Diagnosa Lab.MikrobiologiPenyakit Demam Berdarah dengue (DHF)

TIKMAMPU MENJELASKAN CARA MENEGAKKAN DIAGNOSIS PENYAKIT DHFMAMPU MENJELASKAN CARA PEMERIKSAAN DHF DENGAN CARA SEROLOGI TESMAMPU MENGINTERPRETASIKAN HASIL PEMERIKSAAN SEROLOGIS TES DENGAN TEKNIK ELISA (DENGUE RAPID TEST)

Penyebab DHFVirus dengueGenus Flavivirus4 serotipe: Den-1, Den-2, Den-3 dan Den-4Punya 3 protein struktural: envelope, premembran, core dan 7 protein nonstruktural

PenyebaranNyamuk aedes aegypti: adesi virus netralisasi gangguan pembekuan darah kebocoran pembuluh darah

Diagnosis Dengue virusMikroskopik: mikroskop elektronMakroskopik (in vivo)Serologis tes (elisa, PCR, aglutinasi, presipitasi dll)Dengue IgG/IgM Test

C T1 T2 S1 tetes serum + 2 tetes buffer

Interpretasi hasil:C positif: kit yg digunakan validT1 positif: IgM thd antigen envelope positif T2 positif: IgG thd antigen envelope positifTi dan T2 negatif: tdk ada antibodi thd antigen envelopeC, T1 positif, T2 negatif: apa artinya?C, T2 positif, T1 negatif: apa artinya? C, T1, T2 positif: apa artinya?

Langkah2 ELISASampel yang diukurKit (ready for use) Titrasi antigen dan antibodi (optimasi konsentrasi/ checkerboard) kurva standardCoating, pelapisan pada media (mis: microplate 96 wells)Coating solutionBlocking, menghindari reaksi non spesifikBlocking solutionWashing, setiap langkahWashing solutionAntibodi pertamaAntibodi kedua yang telah dilabel/melabel dengan enzimSubstratSubstrat solutionStop reagent: 1N NaOH

The antigen is added to the well and attaches itself to the bottom and sides of the well which is coated with a special substance. Incubated at 40C overnight or 370C for 1 hrAny excess antigen is washed out the antigen which is adsorbed onto the well surface stays in the well. Detergent is used for the wash.Stage One

Stage TwoNeutralisation - a milk protein is added to fill the gaps on the well. It is then incubated for an hour at room temperature. Incubated at RT for 1-11/2 hrAny excess milk protein is washed out using a detergent

Stage ThreeAntibody is added and attaches only to the antigen, not to the neutral milk protein. Not all the sites get taken up and it important that there is more antigen than antibody. This is because it is the level of antibodies that is being measured. All the antibodies in the sample must be attached to an antigen to get an accurate measurement. At this point effectively the antibodies are invisible. Incubated at RT for 1 hrAny excess antibody is washed out, leaving only the antibodies attached to the antigens.

Conjugate is added and binds to the antibodies. At this point there is a biological sandwich.Stage FourThe conjugate contains an enzyme which indicates the presence of the antibody. Incubated at RT for 1 hrAny excess conjugate is washed out leaving the enzyme markers attached to the antibodies.

Stage Five Substrate is added. This part is a chemical reaction. the substrate reacts with the conjugate and forms a colour which you can see. By using a colourimeter and measuring the optical density of the solution, the technician is able to determine the number of antibody sites available. Incubated at RT for 30.A substrate reacts with the conjugate to form a colour

Stage Six The reaction is stopped with sulfuric acid, nitric acid. Acid stops the activity of the enzyme. The timing of this step is critical. If you leave one reaction longer than another, the colour may be more intense because the reaction has had more time to proceed. The process is useful only if each step and concentration of reactants is standardized.

Pemeriksaan IgM, IgGDengue stick IgM, IgG

DOT BLOTT

Reaksi serologisSemikuantitatifDeteksi spesifitas reaksi antigen dan antibodiDapat menentukan imunogenisitas antigenDapat membedakan tinggi rendahnya antibodi yang direaksikan

Membran NC + Ag + Abs primerMembran NC + Ag + Abs primer + Abs sekunderMembran NC + Ag + Abs primer + Abs sekunder + substratAda noda: +Tak ada noda: -Washing sol Washing sol Stop