2 English reference fluorescent antibody technique
3 Overview Coons is equal to 194 1 year, which was successfully labeled with fluorescein for the first time. This technique of locating antigens by labeling antibodies with fluorescent substances is called fluorescent antibody technique. Fluorescence immunoassay is one of the earliest labeled immunoassay. For a long time, some scholars have tried to combine antibody molecules with some tracer substances and use antigen-antibody reaction to locate antigen substances in tissues or cells.
Because of the high background in conventional fluorescence determination, it is difficult to use fluorescence immunoassay for quantitative determination. In recent years, several special fluorescence immunoassay methods, such as enzyme immunoassay and radioimmunoassay, have been developed for clinical examination.
4 fluorescence phenomenon 4. 1 generation of fluorescence-this chemical substance can absorb and store energy (such as light energy and chemical energy). ) enters the excited state from the outside. When it returns from the excited state to the ground state, the excess energy can be radiated in the form of electromagnetic radiation (i.e. luminescence).
The characteristics of fluorescence emission are: molecules or atoms that can produce fluorescence emit light immediately after receiving energy; Once the energy supply is stopped, the luminescence (fluorescence) phenomenon disappears instantly.
There are many kinds of energy that can cause fluorescence, and the fluorescence generated by light excitation is called fluorescence. What is caused by chemical reaction is called chemical fluorescence, and what is caused by X-ray or cathode ray is called X-ray fluorescence or cathode ray fluorescence respectively. Fluorescence immunoassay generally uses fluorescent substances for labeling.
4.2 Fluorescence Efficiency Fluorescent molecules will not convert all the absorbed light energy into fluorescence, but they will always be released in other forms more or less. Fluorescence efficiency refers to the percentage of absorbed light energy converted into fluorescence by fluorescent molecules, which is directly proportional to the quantum value of emitted fluorescence.
Fluorescence efficiency = amount of light emitted (fluorescence intensity)/amount of light absorbed (excitation intensity)
The quantum number of fluorescent light, that is, the fluorescence intensity, is not only affected by the intensity of excitation light, but also related to the wavelength of excitation light. Each fluorescent molecule has its specific absorption spectrum and emission spectrum (fluorescence spectrum), that is, there is a maximum absorption peak and a maximum emission peak at a certain wavelength. When the wavelength of excitation light is selected near the maximum absorption peak wavelength of fluorescent molecules, and when the measuring light wave is near the maximum emission peak, the fluorescence intensity is also the largest.
4.3 Quenching of Fluorescence After being irradiated by excitation light for a long time, the radiation ability of fluorescent molecules will be weakened or even quenched, because the electrons of the excited molecules cannot return to the ground state and the absorbed energy cannot be emitted in the form of fluorescence. Some compounds have natural fluorescence quenching effect and are used as quenchers to eliminate unnecessary fluorescence. Therefore, the preservation of fluorescent substances should pay attention to avoid direct irradiation of light (especially ultraviolet rays) and contact with other compounds. Some non-fluorescent pigments, such as methylene blue and basic magenta, are often used in fluorescent antibody technology. The samples were dyed with Evans blue or low concentration potassium permanganate and iodine solution to weaken the essence of non-specific fluorescence and make the specific fluorescence more prominent.
5 fluorescent substances 5. 1 fluorescent pigments Many substances can produce fluorescence, but not all substances can be used as fluorescent pigments. Only those organic compounds that can produce obvious fluorescence and can be used as dyes can be called immunofluorescent pigments or fluorescent dyes. Commonly used fluorescent pigments are:
1. Fluorescein thiocyanate (FITC) is a yellow or orange crystalline powder, which is easily soluble in solvents such as water or alcohol. The molecular weight is 389.4, the maximum absorption wavelength is 490495nm, and the maximum emission wavelength is 520530nm, showing bright yellow-green fluorescence, and the structural formula is as follows:
There are two isomorphic structures, among which isomer I is the most widely used fluorescein, because it has better efficiency, stability and protein binding ability, and can be stored in cold, dark and dry places for many years. Its main advantages are: ① human eyes are sensitive to yellow-green color; ② The green fluorescence in slice specimens is usually less than that in red.
2. Tetraethyl rhodamine (RIB200) is orange powder, insoluble in water, soluble in ethanol and acetone. It has stable properties and can be preserved for a long time. The structural formula is as follows:
The maximum absorption wavelength is 570nm, and the maximum emission wavelength is 595 ~ 600 nm, showing orange fluorescence.
3. Tetramethyl rhodamine isothiocyanate (TRITC) has the following structural formula:
The maximum absorption wavelength is 550nm, and the maximum emission wavelength is 620nm, showing orange-red fluorescence. Compared with the emerald green fluorescence of FITC, it can be used for double labeling or contrast dyeing. Its isothiocyano group can bind to protein, but its fluorescence efficiency is low.
5.2 Other fluorescent substances 1. Substances that produce fluorescence after the action of enzymes Some compounds have no fluorescence by themselves, and once they are acted by enzymes, they will form substances with strong fluorescence. For example, βD galactoside of 4 methyl umbrella ketone is decomposed by β galactosidase into 4 methyl umbrella ketone, which can emit fluorescence with excitation wavelength of 360nm and emission wavelength of 450nm. Others, such as 4-methyl umbelliform phosphoric acid as the substrate of alkaline acid enzyme and 4-hydroxyphenylacetic acid as the substrate of horseradish peroxidase, etc.
2. Lanthanide chelates Some trivalent rare earth lanthanide chelates, such as europium (Eu3+), terbium (Tb3+) and cerium (Ce3+), can also emit characteristic fluorescence after excitation, among which Eu3+ is the most widely used. Eu3+ chelate has a wide excitation wavelength range, a narrow emission wavelength range and a long fluorescence decay time, so it is most suitable for fluorescence immunoassay.
6 Preparation of Fluorescent Antibodies 6. 1 Fluorescein labeling of antibodies used to label antibodies requires high specificity and affinity. The antiserum used should not contain antibodies against normal tissues in the specimen. Generally speaking, IgG should be purified and extracted before labeling. As a marker, fluorescein should meet the following requirements: ① It should have chemical groups that can form valence bonds with protein molecules, and it is not easy to dissociate after binding with protein, while unbound pigments and their degradation products are easy to remove. ② The fluorescence efficiency is high, and it can still maintain high fluorescence efficiency after being combined with protein. ③ The color of fluorescence is in sharp contrast with the color of background tissue. ④ The combination with protein does not affect the original biochemical and immune characteristics of protein. ⑤ The labeling method is simple, safe and non-toxic. ⑥ The conjugate with protein is stable and easy to store.
There are two commonly used methods for protein labeling: stirring method and dialysis method. Taking FITC labeling as an example, the stirring labeling method is as follows: firstly, the protein solution to be labeled is balanced with 0.5ml/LpH9.0 carbonate buffer, then the FITC solution is added dropwise under magnetic stirring, and after continuous stirring at room temperature for 4-6 hours, the supernatant is taken as the label. This method is suitable for labeling antibody solution with large volume and high protein content. Its advantages are short labeling time and low dosage of fluorescein. However, there are many factors influencing this method, and if it is not operated properly, it will cause strong nonspecific fluorescence staining.
Dialysis method is suitable for antibody solution with few labeled samples and low protein content. This method has relatively uniform labeling and low nonspecific staining. The method is as follows: firstly, put the protein solution to be labeled into a dialysis bag, put it in 0.0 1mol/LpH9.4 carbonate buffer containing FITC to react overnight, and then dialyze with PBS to remove the free pigment. Centrifuge at low speed and collect supernatant.
After labeling, the labeled antibody should be further purified to remove unbound free fluorescein and antibodies that bind fluorescein too much. The purification method can be dialysis or chromatographic separation.
6.2 Identification of Fluorescent Antibodies Fluorescent antibodies should be identified before use. Identification indexes include titer and binding rate of fluorescein to protein. Antibody titer can be titrated by agar double diffusion method, and the titer is better than 1: 16. The basic method to measure the binding rate (F/P) between fluorescein and protein is: dilute the prepared fluorescent antibody to A280 1≈ 1.0, measure the specific absorption peaks of A280 (protein specific absorption peak) and labeled fluorescein respectively, and calculate according to the formula.
The higher the F/P value, the more fluorescein binds to antibody molecules, and vice versa. Generally, F/P = 1.5 is the suitable fluorescent antibody for fixing the specimen, and F/P = 2.4 is the living cell staining.
The determination method of antibody working concentration is similar to the titration of enzyme-labeled antibody in ELISA indirect method. The fluorescent antibody was diluted from 1: 4 to 1: 256 times, and the slice samples were stained with fluorescent antibody. The working concentration of fluorescent antibody is the highest dilution that can clearly show specific fluorescence and non-specific staining is weak.
The preservation of fluorescent antibodies should pay attention to prevent antibody inactivation and fluorescence quenching. It is best to pack in small batches and freeze at -20℃, which can be preserved for 3 ~ 4 years. Generally, it can be stored at 4℃ 1 ~ 2 years.
7 Immunofluorescence Microscopy The basic principle of immunofluorescence microscopy is: make the fluorescent antibody react with the antigen on the tissue or cell surface in the specimen section, wash off the free fluorescent antibody, and observe under the fluorescence microscope, and you can see bright specific fluorescence on a dark background.
7. 1 Specimen making fluorescence microscopy mainly relies on observing the staining results of fluorescent antibodies on sliced specimens as antigen identification and localization. Therefore, the quality of sample preparation directly affects the test results. In the process of specimen preparation, efforts should be made to maintain the integrity of antigen, which will not dissolve, denature and spread to adjacent cells or tissues during dyeing, washing and sealing. Specimen sections should be as thin as possible to facilitate antigen-antibody contact and microscopic examination. Substances that interfere with antigen-antibody reaction in the specimen should be washed off fully, and infectious specimens should pay attention to safety.
Common clinical specimens mainly include tissues, cells and bacteria. Smears, prints or slices can be made according to different specimens. Tissue materials can be made into paraffin sections or frozen sections. Paraffin section is rarely used because of its complicated operation, unstable results and strong non-specific reaction. Tissue specimens can also be made into printed matter. The method is to gently press the tissue section with the cleaned glass slide to make the glass slide stick with 1 ~ 2 layers of tissue cells. Cells or bacteria can be made into smears, which should be thin and uniform. After the smear or printed matter is made, it should be quickly dried and packaged. Store at-10℃ or use immediately.
7.2 Add fluorescent antibody staining to the fixed sample by dropping fluorescent antibody with proper dilution. Incubate in a wet box at a certain temperature for a certain period of time, generally at 25 ~ 37℃ for 30 min, and at 4℃ for overnight detection of heat-labile antigens. Thoroughly washed with PBS and dried.
7.3 The samples stained with fluorescent antibodies need to be observed under a fluorescent microscope. It is best to do microscopic examination on the day of dyeing to prevent the fading of fluorescence from affecting the results.
Fluorescence microscopy should be carried out in a well-ventilated dark room. First, choose a good light source or filter. The correct selection of filter is an important condition for obtaining good fluorescence observation effect. A set of excitation filters in front of the light source is used to provide suitable excitation light. There are two types of excitation filters. MG is an ultraviolet filter, which only allows ultraviolet light with a wavelength of 275 ~ 400 nm to pass through, with a maximum transmittance of 365nm. BG is a blue ultraviolet filter, which only allows blue light with the wavelength of 325 ~ 500 nm to pass through, and the maximum transmittance is 4 10nm. A set of blocking filters (also called absorption filters or suppression filters) near the eyepiece are used to filter out excitation light and only allow fluorescence to pass through. The light transmission range is 4 10 ~ 650 nm, and the codes are OG (orange yellow) and GG (light green yellow). The excitation filter BG 12 and the absorption filter OG4 or GG9 can be used to observe the FITC mark. When observing RB200 marker, BG 12 can be selected to cooperate with OG5.
7.4 Experimental type: 1. Direct method: the specific fluorescent antibody is directly dropped on the specimen to make it specifically bind to the antigen (figure 17 1). This method is simple to operate, with high specificity and few nonspecific fluorescent staining factors. The disadvantage is low sensitivity, and it is necessary to prepare corresponding specific fluorescent antibodies for each antigen.
Fig. 17 1 schematic diagram of direct immunofluorescence.
2. Indirect method can be used to detect antigens and antibodies, and its principle is shown in figure 172. There are two kinds of antibodies in this method. The first antibody is a specific antibody against antigen, and the second antibody (fluorescent antibody) is an anti-antibody against the first antibody. This method has high sensitivity, and only one fluorescent antibody is needed to detect different antigens.
Fig. 172 schematic diagram of indirect immunofluorescence.
Fig. 173 schematic diagram of complement binding immunofluorescence principle
3. Complement binding method This method is the first step of indirect method. Complement (mostly guinea pig complement) was added in the antigen-antibody reaction, and then the fluorescent labeled anti-complement antibody was used for tracing (Figure 173). This method has high sensitivity and only needs one antibody. However, it is prone to nonspecific staining and unstable complement, and it is complicated to collect fresh guinea pig serum every time. So it is used less.
4. Labeling method In this method, FITC and rhodamine were used to label different antibodies, and the same specimen was stained with fluorescence. In the presence of two corresponding antigens, orange-red and yellow-green fluorescent colors can be seen simultaneously.
Application of fluorescent antibody technology in medical laboratory Fluorescent antibody technology has been used in the detection of bacteria, viruses and parasites and the diagnosis of autoimmune diseases in clinical laboratories. It is mainly used to identify strains in bacteriological examination. Specimen materials can be cultures, infected tissues, excreta secreted by patients, etc. Compared with other serological methods, this method is faster, simpler and more sensitive, but it can only be used as an auxiliary means in bacterial test diagnosis and cannot replace conventional diagnosis. Fluorescent antibody staining has a good effect on the experimental diagnosis of Neisseria meningitidis, Shigella dysenteriae, Vibrio cholerae, Brucella and Bacillus anthracis. Indirect fluorescence staining can be used to determine antibodies in serum, and can be used for epidemiological investigation and clinical retrospective diagnosis. Immunofluorescence detection of Treponema pallidum antibody is one of the commonly used methods for specific diagnosis of syphilis. Immunofluorescence technique is of great significance in virology examination, because the virus can not be seen by ordinary optical microscope, but the virus and its propagator can be detected by fluorescent antibody staining.
Indirect fluorescent antibody staining is widely used in the diagnosis of parasitic infection. Indirect immunofluorescence test (IFAT) is currently recognized as the most effective method to detect malaria antibodies. The common antigen is erythroblastoid antigen in the blood of malaria patients. IFAT also has a high diagnostic value for extraintestinal amoeba, especially for amebic liver abscess. The antigen used is amoeba culture suspension or extracted soluble antigen.
Immunofluorescence is also a good tool to detect autoantibodies, which is widely used in the experimental diagnosis of autoimmune diseases. Its outstanding advantage is that it can simultaneously detect antibodies and tissue components that react specifically with antibodies in a simple way, and can simultaneously detect antibodies against different tissue components in the same tissue. There are mainly anti-nuclear antibodies, anti-smooth muscle antibodies and anti-mitochondrial antibodies. Mouse liver is the most commonly used nuclear antigen in the detection of antinuclear antibodies, which can be made into frozen sections, prints or homogenates. Anti-centromere antibody and anti-neutrophil plasma antibody can also be detected by tissue culture cells such as Hep2 cells or Hela cells smear. Other autoantibodies that can be detected by immunofluorescence technique include anti-parietal cell antibody, anti-double-stranded DNA antibody, anti-thyroglobulin antibody, anti-thyroid microsome antibody, anti-bone marrow muscle antibody and anti-adrenal antibody.