1. Agglutination Reaction
Classical Ag & Ab Reaction
Agglutination Reaction
Lattice Formation 1

2. Introduction of agglutination reaction
Concept :
Granular Ag or Ag attached on granules can bind with corresponding Ab , followed with the formation of agglutinated clots , which is called agglutination reaction.
Particulate Ag
Corresponding Ab +
Visible agglomerate
Classification:
Agglutination-is the interaction 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.
Involve particulate antigens and antibodies
Antigens may be:
(1) on a cell surface (direct agglutination)
(2) attached to latex spheres (indirect or passive agglutination)
Direct agglutination reaction:
 Slide test  qualitative
 Tube test  semi-quantitative
Indirect agglutination reaction

3. Direct agglutination reaction
Particulate Ag
Corresponding Ab +
Visible agglomerate
Direct agglutination reaction
Soluble Ag +
Sensitized particulate
Corresponding Ab
Visible agglomerate
Indirect agglutination reaction

4. Ⅰ. Slide agglutination test
This test is designed to observe the direct agglutination reaction of known antibody and unknown particle antigen on slide.
The reaction is simple and rapid.
Usually it is used to detect qualitatively the antigen or antibody. Such as bacteria identify and ABO blood type assay.

5. ？ Purpose Test 1 Assay of blood type (ABO)
Determine the ABO Blood type of individual .
Anti-A serum OR
Anti-B serum ？ +

6. 2.Material Test 1 Assay of blood type (ABO)
Blood sample: Human peripheral blood
Standard Sera:
Anti-A serum
Anti-B serum
Slide
Aseptic needle
Cotton stick
Indophor

7. Procedure Test 1 Assay of blood type (ABO) a. Slide b. Pick blood
Anti-A
Anti-B
Disinfect the finger
b. Pick blood
Stab the finger
Take blood with two corners of slide. Mix with anti-A and anti-B respectively
c. Reaction
Rock the agglutination test slide for one to five minutes
d. Observation

8. Anticipated results Test 1 Assay of blood type (ABO) A B AB O
Positive result(+):clump
Negative result(-):turbid

9. Blood Type Result Blood type Serum A B AB O Anti-A serum + -
Anti-B serum

10. Test 1 Assay of blood type (ABO)
Attentions
Keep the serum fluid separated.
Disinfect finger before taking blood.
Do not confuse the two corners of slide.
Observe result in time.
Discard the slides into the glass can on the bench.
Discard the stick, needle into garbage bin.

11. Ⅱ Tube agglutination test
1.Principle
Tube agglutination test is usually used in the semi-quantitative test. Directly mix known particle Ag with serial dilution of patient’s serum in the tubes. Observe the appeared agglutinates in the tubes after certain time. Determine the level of the Ab in the serum and its titer according to its relative agglutination amount.

12. Dilute the examinee’s serial dilution serum consecutively
Dilute the examinee’s serial dilution serum consecutively. Mix it with antigen suspension of known concentration in test tube. Observe the agglutination phenomenon in certain reaction time.
In this experiment, typhoid bacterium of certain quantity acts as antigen. Whether serum contains corresponding antibody or not is up to the agglutination reaction to decide. Furthermore, we could decide the titer of antibody by the agglutination extent in different tubes. We could diagnose and tell patient condition and prognosis by this principle.

13. II Tube agglutination test
2. Purpose
Determine the titer of Anti- S. typhi H -Ab in serum.
3. Reagents & Instruments
(1) Ag: 1% S. typhi H antigen solution.
(2) Unknown serum : 1:10 diluted anti-H serum (inactivate complements at 56℃ for 30 min).
(3) Normal saline.
(4) Transfer pipette , incubator, test tube , test tube rack .

14. II Tube agglutination test
4. Procedures
4 persons/group
a. Array 7 tubes in the rack , and mark them in turn.
b. Add NS 0.5ml in each tube with 1ml pipette.
c. Add 0.5ml 1:10 diluted anti-H serum to the 1st tube by 1ml pipette. After sufficient mixing, transfer 0.5ml of this diluted serum to the 2nd tube.
Repeat dilution in the same way (serial twofold dilution of serum )until 6th tube, and discard 0.5ml from 6th tube. Leave the 7th tube (control tube) free from serum.
强调：
1.滴管的使用方法，每组发4支，其中2支为公用，用来取生理盐水和菌液，用完后插入相应试剂管中。
2.一定要混匀，用滴管混匀时，吸时不要用力过猛，以免将液体吸入橡皮头内，吹时不可将液体完全吹净，以免产生过多气泡，影响定量。
3.按顺序加样，不可颠倒
4.伤寒菌液尽量避免洒出。
5.各组记好时间
6.取出时，不可晃动试管，轻拿轻放。

15. II Tube agglutination test
NS（ml）  0.5        0.5     0.5
1:10 serum       0.5     0.5    —
(discard 0.5)
Dilution :20 1:40 1:80 1:160 1:320 1: —
d. Add 0.5ml S.typhi H antigen in each tube(from tube 1 to 7).

16. II Tube agglutination test
NS（ml）  0.5        0.5     0.5
1:10 serum       0.5     0.5    —
(discard 0.5)
dilution :20 1:40 1:80 1:160 1:320 1: —
S.typhi Ag  0.5        0.5     0.5
Final dilution 1:40 1:80 1: :320 1:640 1: —
e. Mix each tube and incubate at 37℃ for overnight. (observe the results at tomorrow ( :30)

17. II Tube agglutination test
5. Anticipated results
Supernatant
Sediment
Degree of agglutination
Clear/transparent
Large and depositing
++++
Slightly turbid
Large and depositing mostly
+++
Obviously turbid
Small and depositing partly ++
turbid
Very small agglutination +
turbid
(Same as control)
No agglutination -

18. Titer of the anti-serum
Sediment
Agglutination degree
The highest dilution ratio
Titer of the anti-serum
The titer is customarily reported as the reciprocal of the highest dilution that causes an agglutination(++) .
The highest dilution of serum causing obvious agglutination of bacteria(++) is defined as the titer of the serum.

19. II Tube agglutination test
6. Attentions
Mark the tube in turn .
Use the graduated pipette correctly.
Mix thoroughly.
Take the tube gently , in case agglutination would disperse.
Observe the control tube to decide the experiment credibility . Then observe the experiment tubes.