Techniques in HIV Research

MT-2 Dye Assay:  (Cell Proliferation Kit)

10 ul to each well with MTT Labeling Reagent

Incubate at 37.5 C for 4 hours

100 ul

Replica Platting:

100 ul of sample to replica plate

Add 15 ul of ____________________ and incubate 37C for 1 hour

put plate at -80

p24 Antigen Capture Assay

A. Lyse sample by adding 1/10 volume of lysing solution to sample. Mix well!

B. Equilibrate all reagents to room temp before use (about 30 min).

• Lysing solution: 10% Triton X-100 (Sigma)

• Wash buffer: 20X wash solution concentrate (KPL cat: 50-63-01), dilute to 1x with milli-Q H2O prior to use

• Sample diluent: 1% BSA, 0.2% Tween 20 in PMS (To make 100 ml: (1) 10 ml 10% BSA diluent (KPL cat# 50-61-01) (2) 10 ml 10X PBS (Dulbecco's without Ca and Mg) (3) 0.2 ml Tween 20 (Sigma Cat# P-1379) (79,8 ml milli-Q H2O (4) filter through a 0.45 micron filter.   Store at 4C

• Normal Mouse Serum (NMS) (Sigma Cat# M-5905)

• Primary antibody diluent: 2% NMS in Sample Diluent. For one plate: Dilute 0.55 ml NMS in 11 ml sample dilutent

• Normal Goat Serum (NGS)

• Secondary antibody dilution: 2% NMS, 5% NGS, 0.01% Tween in PBS. (to make 100 ml: Dilute 5 ml NGS, 2 ml NMS and 0.01 ml Tween 20 in 1X PBS. Filter Trhough a 0.45 micron filter) store at 4C

• Substrate: TMB Peroxidase substrate system

• stop solution (HCl)

C. Prepare serial 2-fold dilutions of standard using sample diluent:

Tube A-500 ul standard   

Tube B--25 ul from Tube A + 250 ul diluent                        Tube C - 250 ul from Tube B + 250 ul diluent

Tube D--250 ul from Tube C + 250 ul diluent                    Tube E - 250 ul from Tube D + 250 ul diluent

Tube F -- 250 ul from Tube E + 250 ul diluent

D. If necessary prepare serial dilutions of samples in sample diluent

E. Antigen incubation: 1. Wash plate in washer by running 2 5 cycle washes and turning plate 180 degrees between cycles. Tap dry on paper towel. 

2. Pipette 100 ul of standards and samples into the plate as follows:

A1 & A2:  Standard Tube A        B1 & B2:  Standard Tube B        C1 & C2:  Standard Tube C    D1 & D2:  Standard Tube D

E1 & E2:  Standard Tube E        F1 & F2:  Standard Tube F        G1 & G2:  (negative control: Sample Diluent)

H1 & H2:  Leave Blank        A3-H12:  Samples

3. seal plate and incubate at 37 C for 2 hours

F. Wash plate as above

G. Primary Antibody Incubation: 1. dilute primary Ab in Primary Ab diluent . dilute 8 ul in 10.4 ml Primary Ab diluent per plate just prior to sue (use at 1:1,300)

[The primary antibody is Rabbit anti-HIV-1 p24 serum, lot No: SP451B and volume 100 ml]

2. Pipette 100 ul into all wells Except the blank wells

3. seal plate and incubate at 37C for one hour

H. Wash plate as above

I. Secondary Antibody incubation: 1. Dilute Secondary Ab in secondary Ab diluent. Dilute 30 ul in 12 ml secondary Ab diluent per plate just prior to use (use at 1:400)

[Secondary antibody is Goat anti-Rabbit IgG peroxidase labeled antibody)]

J. Wash plate as above

Substrate Incubation: This may be prepared previously.

[Take steril flash and draw line up to the 100 ul on it and add Nonopure water which has been filtered (use filter under hood, filter water twice the second time hook filter up to vacuum to pull water through)

(2) add one tablet phosphate-citrate (need to open protective capsule and pour in)

(3) shake flask with hands until dissolved

(4) Pour 20 ml of that solution into fat conical tube and add substrate tablet (tetramethylbenzidine).

(5) pour solution into steril trough. using multi-pipette dispense 100 ul into all wells. Cover plate from light and incubate for about 10-20 min until a blue color appears which incidates substrate reacting with the peroxidate enzyme conjugate)]

Do not wash plate! Stop reaction by adding 100 ul HCL to all wells. Read plate at 450 nm as you would with regular ELISA.