Bio-rad 680 User Manual

Model 680

Microplate Reader

Instruction Manual

Catalog Numbers	168-1000
	168-1001
	168-1002
	168-1003

Warranty Statement

This warranty may vary outside of the continental United States. Contact your local Bio-Rad office for exact terms of your warranty.

Bio-Rad Laboratories warrants to the customer that the Model 680 Microplate Reader (catalog number 168-1000, 168-1001, 168-1002, and 168-1003) will be free from defects in material and workmanship, and will meet all performance specification for period of one year from the date of shipment. This warranty covers all parts and labor.

In the event that the instrument must be returned to the factory for repair under warranty, the instrument must be packed for return in original packaging.

Bio-Rad shall not be liable for any incidental, special, or consequential loss, damage, or expense directly or indirectly arising from the use of the Model 680 Microplate Reader.

Bio-Rad makes no warranty whatsoever in regard to products or parts furnished by third parties, such being subject to the warranty of their respective manufacturers. Service under this warranty shall be requested by contacting your nearest Bio-Rad office.

The following items are considered Customer-installable consumables: thermal printer paper and light bulbs. These parts are not covered by this warranty. All customer-installed parts are warranted only to be free from defects in workmanship.

This warranty does not extend to any instrument or parts thereof that have been subject to misuse, neglect, or accident, or that have been modified by anyone other than Bio-Rad or that have been used in violation of Bio-Rad instructions.

The foregoing obligation are in lieu of all other obligations and liabilities including negligence and all warranties, of merchantability, fitness for a particular purpose or otherwise, expressed or implied in fact or by law, and state Bio-Rad’s entire and exclusive liability and buyer’s exclusive remedy for any claims or damages in connection with the furnishing of goods or parts, their design, suitability for use, installation or operation. Bio-Rad will in no event be liable for any special, incidental or consequential damages whatsoever, and Bio-Rad’s liability under no circumstances will exceed the contract price for the goods for which liability is claimed.

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Regulatory Notices

Electro Magnetic Compatibility:

This instrument is designed to meet the Class-A emissions and immunity levels of the EN61326 product family standard for the CE mark. This rating requires that it only be used in a laboratory environment.

Note: This instrument has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his/her own expense.

Safety:

This instrument is designed to meet EN61010-1 requirements for the CE mark, which are internationally accepted safety standards for electrical equipment for measurement, control and laboratory use. EN61010-1 applies to equipment which is used under the following conditions;

Indoor use

Altitude up to 2,000 m

MAINS supply voltage fluctuations up to ±10%

This instrument has been tested, and its compliance has been confirmed under the following environmental conditions;

Overvoltage category II Pollution degree 2

If the instrument is used in a manner not specified in this manual, the protection provided by the instrument may be impaired. This instrument should not be modified or altered in any way. Alteration of this instrument will result in the following:

Void the manufacture’s warranty. Void regulatory compliance.

Create a potential safety hazard.

Protective Ground: This instrument has detachable, three-wire power cord for connection to both the power source and protective ground. The protective ground contact is connected to the accessible metal parts of the instrument. To prevent electrical shock, always use a power source outlet that has a properly grounded protective-ground contact.

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Table of Contents

Section 1. ........		Introduction ................................................................................................	1
1.1	Description and accessories for Model 680 Microplate Reader ..........................		2
Section 2. ........		Product Description .....................................................................................	3
2.1	Contents of Shipping Carton ..........................................................................		3
2.2	External Features..........................................................................................		4
2.3	Membrane Keypad ........................................................................................		5
Section 3. ........		Instrument Set-up ........................................................................................	6
3.1	Initial Start-up................................................................................................		6
Section 4. ........		Operation ...................................................................................................	7
4.1	Instrument Overview .....................................................................................		7
4.2	Functions Overview.....................................................................................		10
4.2.1		Calculations .....................................................................................................	10
4.2.2		Security password ..........................................................................................	10
4.2.3		Memory Back-up.............................................................................................	10
4.2.4		Limits.................................................................................................................	11
4.2.5		Reports.............................................................................................................	11
4.2.6		RS-232C Interface..........................................................................................	16
4.2.7		Printer ...............................................................................................................	17
4.3	Detailed Operation ......................................................................................		18
4.3.1.		Initial Start-up ..................................................................................................	18
4.3.2.		Configuring the system ..................................................................................	19
4.3.3.		Printing Reports and Protocol information..................................................	21
4.3.4.		Memory Recall operation...............................................................................	22
4.3.5.		Editing Kinetic protocol ..................................................................................	23
4.3.6.		Checkmark value setting (by Administrator only) ......................................	25
4.3.7.		Editing End Point Protocol ............................................................................	26
4.3.8.		Cutoff setting procedure ................................................................................	27
4.3.9.		Standard setting procedure...........................................................................	31
4.3.10.		Mode setting procedure.................................................................................	34
4.3.11.		Plate Map setting procedure.........................................................................	36
4.4	Quick Guide – Reading a plate .....................................................................		39
4.5	Language choice.........................................................................................		40
Section 5. ........		Instrument service by the user.....................................................................	42
5.1	Installing Interference Filters ........................................................................		42
5.2	Changing the Lamp .....................................................................................		42
Section 6. ........		Troubleshooting and Error Messages...........................................................	43
Section 7. ........		Specifications ...........................................................................................	45
7.1	Instrument Specifications .............................................................................		45
7.2	Interface Specifications................................................................................		47
7.3	Command Language for Model 680 Microplate Reader ..................................		48
7.3.1		Command Language for Remote control mode ........................................	48
7.3.2		Error Codes .....................................................................................................	63

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Section 1. Introduction

The Model 680 Microplate Reader is an eight-channel, vertical pathlength photometer that measures the absorbance of the contents in the wells of a 96-well microtitration plates. It can perform single or dual wavelength measurements and can report absorbance values to three decimal places.

The Model 680 reader can be programmed by entering commands through the membrane keypad to define plate reading and data analysis parameters as testing protocols and select report types. Hard copy reports can be produced by an optional on-board thermal printer or external printer.

The Model 680 reader can also be controlled by the Windows’ based Microplate Manager software (catalog number 170-9520) program, through its built-in RS232C serial interface device. Microplate Manager software program provides a complete, user-friendly package for protocol programming as well as data analysis, for all absorbance microplate readers from Bio-Rad Laboratories.

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1.1Description and accessories for Model 680 Microplate Reader

Catalog Number	Description
168-1000	Model 680 microplate reader
168-1001	Model 680 microplate reader with temperature control
168-1002	Model 680 microplate reader with internal printer
168-1003	Model 680 microplate reader with temperature control and internal
	printer
168-1011	405 nm filter
168-1013	415 nm filter
168-1020	450 nm filter
168-1028	490 nm filter
168-1038	540 nm filter
168-1040	550 nm filter
168-1044	570 nm filter
168-1049	595 nm filter
168-1056	630 nm filter
168-1061	655 nm filter
168-1080	750 nm filter
168-1005	Serial PC Cable
168-1006	Replacement lamp for Model 680 microplate reader
168-1007	Printer paper, 2 rolls

Custom filters between 400 and 750 nm may be ordered, corresponding catalog numbers can be found on www.bio-rad.com, or you can order by specifying the wavelength and the model number of the reader.

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Section 2. Product Description

2.1Contents of Shipping Carton

The shipping carton contains the following items:

•Model 680 Microplate Reader, with 415nm, 450nm, 490nm and 655nm interference filters installed on the filter wheel.

•Power cord

•Serial PC Cable

•Spare fuse

•Dust cover

•Instruction manual

•Warranty card

•One roll of thermal printer paper (for units equipped with internal printer)

Inspect the exterior of the instrument for any signs of shipping damage. Contact your local Bio-Rad representative if any of these items are damaged or missing.

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2.2External Features

1

4

5

2

3

6

1. Release latch for rear cover		4. Rear cover
2.	Liquid crystal display (LCD)	5.	Printer slot (when optional built-in printer is installed)
3.	Membrane keypad	6.	Reading chamber door

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8

14

11

10

		9
				12		13
7.	RS-232C serial interface	11. Printer port (only when optional built-in printer is not installed)
8.	Serial number Label	12.		Power cord receptacle
9.	Fuse	13.		Cooling Fan
10. Power switch		14.		Cooling Vents

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2.3Membrane Keypad

1

2

3

4

5

				Memory	E dit
	Sta rt/ Sto p	Main		Recall
				P aper	P rin t
				Feed
			7	8	9
			CP	CN	CW
	A Z		4	5	6
			CO	Q C	CAL
	Back	Change	1	2	3
			SMP	BLK	S TD
	Z A			.
			0
			EMP	FUNC

13

12

11

10

				6	7
							8		9
1. Main		Returns to MAIN SCREEN.
2.	Start/Stop	Initiates plate reading using current active protocol.
		Stops plate reading and printing.
3.	Paper Feed	Advances paper strip in internal printer.
4. Up Arrow		Moves the cursor upward.				Selects the alphabet or the symbol.
5.	Left Arrow	Returns to the previous screen. Moves the cursor to the left.
6. Down Arrow		Moves the cursor downward.					Selects the alphabet or the symbol.
7.	Right Arrow	Moves the cursor to the right.					Changes or selects the value and type.
8.	Dot/Function	Inputs the dot.			Changes the input mode.
9.	Enter	Completes or seals a field entry.
10. Ten Keys		Inputs numbers or well type in plate mapping.
		0	/ EMP : Empty			5 / QC			: QC control
		1	/ SMP : Sample			6 / CAL			: Calibrator
		2	/ BLK	: Blank		7 / CP			: Positive Control
		3	/ STD	: Standard		8 / CN			: Negative Control
		4	/ CO	: Cutoff control		9 / CW			: Weak Positive Control
11.	Print	Prints out the plate data and protocol information.
12. Edit		Enter the Edit Menu, and set up the instrument.
13. Memory Recall		Read out protocol and plate data.

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Section 3. Instrument Set-up

3.1Initial Start-up

1Place the instrument on a clean, sturdy table or bench. It is important to keep the instrument in a clean, relatively dust free environment to ensure optimal performance.

2Connect the power cord to the back of the instrument. Before connecting the instrument to the main electrical supply, check that the AC voltage is appropriate for the instrument.

3Turn on the power switch on the rear panel. The LCD will display the version number of the on-board firmware. After about three seconds, the instrument will perform an initial self-diagnosis that requires about fifteen seconds. The Login Screen will now be displayed for you to choose your user level (administrator or common user), and enter the password (Initial password: 00000). Please allow three minutes for the instrument to warm up (reach thermal equilibrium) before reading plates.

4Install the printer paper (if the optional internal printer is installed). The thermal printer paper is treated on one side only and must be properly installed for the printer to function. The side of the paper that faces out from the roll is the treated side.

a.Open the rear cover of the instrument.

b.Tear off a small piece at the beginning of a new roll at an angle to form a point.

c.Place the roll of paper in the small pan-like holder positioned below the printer.

The roll should be positioned so that it will feed from the bottom.

d.While pressing the PAPER FEED key, feed the pointed end of the paper into the paper feed slot in the underside of the printer until the printer grabs the paper and feeds it through the slot in the top of the instrument.

e.Securely close the rear compartment.

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Section 4. Operation

4.1Instrument Overview

The Model 680 Microplate Reader has built-in software that allows the user to set the plate reading and data analysis conditions as a protocol data, read a microplate under the defined reading conditions, and get the printout reports from the internal or external printer. The software communicates through the 4-line, 20-character LCD and is controlled through the instrument's membrane keypad.

The display shows the current mode of the on-board software. The software has three different protocol types: End-point analysis, Kinetic analysis, and the Checkmark validation protocols. The user presses the Memory Recall key to select analysis mode passing through the choice of the protocol or the collected data.

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Operation flow when End point protocol selected

Not Use

Edit

Constant

Ranged Cutoff

Cutoff Values setting

Single Cutoff

Gray zone setting when single Cutoff

Control

Ranged Cutoff

Cutoff Control setting

Cutoff

Single Cutoff

Gray zone setting when single Cutoff

Formula

12 choices of

Coefficient setting of selected formula

cutoff formula

0 - 9999

Ratio

Concentration

17 choices of unit

Unit

Cutoff value

Ranged

Cutoff Values setting

Cutoff

Gray zone setting when single Cutoff

Single

Cutoff

Report

Raw(Raw data), Abs(Absorbance), Lim(Limit), Mtx(Matrix), Cut(Cutoff)

Crv(Curve fit), Cnc(Concentration), Dif(Difference)

Upper Limit

Up to 12 Standards

Protocol

Limit

Number of STD

Lower Limit

Concentration

0 to 9999

For Limit Report

Unit

17 choices

12 choices of curve fit type

Standard information

Curvefit type

Graph axis

4 choices of graph axis

STDs

Curvefit

combination of Log and Linear

Recall STD Curve

Recall Setting

Recall Yes/No Selection

Select STD Curve

5 choices of memorized STD

curve

Single/Dual Wavelength

Set Photo mode

Mode

Set Shaking

Shake (Yes, No), Speed (High, Low, Mid), Time

Set Read mode

Read Speed (Fast//Step) and Read Mode (Normal/Eval)

Incubation

Disable/Enable, Temperature

.Func key to switch [N] to [F]

Manual mapping

Mapping

Automatic mapping

[N] for sample number, [F] for sample type

Blank,CN,CW,CP,CO,QC,Calibrator,Sample,Replicate well

Kit name

.Func key to switch from [ABC] to [abc] to [sym]

key to move cursor

For current user mode

Change password

Security

Change user

Administrator or Common user

Lock/Unlock

Administrator has power to switch

Filters

Filter wavelength setting for 1-8 positions, “0” for no filter

Date Set

Day/Month/Year, Hour:Min

Lab name

.Func key to switch from [ABC] to [abc] to [sym]

key to move cursor

5 choices of storage number

SaveSTDC

Save STD Curve

Rename STD Curve

5 choices of STD

.Func key to switch from [ABC] to [abc] to [sym]

curve

key to move cursor

Printer

Select Ext.Printer

ASCII / ESC/P Printer Selection for external printer

ASCII / ESC/P

Italic for program pages

Bold

for menu

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Operation flow when Kinetic protocol selected

Edit

Protocol

Security

Filters

Date Set

Lab Name

SaveSTDC

Printer

			Reading		Number of reads (2-30), Start delay time (0-999)
			Inter		Reading interval time
Mode			Shaking		Shake (First/Every/disable), Speed (High, Low, Mid), Time
			Ph.mode		Single / Dual Wavelength
			Speed		Read Speed (Fast/Step)
			Incu		Disable/Enable Temperature
			CC Limit (correlation coefficient limit)							GALT factor k input (0- 9999)
Calc			Calc.range (calculation range of reading number)							Result = (2nd read – 1st read)*k
			for Linear regression Report							for GALT Report
									.Func key to switch [N] to [F]
Mapping			Manual mapping
			Automatic mapping						[N] for sample number, [F] for sample type
									Blank,CN,CW,CP,CO,QC,Calibrator,Sample,Replicate well
Assay			General Kinetic or GALT assay
			assay type selection
Report			Absorbance, KINETIC Plots, Linear regression when General Kinetic Assay selected
			GALT report when GALT Kinetic assay selected
Kit name									.Func key to switch from [ABC] to [abc] to [sym]
									key to move cursor

Change password For current user mode

Change user Administrator or Common user

Lock/Unlock Administrator has power to switch

			Filter wavelength setting for 1-8 positions, “0” for no filter
			Day/Month/Year, Hour:Min
			.Func key to switch from [ABC] to [abc] to [sym]
			key to move cursor
		5 choices of storage number
	Save STD Curve
	Rename STD Curve	5 choices of STD		.Func key to switch from [ABC] to [abc] to [sym]
		curve		key to move cursor
	Select Ext.Printer	ASCII / ESC/P Printer Selection for external printer
	ASCII / ESC/P

Special Buttons

Memory	Protocol	End point	1-64 End point protocol storage
Recall	Plate Data	Kinetic	1-2 Kinetic protocol storage
		Checkmark	Checkmark reader test
Print	Report		Printout
	Protocol
Main			Returns to MAIN screen

Bold for menu Italic for program pages

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4.2Functions Overview

4.2.1Calculations

The Microplate Reader uses Beer's Law to calculate the absorbance value of each well. Beer's Law states that absorbance is equal to the log10 of the ratio of the baseline measurement intensity (Io) to the sample measurement intensity (I).

Beer's Law

Absorbance = Log10 (Io/I)

Before measuring the plate, the reader takes a reading for all eight photodiode channels. These values are recorded as the baseline measurement (Io) values for each channel, respectively. The reader then records the sample measurement (I) value for each well, and calculates the absorbance using these values. Channel-to-channel error is significantly reduced because the Io value for a given channel is used only in determining the absorbance of the wells of that channel.

4.2.2Security password

The Model 680 Microplate Reader requires user login with password. This security feature helps to prevent any unwarranted modifications to the analysis conditions/protocols and data stored in the instrument, and identifies operator on reports. The reader has two user settings, the Administrator and the Common user. Each user level has its own security password.

4.2.3Memory Back-up

The battery back-up provides memory even after the reader is turned off.

The following information will be saved in the memory until new reading parameters are set by the user or a new plate is read by the instrument. Note that if a run is aborted before it is finished, all the previous plate reading data remain in memory and none of the data from the aborted reading is stored.

1.10 latest plate reading result for End-point protocol

2.2 latest sets of Kinetic plate reading result (Max 30 series of readings for each

Kinetic)

3.64 End-point Protocol settings and 2 Kinetic Protocol settings

4.A set of Checkmark reference data and one latest set of Checkmark reading result

5.5 set of Standard curve data for the storage of standard curves and graphs

6.8 wavelength values for filters

7.Laboratory name

8.Two security passwords (for the Administrator and the Common user)

9.External printer type (ASCII or ESC/P type)

In addition to the battery back-up memory, the instrument has a small amount of none volatile memory to keep the system information after the battery is dead. The Serial Number is stored

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in this memory.

The first time the instrument is turned on, or after a battery failure, the following default information will be held in the memory.

1.Both of Administrator and Common user security password are set to "00000".

2.Laboratory name is "Bio-Rad Laboratories".

3.All wavelength values of the filters are set to "---" nm.

4.The date and time of the calendar chip are set to 1 day, 1 month, 00 year, 0 hour and 0 minute.

5.None of plate reading data is available.

6.End-point protocol is chosen and the protocol number is set to #1.

7.None of Standard curve data for the storage of standard curves and graphs is available.

8.External printer type is set to ASCII type.

4.2.4Limits

The Microplate Reader displays absorbance readings with absolute values as 3.500.

Out-of-range absorbance values, i.e. those with absolute values greater than 3.500, are displayed as either "*.***" or "-*.***". For example, if the absorbance is 4.500, then the display will read "*.***", and if the absorbance is -4.500, the display will read "-*.***".

4.2.5Reports

The Microplate Reader can generate nine types of reports for End-point protocol: Raw data,

Absorbance, Limit, Matrix, Cutoff, Curve fit, Concentration, Difference, and TeSeE Screening report. And four types of Kinetic protocol can be generated by the reader: Absorbance, Kinetic Plots, Linear regression, and GALT report.

Reports of End-point Protocol

(1)Raw data report

The Raw data report is the uncorrected absorbance values (without blank subtraction). In single-wavelength mode, the reported value is the measured absorbance. In dual-wavelength mode, the reported value is the difference between the uncorrected readings taken with the measurement filter and with the reference filter.

(2)Absorbance report

The Absorbance report is the blank-corrected absorbance values. The mean absorbance value of all of the wells designated as assay blanks is calculated and then subtracted from all 96 values of the raw data set to produce the Absorbance report.

Abs = Raw – Blank mean

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Blank mean = X/n

S.D. = [{X^2 – n*(Blank mean)^2}/{n-1}]^1/2 Where:

S.D. = Standard deviation

X = Sum total of the raw absorbance for each blank

X^2 = Sum total of the squared raw absorbance for each blank n = Number of blanks

(3)Limit report

The Limit report provides a qualitative YES/NO report. Wells with blank-subtracted absorbance values between the upper and lower limits are represented with an asterisk (*), wells with absorbance values below the lower limit by minus signs (-), and wells with absorbance values greater than the upper limit by positive signs (+).

(4)Matrix report

The Matrix report provides a qualitative report of the relative magnitude of the absorbance values on the plate. The absorbance range defined by the upper and lower limits is divided into 10 equal partitions, numbered 0 through 9. The blank-subtracted absorbance value of each well is classified according to the partition of the matrix to which it corresponds, and is reported as a signal digit. Wells with absorbance values greater than the upper limit are reported by plus signs (+), and wells with absorbance values less than the lower limit by minus signs (-).

(5)Cutoff report

The Cutoff report provides a qualitative report of the relative magnitude of the absorbance values or converted concentrations on the plate.

Four types of cutoff report are supported as listed below.

a. Cutoff Constant

Ranged Cutoff Constant

The user inputs Positive and Negative values to be used as the cutoff.

For units designated "Abs", if the absorbance of a well is within the positive and negative cutoff values, the well is scored "*". If the absorbance of a well is greater than the positive value, the well is scored "+", and if the absorbance of a well is below the negative value, the well is scored "-".

For units not designated "Abs", the absorbance value of each well is converted to the concentration value using the curve fit constants of the curve fit report. If the concentration of a well is within the positive and negative cutoff values, the well is scored "*". If the concentration of a well is greater than the positive value, the well is scored "+", and if the concentration of a well is below the negative value, the well is scored "-".

Single Cutoff Constant

The user inputs Positive and Gray zone values to be used as the cutoff.

For units designated "Abs", if the absorbance of a well is within the gray zone value of the positive cutoff value, the well is scored "*". If the absorbance of a well is more than the gray zone value and greater than the positive value, the well is scored "+", and if the absorbance of a well is more than the gray zone value below the positive value, the well is scored "-".

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Upper cutoff absorbance = Positive absorbance + ((Gray zone/100) * Positive absorbance) Lower cutoff absorbance = Positive absorbance – ((Gray zone/100) * Positive absorbance)

For units not designated "Abs", the absorbance value of each well is converted to the concentration value using the curve fit constants of the curve fit report. If the concentration of a well is within the gray zone value of the positive cutoff value, the well is scored "*". If the concentration of a well is more than the gray zone value greater than the positive value, the well is scored "+", and if the concentration of a well is more than the gray zone value below the positive value, the well is scored "-".

Upper cutoff conc. = Conc. of positive controls + ((Gray zone/100) * Conc. of positive controls) Lower cutoff conc. = Conc. of positive controls - ((Gray zone/100) * Conc. of positive controls)

b. Cutoff Control Ranged Cutoff Control

The mean absorbance values of the Positive and Negative wells defined in the plate map are used for the cutoff.

The absorbance range of the Positive and Negative is divided into 10 equal partitions, numbered 0 through 9. The absorbance value of each well is classified according to the partition of the matrix to which it corresponds, and is reported as a signal digit. Wells with absorbance values greater than the Positive are reported by plus signs (+), and wells with absorbance values less than the Negative by minus signs (-).

Single Cutoff Control

The mean absorbance value of the Negative wells that defined in the plate map and the gray zone value that is entered by the user are used to the cutoff. The upper and lower cutoff values are:

Upper cutoff absorbance = Mean of negative controls + ((Gray zone/100) * Mean of negative controls) Lower cutoff absorbance = Mean of negative controls – ((Gray zone/100) * Mean of negative controls)

The absorbance range of the upper and lower cutoff is divided into 10 equal partitions, numbered 0 through 9. The absorbance value of each well is classified according to the partition of the matrix to which it corresponds, and is reported as a signal digit. Wells with absorbance values greater than the upper cutoff are reported by plus signs (+), and wells with absorbance values less than the lower cutoff by minus signs (-).

c. Cutoff Formula

The mean absorbance values of the Positive and Negative wells defined in the plate map are used for the cutoff formula calculation.

The 12 types of formula supported are:

i.	k * CNx	vii.	k + CNx
ii.	k * CPx	viii.	k + CPx
iii.	k * COx	ix.	k + COx
iv.	CNx / k	x.	k*CNx + CPx
v.	CPx / k	xi.	(CNx + CPx) / k
vi.	COx / k	xii.	k1*CNx + k2*CPx
		13

The result absorbance value of the formula calculation and the gray zone value that is entered by the user are used for the cutoff. The upper and lower cutoff values are:

Upper cutoff absorbance = Result of a Formula + ((Gray zone/100) * Result of a Formula) Lower cutoff absorbance = Result of a Formula - ((Gray zone/100) * Result of a Formula)

The absorbance range of the upper and lower cutoff is divided into 10 equal partitions, numbered 0 through 9. The absorbance value of each well is classified according to the partition of the matrix to which it corresponds and is reported as a signal digit. Wells with absorbance values greater than the upper cutoff are reported by plus signs (+), and wells with absorbance values less than the lower cutoff by minus signs (-).

d. Cutoff Ratio

The mean absorbance value of the Calibrator wells defined in the plate map and the concentration value entered by the user are used for the cutoff ratio. Before the cutoff, the absorbance value of each well is converted into the concentration value. The ratio of Concentration/Absorbance of Calibrator is applied to the conversion. Then, the cutoff is done with the defined Positive and Negative or the defined Positive and Gray zone values.

Ranged Cutoff Control

The concentration range of the Positive and Negative is divided into 10 equal partitions, numbered 0 through 9. The concentration value of each well is classified according to the partition of the matrix to which it corresponds, and is reported as a signal digit. Wells with concentration greater than the Positive are reported by plus signs (+), and wells with concentration less than the Negative by minus signs (-).

Single Cutoff Control

The concentration value of the Negative and the gray zone value entered by the user are used for the cutoff. The upper and lower cutoff values are:

Upper cutoff concentration = Positive + ((Gray zone/100) * Positive)

Lower cutoff concentration = Positive - ((Gray zone/100) * Positive)

The concentration range of the upper and lower cutoff is divided into 10 equal partitions, numbered 0 through 9. The concentration value of each well is classified according to the partition of the matrix to which it corresponds, and is reported as a signal digit. Wells with concentrations greater than the upper cutoff are reported by plus signs (+), and wells with concentrations less than the lower cutoff by minus signs (-).

(6)Curve fit report

The Curve fit report provides a regression analysis based on the absorbance values of a series of standards. There are ten types of curve fits supported with the onboard software on Model 680 microplate readers:

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i.5P Logistic Rodbard

ii.4P Logistic Rodbard

iii.5P Logistic Cook-Wilkenson

iv.4P Logistic Cook-Wilkenson

v.5P Exponential

vi.Sigmoid Logistic

vii.Linear regression

viii.Quadratic regression

ix.Cubic spline

x.Point To Point regression

The onboard software calculates the best fit of the defined curve between each set of two consecutive data points in the standard curve of absorbance vs. concentration. And then it generates the report with the coefficients of regression, the correlation coefficient, and the standard error.

For readers with the optional internal printer or with an external ESC/P printer which accepts ESC/P code, a graph of the standard curve may be generated when selected in the report parameter.

(7)Concentration report

In case of the curve fit, the Concentration report also provides the regression analysis. After the calculation of the curve fit, it calculates the concentrations values of the samples, and generates the report.

In case of the cutoff constant with the unit not designated "Abs" and in case of the cutoff ratio, the reader generates the list of concentration of each well.

(8)Difference report

The Difference report is the subtraction report between the neighboring columns or row.

(9)TeSeE Screening report

Analysis of the TeSeE screening assay results can be done with two pre-programmed protocols: “TeSeE” protocol is stored in Endpoint Protocol position #5 and “TeSeE eq” is stored in Endpoint Protocol position #6 as factory default setting. Both protocols are locked.

TeSeE screening report is generated only if these protocols are selected as current active protocol. Plate is first validated according to negative and positive controls validation criteria; samples are then compared to the calculated cut-off.

Criteria for plate validation are set as follow:

Criteria #1:	Individual Negative Control must be < 0.150
	Number of valid Negative Control must be >= 3
Criteria #2:	Individual Negative Control must be < 1.400 * Negative Control average
	Number of valid Negative Control must be >= 2
Criteria #3:	Positive Control average must be >= 1.000

Cutoff is calculated as follow:

TeSeE:

Positive cutoff = Negative Control average + 0.210

Negative cutoff = (Negative Control average + 0.210) * 0.900

TeSeE eq:

Positive cutoff = Negative Control average + 0.210

Negative cutoff = Negative Control average + 0.090

Samples are interpreted as follow:

“POS”:	Positive when sample OD >= Positive cutoff
“NEG”:	Negative when sample OD < Negative cutoff
“???”:	Gray zone: NEG cutoff =< sample OD < POS cutoff

Reports of Kinetic Protocol

(1)Absorbance report

The Kinetic Absorbance report is the series of absorbance data in the kinetic sequence.

(2)Kinetic Plots

The Kinetic plots are the absorbance plots of each well in the plate. It is available for readers with the optional internal printer or with an external ESC/P printer which accepts ESC/P code.

(3)Linear Regression report

The Linear Regression report provides the calculation of Kinetic reaction rate for each well using the linear regression.

(4)GALT report

The GALT report calculates the following formula for each well between two readings of a plate.

GALT = (R2 – R1) * k

Where: R1 = Absorbance value of 1st reading R2 = Absorbance value of 2nd reading k = GALT factor

4.2.6RS-232C Interface

The Model 680 Microplate Reader has a built-in bi-directional RS-232C interface (9 pin D-sub connector, Bio-Rad catalog number: 168-1005). This allows external computers to control the instrument. When an external computer is in control of the microplate reader, the printer and the membrane keys, except for the "Start/Stop" key, are automatically deactivated and the LCD display as below.

Remote mode

The reader will remain in remote control mode until it receives "RL" command from the host computer or until the "Start/Stop" key on the microplate reader is pressed.

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4.2.7Printer

The Model 680 Microplate Reader has a built-in printer interface connector (25 pin D-sub) or an optional factory built-in printer.

The printer interface connector allows the user to connect an external printer for the printout of reports or protocol. This external printer interface is based upon Centronics interface standards.

Two types of printer, ASCII type and ESC/P type, are allowable to connect. The ASCII type printer can only accept ASCII character based codes which does not include any graphic codes. Next, the ESC/P type printer can accept ESC/P codes which include graphic codes.

The optional factory built-in internal printer and the external ESC/P type printer can generate all types of report including graphs and kinetic plots.

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