凝集與沉淀教學課件

1、Exp.1 Agglutination and PrecipitationExp.1 Agglutination and Precipitation Exp. 1.1 Direct agglutination reaction - slide agglutination test Exp. 1.2 Indirect Agglutination Reaction -Latex Agglutination Test For Detection of RF Exp. 1.3 Countercurrent Electrophoresis Test Exp. 1.1 Direct agglutinati

2、on reaction - slide agglutination test Exp. 1.2 Indirect Agglutination Reaction -Latex Agglutination Test For Detection of RF Exp. 1.3 Countercurrent Electrophoresis Test Gannan Medical UniversityExp. 1.1 Direct agglutination reaction - slide agglutination testPrinciple Agglutination is the interact

3、ion of insoluble particles (e.g. intact bacteria and cells) and specific antibodies to these antigens that results in some visible agglutinates after certain time and under certain concentration of electrolyte. Slide agglutination test is the direct agglutination carried out on the slides. Directly

4、mix an antibody with a particle antigen under the certain concentration of electrolyte. The result is positive when there is visible agglutinate, otherwise it is negative. Application Slide agglutination is a qualitative test. It may be used mostly to determine blood type (ABO types) or bacterial in

5、fection. This experiment is to determine undetected bacteria by the known anti-bacterial antibody. Material 1. Antigen: undetected bacteria 2. Antibody: the anti-Salmonella antibody (antibody 1); the anti-Escherichia coli antibody (antibody 2) 3. Saline (0.9%NaCl); slide, etc.Exp. 1.1 Direct aggluti

6、nation reaction - slide agglutination test123+-Procedures1. Label the clean slide as “1”, “2” and “3”. 2. Drop the anti-Salmonella antibody (1drop) on the slide “1”. Drop the anti-Escherichia coli antibody (1drop) on the slide “2”. Drop the Saline (1drop) on the slide “3”. 3. Drop the undetected bac

7、teria (1drop) on the slide “1”, “2” and “3” respectively. Mix it well by the toothpick respectively.4. Shake the slide gently and observe the results. Results Positive result appears agglutination of white particle; Negative result keeps turbid. Record the results and discard the slide into the beak

8、er containing saponated cresol solution after the experiment.Exp. 1.1 Direct agglutination reaction - slide agglutination testExp. 1.2 Indirect Agglutination ReactionLatex Agglutination Test For Detection of RF Rheumatoid factor (RF) is a group of abnormal Igs in the sera from patients of rheumatoid

9、 arthritis. Their antibody activity is directed against antigenic sites in the Fc region of human IgG. The principle of the test is to coat latex particle with human IgG, if there is agglutination after mixed with the test sera, that indicates presence of RF. PrincipleDenaturated IgG B cells anti-de

10、naturated-IgG IgMApplication Indirect agglutination test is used to detect specific antibody for the soluble antigen. This assay here is to detect RF in the human sera using known human IgG sensitized latex particle.Material 1. Human IgG sensitized latex particle 2. RF positive control sera, the Sal

11、ine 3. test sera from patient 4. Glass slideExp. 1.2 Indirect Agglutination ReactionLatex Agglutination Test For Detection of RF 123+-Procedures1. Label the clean slide as “1”, “2” and “3”. 2. Drop the RF positive control sera (1drop) on the slide “1”. Drop the test sera from patient (1drop) on the

12、slide “2”. Drop the Saline (1drop) on the slide “3”.3. Add human IgG sensitized latex particles into the above sera respectively. Mix it well by the toothpick respectively.4. Shake the slide gently and observe the results. 2021/7/9119、 人的價值，在招收誘惑的一瞬間被決定。2022-3-22022-3-2Wednesday, March 02, 202210、低頭

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14、。2022年3月2022-3-22022-3-22022-3-23/2/202216、業(yè)余生活要有意義，不要越軌。2022-3-22022-3-2March 2, 202217、一個人即使已登上頂峰，也仍要自強不息。2022-3-22022-3-22022-3-22022-3-2Results Positive: should exhibit strong agglutination, which will appear as white clumps in a white suspension against a blank background. Negative: should not

15、which will appear as a white homogeneous suspension against a blank background. Exp. 1.2 Indirect Agglutination ReactionLatex Agglutination Test For Detection of RF Principle Countercurrent electrophoresis is performed in the agar gels where the PH is chosen so that the antibody is positively charge

16、d and the antigen being tested is negatively charged. By applying a voltage across the gel, the antigen and antibody move towards each other and precipitate. Antibody is usually put at anode (+) side, while antigen is put at cathode (-) side. The antibodies (IgGs) usually carry low negative charge a

17、t the pH 8.6 of electrophoresis buffer since the values of their pIs is usually at 6-7. The molecular weight of the antibodies is high. So the electrophoresis mobility of the antibodies is lower than its electro-osmotic mobility. While antigens usually carry high negative charges at the pH 8.6 of el

18、ectrophoresis buffer since the values of their pIs are usually at 4-5. Moreover, the molecular weight of the antigens is small. So the electrophoresis mobility of the antigens is larger than its electro-osmotic mobility. Exp. 1.3 Countercurrent Electrophoresis Test AgAb-+Application Its mostly used

19、to analyze the characteristics of antigen or antibody, to check the purity of antigen and antibody, and to measure the titer of antibody.Material 1. Antigen (Ag): unknown human serum, AFP (alpha fetoprotein) positive human serum 2. Antibody (Ab): AFP diagnostic serum (anti-AFP) 3. Barbital buffer (0

20、.05mol/L, PH 8.6), 0.4% barbital agar, 1.5% barbital agar 4. Others: electrophoresis apparatus, slide, etcExp. 1.3 Countercurrent Electrophoresis Test Procedures 1. Mark the agar plate 1234+-2. Add samples: add 10l unknown human serum to the well “1” at cathode (-) side with micropipette. Add 10l AFP positive human serum, e.g. hepatitis positive patient serum, to the well “2” as control well at cathode (-) side with micropipette. Add 10l AFP diagnostic serum to the wells “3”