This kit is an enzyme-linked immunosorbent assay (ELISA) for qualitative detection of hemagglutinin (HA) of Influenza Type-A viruses, H5 strain (also known as highly pathogenic avian influenza) in avian excretes, cloacae swabs, human nasopharyngeal aspirates, swabs, or nasal wash, chicken embryo whole virus inoculation or viral lysates, etc. It is intended for clinical identification of early infections with type-A influenza, H5 strain viruses.
Influenza infection is an acute fever-like virus infection of the respiratory tract. The influenza virus and its toxin can lead to serious inflammation of the bronchial mucosa and damage of the tract. The influenza viruses belong to the family of Orthomyxoviridae that have linear segmented (8) negative ssRNA genome with lipid envelope. Total genome length is 12000-15000 nucleotides (nt). The largest segment is 2300-2500 nt; the second largest is 2300-2500 nt; the third is 2200-2300 nt; the fourth is 1700-1800 nt; the fifth is 1500-1600 nt; the sixth is 1400-1500 nt; the seventh is 1000-1100 nt; the eighth is 800-900 nt. The genome sequence has terminal repeated sequences; repeated at both ends. The virion envelope could be spherical, or filamentous with diameter of 50-120 nm, or 20 nm and 200-300(-3000) nm long. About 500 spikes are dispersed evenly over all the surface (i.e. hemagglutininesterase (HEF)), or dispersed equally over all the surface, but the various types are in clusters (i.e. hemagglutinin (HA) the major glycoprotein is interposed irregularly by clusters of neuraminidase (NA), with ratio of HA to NA about 4-5 to 1).
The Orthomyxoviridae family is divided into three types; A,B,C. Type A influenza viruses are further divided into subtypes according to their Hemagglutinin (HA) and Neuraminidase (NA) proteins. Currently 15 (HA) subtypes and 9(NA) subtypes have been identified. Type B influenza viruses produce less serious disease than does influenza type A and are not categorized as by H or N type as Influenza A is. Type C influenza viruses were first isolated in 1949 and are not known to be responsible for epidemics. The infection in human mostly results from a droplet infection and appears as an epidemic which sometimes can be of pandemic proportions. After an incubation time of 1-3 days the symptoms appear suddenly- fast rise of temperature, often accompanied by shivering, the leading symptom of catarrhal inflammation appears, contributing to the clinical course of painful dry cough, tracheitis, laryngitis and frequent rhinitis and conjunctivitis.
The appearance of new Influenza epidemics and pandemics are facilitated by an antigen variability mainly in the HA and NA. In the past century, three major influenza epidemics have occurred;
1918-1919, (Spanish Flu, A (H1N1), 20-50 million deaths worldwide, nearly half were young, healthy adults.
1957-1958, Asian Flu (A (H2N2), 1st identified in China Feb. 1957, 70,000 deaths in the United States.
1968-1969, Hong Kong Flu (A (H3N2), 1st detected in Hong Kong early 1968, virus still circulating today.
The 1997 outbreak of H5N1 avian influenza (or bird flu) in humans in Hong Kong caused alarm because it involved highly pathogenic strains of an influenza subtype A to which humans lack immunity. The H5N1 infected 18 humans, 6 of whom died (death rate of about 70 percent). Most of these cases occurred from contact with infected poultry or contaminated surfaces; however, it is thought that a few cases of human-to-human spread of H5N1 have occurred.
Avian influenza is an infectious disease of birds caused by type A strains of the influenza virus. The disease, first identified in Italy more than 100 years ago, now occurs worldwide. Infection triggers a wide spectrum of symptoms in birds, ranging from mild illness to a highly contagious and rapidly fatal disease resulting in severe epidemics. In the H5N1 bird flu in Hong Kong in 1997, patients had developed symptoms of fever, sore throat, cough and, in several of the fatal cases, severe respiratory distress secondary to viral pneumonia. Previously healthy adults and children, some with chronic medical conditions, were affected. More recently, outbreaks of avian influenza H5N1 occurred among poultry in eight countries in Asia (Cambodia, China, Indonesia, Japan, Laos, South Korea, Thailand, and Vietnam) during late 2003 and early 2004. At that time, more than 100 million birds in the affected countries either died from the disease or were killed in order to try to control the outbreak. By March 2004, the outbreak was reported to be under control. Beginning in late June 2004, however, new deadly outbreaks of influenza H5N1 among poultry were reported by several countries in Asia (Cambodia, China, Indonesia, Malaysia [first-time reports], Thailand, and Vietnam ). It is believed that these outbreaks are ongoing.
Human infections of avian influenza H5N1 however, have been reported in Cambodia (1case/1death) Thailand (17cases/1 death) and Vietnam (51cases/ 33deaths) during both of these outbreak periods. Hemagglutinin (HA) is a surface glycoprotein on Influenza A responsible for binding to N-AcetylNeuraminic Acid (NeuNAc) or commonly Sialic acid on host cell surface receptors. The Influenza viruses form the A virus group have principally similar morphological, chemical and biological features. The differentiation of the types is possible by the different antigenicity of their nucleo- and matrix proteins that have type-specific antigenicity. However, the essential immunodominant antigens and primary targets in diagnosis are the hemagglutinin (HA) and the neuraminidase (NA) antigens. Screening for type-specific anti-HA or anti-NA antibodies has also been proved to be useful method in clinical identification of different influenza strains.
Principle of Assay:
This H5 HA Antigen ELISA kit uses polystyrene microtiter strips pre-coated with antibodies specific to the hemagglutinin (HA) of the H5 influenza strain. The sample is added to the wells of the microtiter plate. During the first incubation step, the specfic anti-HA antibody-HA immunocomplex which forms in the presence of H5 HA in the sample, is captured on the solid phase. After washing to remove sample proteins or unbound material, a second H5 HA-specific antibody conjugated to horseradish peroxidase (HRP) is added. During this second incubation step, this HRP-conjugated anti-HA will specifically bind to the anti-HA-HA immunocomplex previously formed inside the wells, forming a sandwich immunocomplex. Unbound HRP-conjugated anti-HA is removed after washing. Chromogen solutions containing tetramethylbenzidine (TMB) and urea peroxide are added to the wells. In the presence of the sandwich immunocomplex, the colorless chromogens are hydrolyzed by the bound HRP-conjugate to a blue colored product. The blue color turns yellow after stopping the reaction with sulfuric acid. The amount of color can be measured and is proportional to the amount of HA in the sample. Wells containing samples negative for H5 HA remain colorless.
I7649-44A: Microtiter Plate, 1x96wells. Coated with HA Antibody. Blank microwell strips fixed on white strip holder. 8x12 or 12 x8-well strips per plate; strips can be broken to be used separately. Place unused wells in the plastic sealable storage bag together with the desiccant and return to 4 degrees C.
I7649-44B: Negative Control, 1x1ml. Protein-stabilized buffer tested non-reactive for HA of H5 influenza-A. Contains 0.1% ProClin 300 as preservative. Blue liquid in a vial with green screw cap. Ready to use as supplied. Stable for one month at 4 degrees C once opened.
I7649-44C: Positive Control, 1x1ml. Protein-stabilized buffer tested reactive for HA of H5 influenza-A. Contains 0.1% ProClin 300 as preservative. Red liquid in a vial with red screw cap. Ready to use as supplied. Stable for one month at 4 degrees C once opened.
I7649-44D: Mab (HRP), 1x12ml. Horseradish peroxidase-conjugated H5 specific anti-HA monoclonal antibody. Red liquid in a white dropper vial with red screw cap. Ready to use as supplied. Stable for one month at 4 degrees C once opened.
I7649-44E: Lysis Bufer, 2x50ml.
I7649-44F: Wash Buffer (20X), 1x50ml. PBS, pH 7.4, containing Tween-20. Colorless liquid in a clear bottle with white screw cap. Dilute 1:20 before use. Once diluted, stable for one week at room temperature or for two weeks at 4 degrees C.
I7649-44G: Chromogen Solution A, 1x6ml. Urea peroxide solution. Colorless liquid in white dropper vial with green screw cap. Ready to use as supplied. Stable for one month at 4 degrees C once opened.
I7649-44H: Chromogen Solution B, 1x6ml. Tetramethylbenzidine (TMB) dissolved in citric acid. Ready to use as supplied. Colorless liquid in black dropper vial with black screw cap. Stable for one month at 4 degrees C once opened.
I7649-44J: Stop Solution, 1x6ml. 2M H2SO4.
Storage and Stability:
May be stored at 4 degrees C. Do not freeze. Stable for 6 months. Protect the reagents from contamination with microorganisms or chemicals