x 12 x 8 MTP The microporous reaction plate is ready for use and can be separated. Pre-coated with recombinant Hantaan antigen (strain: CG 76-118) 1 x 750 μL ANTI IgG CONJ CONC Enzyme complex (20 times concentrated)
Peroxidase-labeled anti-IgG 1 x 750 μL ANTI IgM CONJ CONC Enzyme complex (20 times concentrated)
Peroxidase labeling anti-IgM 1 x 1.5 mL CONTROL + IgG Positive control IgG
Ready to use. Contains: human serum, stabilizer, preservative 1 x 1.5 mL REFCONTROL IgG Reference control IgG
Ready to use. Contains: human serum, stabilizer, preservative 1 x 1.5 mL CONTROL + IgM Positive control IgM
Ready to use. Contains: human serum, stabilizer, preservative 1 x 15 mL DILBUF CONC Sample dilution buffer (20 times concentrated)
Red cap. Contains: PBS, pH 7.4, detergent and 0.01% thimerosal 1 x 1.5 mL REFCONTROL IgM Reference control quality control IgM
Ready to use. Contains: human serum, stabilizer, preservative 1 x 1.9 mL CONTROL- Yin nature control ready to use. Contains: human serum, stabilizers, preservatives 1 x 100 mL WASHBUF CONC Washing solution (10 times concentrated)
Contains: PBS buffer 1 x 1.5 mL RE-AB Rheumatoid factor adsorbent ready for use. Contains anti-human IgG, stabilizer, preservative 1 x 12 mL TMB SUBS TMB substrate liquid is ready to use. Contains: TMB 1 x 12 mL TMB STOP TMB stop solution ready for use. Contains 0.5 M sulfuric acid 1 x FOIL Adhesive film
The experiment required equipment but the kit did not provide
Pre- Experiment Preparation <br>The kit is equilibrated to room temperature (18-25 °C) before the experiment. The buffer concentrate containing crystals can be quickly dissolved at 37 ° C. It is cooled to room temperature before the experiment (18- 25°C)
Preparation for dilution of concentrated components (take 32 holes as an example)
Tip: Take the required amount of reagent before the experiment and use it quickly for dilution. Concentrate
(example) Component volume Diluent proportion Remarks storage stability 3mL DILBUF CONC 57mL Deionized water 1:20 Mix thoroughly 2-8 ° C 1 week 10 mL WASHBUF CONC 90 mL Deionized water 1:10 Mix thoroughly 2-8 ° C 8 weeks 200 μL ANTI IgG CONJ CONC
OR
ANTI IgM CONJ CONC 3.8 mL Dilution washing solution 1:20 Mix thoroughly - Pour off after the experiment
Sample dilution
Tip: Undiluted samples can be kept at -20 ° C for several months dilution proportion Remarks stability IgG sample conventional Dilute sample dilution 1:201 10 μL + 2000 μL 2-8 ° C 6 weeks IgM sample conventional Dilute sample dilution 1:201 10 μL + 2000 μL
( Add 15 μL of rheumatoid factor adsorbent to 250 μL of diluted serum sample and incubate at 18-25 ° C for 30 minutes) 2-8 ° C 6 weeks
Experimental procedure
1 Add 100 μl of ready-to-use positive control, negative control, reference control, and diluted serum samples (eg, IgM samples, pre-treatment with rheumatoid factor adsorbents) to the corresponding microwells.
2 Adhesive film cover plate, incubated at 37 ° C for 45 minutes.
3 Remove the adhesive film, discard the reaction solution in the well. Add 300 μl of the diluted washing solution to each well, repeat the plate washing 4 times, and pat dry on the absorbent paper to remove residual droplets.
4 Add 100 μl of diluted enzyme conjugate (IgG or IgM) to each well.
5 adhesive film cover. Incubate for 45 minutes at 37 °C.
6 Remove the adhesive film, discard the reaction solution in the well. Add 300 μl of the diluted washing solution to each well, repeat the washing 4 times, and pat dry on the absorbent paper to remove residual droplets.
7 Add substrate solution and stop solution. It is recommended to use 8 micropipettes to ensure the time interval of adding liquid phase. Avoid creating bubbles.
8 Add 100 μl of TMB substrate solution to each well
9 incubation at room temperature (18-25 ° C) for 10 minutes
10 The substrate solution was terminated by adding 100 μl of stop solution to each well.
11 Read the OD value at 450 nm in the microplate reader within 20 minutes. (Reference wavelength: 650nm)
8. Calculation of results <br>The test result is calculated by using the OD value of the patient sample compared with the OD value of the reference control quality control.
A patient sample / A reference control quality control = Q
IgG antibody: Q<1 Negative: no anti-Hantan Hantavirus IgG antibody was detected 1≤Q≤1.5 Suspicious gray areas, the results cannot be clearly stated, and the course of the disease should be retested after 10 days. If the suspected patient is infected with Hantavirus, we recommend that you also test Hantaan IgM antibodies or detect Puumala antibodies for the samples tested. Q>1.5 Positive: anti-Hantan virus-specific IgG antibody detected Q<1 Negative: no anti-Hantan Hantavirus IgM antibody was detected 1≤Q≤2 Suspicious gray areas, the results cannot be clearly stated, and the course of the disease should be retested after 10 days. If a suspected patient is infected with Hantavirus, we recommend that you also test Pumuala antibodies for the samples tested. Q>2 Positive: Anti-Hantavirus-specific IgM antibody detected
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