Counterimmunoelectrophoresis (CIE): Principle, Procedure, Uses

By Acharya Tankeshwar •  Updated: 06/23/21 •  2 min read

Counterimmunoelectrophoresis (CIE) is a modification of the Ouchterlony method that speeds up the migration of an antigen and antibody by applying an electrical current.

Most bacterial antigens are negatively charged in a slightly alkaline environment, whereas antibodies are neutral. When an electric field is applied in an electrophoresis apparatus filled with buffer (pH 8.4) and containing known antibodies and unknown antigens in the agarose well, the antibodies will migrate towards the negative end (cathode) whereas antigens will migrate towards the positive end (anode). 

As the antibody and antigen move towards each other in an electric field, they will soon meet in optimal proportion (zone of equivalence is formed) at some points between the well and visible precipitation occurs.


Salient features

  1. Modification of the Ouchterlony method.
  2. Unknown bacterial antigen and a known specific antibody move towards each other in an electrical field.
  3. Most bacterial antigens are negatively charged  (exceptions Streptococcus pneumoniae serotypes 7 and 14) in a slightly alkaline environment.
  4. When Ag and Ab meet in optimal proportions, a line of precipitation appears within the agar matrix.
  5. The entire procedure takes about one hour.
  6. Any antigen for which antisera is available can be tested by CIE
  7. Sensitivity less than that of particle agglutination (0.01-0.05 mg/ml) of antigen.

Uses of Counterimmunoelectrophoresis

Counterimmunoelectrophoresis is used to detect presence of capsular antigens in CSF of

Demerits of the test:

  1. CIE is more expensive than agglutination-based tests.
    • Initial capital outlay for the apparatus
    • Need of large quantity of Ag and Ab

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please email at

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36 responses to “TPHA: Principle, Procedure, Results, Interpretations”

  1. Rahul Shah says:

    Hello Sir
    My TPHA is Negative & VDRL is reactive . can u explain about it.

  2. Jakisa Emmanuel says:

    Great information well explained

  3. shameen says:

    can you plz tell about the specificity and sensitivity of tpha test?

  4. Tarek says:

    My TPHA Is Positive.. So How I Solv This Problem Now

  5. Exequiel Botardo says:

    My TPHA result is non reactive gray zone.COV 0.212 TITER 0.191HCV non reactive what is that means.what should I do?thanks

  6. Vasu says:

    Hi sir.. Recently ive tested for tpha titre after secondary infection, now reading shows vdrl negative and tpha positive titre 1:1280, even i got treated with penincilin 4 doses the tpha titre will go down or remain same? Why the tpha titre always remain same? Even after get treated?

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